Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated From Dairy Products

Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated from Dairy Products
by
Imran Javed
Department of Microbiology Quaid-i-Azam University, Islamabad 2009
Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated from Dairy Products
by Imran Javed A thesis submitted in fulfillment of the requirements for the award of degree of
Doctor of Philosophy (Ph.D) In Microbiology
Department of Microbiology Quaid-i-Azam University, Islamabad 2009
DEDICATION
Dedicated to my Mother (Late) & all of my Family
DECLARATION
The material contained in this thesis is my original work and I have not presented any part of this thesis/work elsewhere for any other degree.
Imran Javed
CERTIFICATE This thesis by Imran Javed is accepted in its present form by the Department of Microbiology, Quaid-i-Azam University, Islamabad, and is satisfying the thesis requirements for the award of degree of
Doctor of Philosophy (Ph.D) in Microbiology
External examiner
_____________________ (Prof. Dr. Fauzia Y. Hafeez)
External examiner
_____________________ (Dr. Jahangir Arshad Khan)
Internal examiner
_____________________ (Dr. Safia Ahmed)
Chairman
_____________________ (Prof. Dr. Abdul Hameed)
Dated: 30-04-2009
Table of Contents
Sr. No. Title Page No. Chapter 1 Introduction
1.1. Food borne diseases and public health 1.2. Microbial solutions to microbial problem 1.3. Lactic Acid bacteria 1.4. Use of lactic acid bacteria in food industry 1.5. Enterococcus as a agent for control of Listeria and Clostridia 1.6. Enterococci and health perspective 1.7. Bacteriocins 1.8. Identification of enterococci with modern molecular techniques 1.9. Aims and objectives of the research project 1 2 3 5 6 8 9 12 14
Chapter 2 Review of Literature

2.1. Bacteriocins 2.1.1. History 2.1.2. Classification 2.1.3. Production and characterization of bacteriocins 2.1.4. Application 2.2. Food Safety Issues 15 15 17 21 26 30
Chapter 3 Materials and Methods

3.1. Isolation and Characterization of Lactic Acid Bacteria 3.1.1. Isolation and growth characteristics 3.1.2. Morphological and biochemical characterization 3.1.3. Molecular Characterization 3.1.3.1. Extraction of genomic DNA 3.1.3.2. PCR amplification for 16S rRNA of Enterococcus spp. 3.1.3.3. PCR amplification for 23S rRNA of Enterococcus spp. 3.1.3.4. PCR product purification 3.1.3.5. DNA sequencing and identification of bacterial strains 3.1.4. Safety Assessment of Isolated Enterococcus spp. 33 33 34 35 35 35 36 38 38 40
3.1.4.1. Antibiotic susceptibility of the isolates 3.1.4.2. Qualitative assessment of virulence factors 3.1.4.3. Detection of virulence genes from isolated strains 3.2. Bacteriocin Production by Bacterial Isolates 3.2.1. Screening of isolates for bacteriocin potential 3.2.2. Bacteriocin production trials by selected isolates 3.2.3. Optimization of physical and chemical parameters 3.3. Analytical Methods 3.3.1. Bacteriocin quantification 3.3.2. Study of growth pattern of bacteriocin producing isolates 3.3.3. Protein estimation 3.4. Characterization of Bacteriocin by Selected Isolates 3.4.1. Locus identification of bacteriocin genes
40 40 40 42 42 42 43 44 44 44 46 46 46
3.4.2. Characterization of bacteriocins genes with secondary PCR 46 3.4.3. Cloning and transformation of bacteriocin genes in E. coli 3.4.4. Partial purification of bacteriocins 49 49
3.4.5. Sensitivity of bacteriocins to enzymes, pH and heat treatment 50 3.4.6. Antilisterial activity 3.4.7. Purification by chromatography 3.4.8. Molecular weight estimation by SDS-PAGE 51 51 51
Chapter 4 Results
4.1. Isolation and Characterization of Lactic Acid Bacteria 4.1.1. Isolation and growth characteristics 4.1.2. Morphological and biochemical characterization 4.1.2.1. Preliminary identification of isolated strains 4.1.3. Molecular characterization 4.1.3.1. Extraction of genomic DNA 4.1.3.2. PCR amplification of 16S rRNA of isolated enterococci 4.1.3.3. PCR amplification of 23S rRNA of isolated enterococci 4.1.4. Safety Assessment of Isolated Enterococcus Strains 53 53 53 56 58 58 58 60 65
4.1.4.1. Antibiotic susceptibility of the isolates 4.1.4.2. Assessment of virulence factors by qualitative tests 4.1.4.3. Detection of virulence genes from isolated strains 4.2. Bacteriocin production by selected bacterial isolates 4.2.1. Screening of Isolates for Bacteriocin Production 4.2.2. Study of growth pattern of potential strains 4.2.3. Optimization of growth parameters and media composition 4.2.3.1. Optimization of temperature and incubation time 4.2.3.2. Optimization of media pH 4.2.3.3. Optimization of inoculum size 4.2.3.4. Optimization of glucose and NaCl concentrations
65 65 65 68 68 68 72 72 75 78 78
4.2.3.5. Influence of medium components on bacteriocin production 78 4.3. Characterization of Bacteriocin 4.3.1. Plasmid curing 84 84
4.3.2. Characterization of bacteriocins genes with Secondary PCR 84 4.3.3. Cloning and transformation of bacteriocin genes in E. coli 4.3.4. Isolation and partial purification of enterocins 4.3.5. Sensitivity of enterocins to enzyme, pH and heat treatment 4.3.6. Mode of inhibition 4.3.7. Inhibitory spectrum 4.3.8. Tricine-SDS-PAGE 4.3.9. Gel filtration using sephadex G-50 88 89 89 93 93 93 94 100 109
Chapter 5 Discussion References
ACKNOWLEDGEMENT
I bow my head in deep gratitude to Allah, The Almighty, Who is the intact source of knowledge and wisdom to me and all mankind. I owe my deep respect to Hazrat Muhammad (Peace Be Upon Him), the city of knowledge and mercy for the entire universe.
Almighty Allah has vastly blessed me through the people who have contributed to the completion of this thesis. The completion of this thesis would not have been possible without their support and patience.
I wish to extend my greatest appreciation to my professor and thesis supervisor, Dr. Safia Ahmed, Associate Professor Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad for her suggestions, guidance, support, and encouragement in the completion of this thesis and during the last four years in providing the basic research facilities and supervision throughout my research project.
Im thankful to Professor Dr. Abdul Hameed, Chairman Department of Microbiology, Quaid-i-Azam University, Islamabad for his kind attitude, ever new cooperation along with non ending valuable guidance.
With pleasure and help deep sense of indebtedness, I acknowledge the invaluable guidance extended to me during entire research work by Dr. Fariha Hasan and Dr. Aamer Ali Shah.
I tender my thanks to M. Ishtiaq Ali, Bashir Ahmad, Arfat Yameen, Ghulam Raza, Tariq Mehmood, Sajad Shakuat Ph.D. research fellows for their energetic cooperation, moral support and sincere help, with out which it would not have been possible for me to make my head way in my research.
I also wish to thank all of my lab fellows especially Pir Bux, Zulfiqar, Fazal, khalid, Masroor, Tatheer, Sadiq, Naima, Zainab, Maryam, and Sadia along with Shazad, Sohail and Payra with whom I have had the opportunity to work, for their patience and willingness to share their great expertise.
As with all things that have or may accomplish, I thank God above all others for continuously blessing my life and giving me the strength to persevere during trying times. One of the greatest blessings He has given me is my family. Without the love and guidance of my father and late mother, have provided throughout my life, I have no doubt that success in any capacity would be unachievable for me. My appreciations are also reserved for my brothers Dr. M. Tayyeb Javed and Rizwan Javed, my sisters Nazia and Munazza.
Last but not the least I am greatly indebted to my better half Abida Parveen for her love, kind and mellifluous affections, which hearten me to achieve success in every sphere of life, without whose encouragement and moral support to perceive and peruse higher studies would a mere dream.
This project was funded by Higher Education Commission of Pakistan (HEC) with Indigenous Research Fellowship and International Research Support Initiative Programme (IRSIP) for characterization work at University of Texas at San Antonio under the kind supervision of Dr. G. Jilani Chaudry, Assistant Professor Department of Biology, University of Texas at San Antonio, USA. Author would like to acknowledge Gent Culture Collection Centre, Belgium for providing the control strains for this study.
Imran Javed
LIST OF ABBREVIATIONS
B. cereus B. subtilis BHI CDC DNA Ent. faecium Ent. faecalis Ent. durans Ent. bovis EU FDA G+C content GRAS GCMS HPLC HSV KAA KDa LAB L. monocytogenes L. innocua M MRS MLDI-TOF
Bacillus cereus Bacillus subtilis Brain Heart Infusion Center for disease control Deoxyribose nucleic acid Enterococcus faecium Enterococcus faecalis Enterococcus durans Enterococcus bovis European Union Federal Drug Administration Guanine + Cytosine contents Generally recognized as safe Gas chromatography mass spectroscopy High performance liquid chromatography Herpes simplex viruses Kanamycin aesculine azide agar medium Kilo Dalton Lactic acid bacteria Listeria monocytogenes Listeria innocua Molarity de Man Rogosa Sharpe Matrix assisted laser desorption/ionizationtime of flight
N NCI-ELISA
Normality Noncompetitive indirect enzyme linked
immunosorbent assay OD Optical density
PAGE RNA rRNA SDS SB Tm TAE UV USDA VRE
Poly acrylamide gel electrophoresis Ribose nucleic acid Ribosomal ribose nucleic acid Sodium dodecyl sulphate Slantz and Bartely medium Melting Temperature Tris acetate EDTA buffer Ultra Violate United States Department of Agriculture Vancomycin resistant enterococci
LIST OF TABLES
Table No. 2.1. 3.1. Title List of characterized enterocin with their amino acid sequences Page No 20
Primers for amplification of 16S and 23S rRNA from isolated 37 Enterococcus spp.
3.2.
Sequencing primers used for 16S and 23S rRNA nucleotide 39 sequencing of isolated Enterococcus strains
3.3.
Primers
for
amplification
of
virulence
determinants
from 41
Enterococcus spp. 3.4. 3.5. Composition of MRS broth 43
Modified MRS media with different levels of peptone, yeast extract, 45 meat extract and tween 80
3.6.
Primers used for amplification of bacteriocins genes from isolated 48 strains of enterococci
4.1. 4.2. 4.3.
Morphological characterization of bacterial isolates Growth characteristic and screening of bacterial isolates
54 55
Identification of isolated enterococci with API 20 Strep system 57 (bioMrieux, Germany)
4.4.
16S rRNA nucleotide BLAST results of isolated strains of 64 enterococci and control
4.5.
Detection of virulence determinants and antibiotics resistance of 66 bacteriocin producing isolates
4.6. 4.7.
Antimicrobial activity of isolated Enterococcus strains
69
Effect of different concentrations of glucose and NaCl on 81 bacteriocin production
4.8.
Influence of initial pH of MRS medium on numbers (log CFU/ml) 82 and bacteriocin production (AU/ml) after growth for 24 h at 37C
4.9
Influence of medium composition on numbers (log CFU/ml) and 83 bacteriocin production (AU/ml) of isolated strains
4.10.
Potential strains for bacteriocins and PCR amplification of 86
bacteriocin gene 4.11. Activity of bacteriocin-producing enterococci against different indicator strains 4.12. Antilisterial activity of selected bacteriocin-producing strains using 91 L. monocytogenes as indicator strains 4.13. Summary of purification of bacteriocin from crude culture filtrate of 98 Enterococcus faecium IJ-31 90
LIST OF FIGURES
Fig. No. 1.1. Title Page No.
The position of enterococci among benefits and risks in medical and food 7 microbiology
4.1.
PCR amplification for 16S rRNA from isolated Enterococcus strains on 59 0.9% agarose gel
4.2.
PCR amplification for 23S rRNA from isolated Enterococcus spp. on 61 0.9% agarose gel
4.3.
4.4.
4.5.
Antibiotics suscptibility of isolated enterococci to commonly used 67 antibiotics.
4.6.
Growth pattern of isolated strains and bacteriocin production by Ent. 70 faecium IJ-21
4.7.
Growth pattern of isolated strains and bacteriocin production by Ent. 70 faecium IJ-31
4.8.
Growth pattern of isolated strains and bacteriocin production by Ent. 71 faecium LMG 11423T
4.9.
Time course of bacteriocin production by selected bacterial isolates at pH 73 7.0 and 30 C
4.10.
4.11.
4.12.
Effect of media pH for bacteriocin production form Ent. faecalis IJ-11 at 76 35 C
4.13.
Effect of media pH for bacteriocin production from Ent. faecium IJ-21 at 76 35 C
4.14.
Effect of media pH for bacteriocin production from Ent. faecium IJ-31 at 77 35 C
4.15.
Effect of media pH for bacteriocin production from Ent. faecium LMG 77 11423T at 35 C
4.16.
Bacteriocin production by selected bacterial isolates with different size of 80 inolulum in batch fermentation
4.17. 4.18. 4.19.
Plasmid isolation of isolated strains with Qiagen plasmid mini prep Amplification of bacteriocin genes from isolates on 2.0% Agarose gel
85 87
Effect of different enzymes on the residual activity of partially purified 92 bacteriocin
4.20. 4.21.
Thermo-stability assay of partially purified bacteriocin
92
Antimicrobial assay SDS-PAGE Gel on Ent. faecalis IJ-11 as indicator 95 strain
4.22.
Antimicrobial assay SDS-PAGE Gel (20 and 40% of a. sulphate 96 saturation) on L. monocytogenes as indicator strain
4.23.
Purification of bacteriocin by Sephadex G-50 column
97
Summary
Aim of this study was to isolate, identify and characterize enterococci from processed dairy products of Pakistan and to investigate their safety level and their potential technological properties. Initially 34 bacterial strains were isolated mainly from cheese, butter and yoghurt samples on de Man Rogosa Sharpe (MRS) agar. Selective enumeration on kanamycin aesculin azide medium revealed seven out of 34 strains as members of genus Enterococcus. Cheese was the best source for the isolation of enterococci followed by butter and yoghurt. Standard biochemical, phenotypic and morphological tests as well as with API 20 Strep system (bioMrieux, Germany) identified four isolates (IJ-03, IJ-07, IJ-11 and IJ-12) as Enterococcus faecalis and three (IJ-06, IJ-21 and IJ-31) as Enterococcus faecium. Molecular characterization of these isolates was done by 16S and 23S rRNA amplification. Approximately 1.5 kb fragment was amplified in case of 16S rRNA, while 2.8 kb fragment was amplified for 23S rRNA. Nucleotide sequencing and molecular characterization confirmed the identification of these isolates. These strains were clonally related having homology in their conserved regions. The 16S and 23S rRNA sequences were deposited in NCBI Gene Bank with following accession numbers of Ent. faecalis IJ-03 (EU547773, EU547781), Ent. faecium IJ-06 (EU547774, EU547782), Ent. faecalis IJ-07 (EU547775, EU547783), Ent. faecalis IJ-11 (EU547776, EU547784), Ent. faecalis IJ-12 (EU547777, EU547785), Ent. faecium IJ-21 (EU547778, EU547786), Ent. faecium IJ-31 (EU547777, EU547787) and control strain Ent. faecium ATCC 27273 (EU547780, EU547788).
Safety investigations revealed that all of the Enterococcus isolates were sensitive to commonly used antibiotics vancomycin, teicoplanin, ampicillin, erythromycin, ciprofloxacin, tetracycline and gentamycin but found to be resistant against methicillin and kanamycin. Minimum inhibitory concentration (MIC) values of the isolated strains against glycopeptide antibiotics was in
range of 1-4 g/ml. Absence of known virulence determinants along with antibiotics resistance was confirmed by amplification through PCR for critical virulence genes mainly cytolysin (cyl), haemolysin, gelatinase (gel), enterococcal surface protein (esp), vancomycin resistance type A (vanA) and type B (vanB). Antimicrobial screening revealed Enterococcus faecium IJ-21 and IJ-31 strains as the potential producer of bacteriocin showing good antimicrobial activity in supernatant while Ent. faecium IJ-06 which activity in stab overlay assay and after partial purification. Isolated strains showed good antimicrobial potential against closely related strains. Ent. faecium IJ-06, Ent. faecalis IJ-11 and control strain showed a narrow spectrum of activity and it was mainly observed against Enterococcus spp. along with Listeria spp. On the other hand Ent. faecium IJ-21 and Ent. faecium IJ-31 were active against a broad range of microorganisms including Listeria spp. Enteroccous spp. Bacillus cereus and Bacillus subtlis. Enterococcus faecium IJ-31 also showed week inhibition against S. aureus. Optimization of growth parameters revealed the maximum bacteriocin was produced at pH 7 and 8 at 35 C after 24hrs of incubation with 1% inoculum. A moderate level of NaCl enhances the bacteriocin production while increasing the concentration of glucose had no profound effect. Molecular characterization of these isolates was done by amplification of previously known bacteriocin genes. Polymerase chain reaction analyses revealed that both enterocin A and P genes were present in the genome of Ent. faecium IJ-06 and Ent. faecium IJ-21. Enterocin P was detected in the genome of Ent. faecium IJ-31 but no signal was found for Ent. faecalis IJ-11 indicating the antimicrobial activity due to enterocin other than checked through PCR.
The enterocins produced by selected strains Ent. faecium IJ-06, IJ-21, IJ-31 and ATCC 27273 were partially purified with ammonium sulphate precipitation (20 and 40%) followed by chloroform and methanol extraction. Enterocin from Ent. faecium IJ-31 was further purified with gel filtration chromatography. Molecular weight of the enterocins was in range of 4.5-5.0 kDa when checked
by SDS-PAGE followed by antimicrobial activity assay. Single zones of inhibition were observed from enterocins of Ent. faecium IJ-06, IJ-21, IJ-31 and control, showing the production of one type of bacteriocin. The partially purified enterocins of Ent. faecium IJ-06, IJ-21 and IJ-31 were highly thermostable, retaining their activity even after treatment with 121 C for 15min, and were stable in a pH range of 4-9 with higher activity at acidic pH. The activity was readily lost after protease treatment while catalase, lipase and lysozyme showed no effect. So the isolated enterococci are promising candidate for further investigation of their technological potential in relation to their use in dairy food products to avoid Listeria contamination.
Chapter 1
Introduction
1.1. Food Borne Diseases and Public Health

Microbiological food safety is a complex, fundamental issue of continuing concern. Contributing to this complexity and emergence of food safety issues are ongoing changes in demographic, geographic origin of food, food production and processing (IFT expert report 2002). There are more than 250 known diseases that are transmitted through food. Most of these diseases are infections, caused by a variety of bacteria, viruses, and parasites that can be foodborne. While other diseases are poisonings, caused by harmful toxins or chemicals that contaminate food, for example, poisonous mushrooms. These different diseases have many different symptoms, so there is no one "syndrome" that is foodborne illness. However, the microbe or toxin that enters the body through the gastrointestinal tract, and causes the symptoms e.g. nausea, vomiting, abdominal cramps, diarrhea etc may be foodborne. (www.cdc.gov/ncidod/dbmd/diseaseinfo.htm) In the United States alone food borne diseases have been estimated to cause approximately 76 million illness, 325,000 hospitalization and 5,000 deaths each year (Mead et al., 1999).
The spectrum of foodborne diseases is constantly changing. A century ago, typhoid fever, tuberculosis and cholera were common foodborne diseases. Improvements in food safety, such as pasteurization of milk, safe canning, and disinfection of water supplies have conquered those diseases. However some other foodborne infections have taken their place e.g. food borne salmonelsis, listeriosis, staplococcal food poisoning etc. (www.cdc.govdbmd/ diseaseinfo/foodborneinfections_g.htm#typeschanging).
Salmonella, Listeria monocytogenes and Toxoplasme gondii are emerging food borne pathogens responsible for more than 78% of total deaths. The issue is further complicated by pathogen Listeria, that cause little or no illness in healthy individuals but causes severe illness and death in sensitive
Characterization of bacteriocin produced by Lactic Acid Bacteria isolated from dairy products
Chapter 1
Introduction
populations including immuno-compromised and immuno-suppressed and the elderly and developing fetus (Mead et al., 1999).
Even though Listeria monocytogenes was first time recognized as a human pathogen in 1929, the route of infections was unknown until the 1980s, when outbreaks of listeriosis were associated with consumption of contaminated food (Fleming et al., 1985; Linnan et al., 1988). Listeria monocytogenes is a ubiquitous microorganism which is widely distributed in nature and has been isolated from a variety of foods, including dairy and meat products. Contamination of food with Listeria monocytogenes continues to be a threat to public health (Gaya et al., 1996; Wan et al., 1997).
Listeria monocytogenes was frequently overlooked as a possible cause of illness due to its unique growth characteristics. First, it is somewhat difficult for laboratories to grow, and when they do so, Listeria can be confused with common harmless contaminants and disregarded. Second, most bacteria grow poorly at temp. 4 C, while Listeria survives in temperatures from below freezing to body temperature as it grows best at -18 to 10 C. As a result, Listeria may be transmitted in ready to eat foods that have been kept properly refrigerated. Its ability to grow in such diverse environments is just one of the many challenges presented by this dangerous bacterium (Dieterich et al., 2006). Listeria infections are relatively uncommon. However, the fatality rate can be as high as 30% among risk people. It is estimated that Listeria causes approximately 1600 cases of listeriosis annually, resulting 400 to 500 deaths (Ramaswamy et al., 2007).
1.2. Microbial Solutions to Microbial Problems

Microbial solutions to the microbial problems are one of the best possible solutions to these emerging food borne problems. The bacteriocins are antimicrobial peptides, naturally produced by Lactic Acid Bacteria (LAB). These compounds vary in spectrum and mode of activity, molecular structure and molecular mass, thermostability, pH range of activity, and genetic determinants (De Vuyst and Vandamme, 1994; Nes et al., 1996; Nissen-
Chapter 1
Introduction
Meyer and Nes, 1997; Moll et al., 1999; Ennahar et al. 2000; Cleveland et al., 2001; McAuliffe et al., 2001; Riley and Wertz, 2002; Eijsink 2002).
Nisin was the first lantibiotic discovered in the 1920s. It is a 34 amino acid peptide produced by some strains of Lactococcus lactis with a wide spectrum of inhibition against Gram-positive bacteria. Nisin has a wide range of applications due to its broad bactericidal mode of action and because it can be easily broken down by digestive proteases without disturbing gut biota. It is the only lantibiotic to date that has been approved for commercial use. It was added to the positive list of European Union (EU) food additives in 1983 as additive number E234 and was approved by the Food and Drug Administration (FDA) in 1988. Nisin is now approved for use as a food preservative in approximately 50 countries (Ennahar et al., 2000; Cleveland et al., 2001; Riley and Wertz, 2002; Rodriguez et al., 2003; FDA, 2003).
1.3. Lactic Acid Bacteria (LAB)

Lactic acid bacteria are a group of related bacteria that produce lactic acid as a result of carbohydrate fermentation. These microbes are broadly used in the production of fermented food products, such as yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.), sauerkraut
(Leuconostoc spp.) and sausage and other fermented foods. Lactic acid bacteria are often associated with animal oral cavities and intestines (e.g. Enterococcus faecalis); plant leaves (Lactobacillus,
Leuconostoc) as well as decaying plant or animal matter such as rotting vegetables, fecal matter, compost, etc. These bacteria usually found in decomposing plants and dairy products and produce lactic acid as the major metabolic end product of carbohydrate fermentation. This trait has historically linked LAB with food fermentations as acidification inhibits the growth of spoilage agents. Furthermore, lactic acid and other metabolic products contribute to the organoleptic and textural profile of a food item. The industrial importance of the LAB is further evidenced by their Generally Regarded As Safe (GRAS) status, due to their ubiquitous appearance in food and their
Chapter 1
Introduction
contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the LAB are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella (Sabia et al., 2002, 2004).
Fermentation of various food stuffs by lactic acid bacteria (LAB) is one of the oldest forms of biopreservation practiced by mankind. Bacterial antagonism has been recognized for over a century but in recent years this phenomenon has received more scientific attention, particularly in the use of various strains of lactic acid bacteria. One important attribute of many LAB is their ability to produce antimicrobial compounds called bacteriocins. In recent years interest in these compounds has grown substantially due to their potential usefulness as natural substitute for chemical food preservatives in the production of foods with enhanced shelf life and safety. There is growing consumer awareness of the link between diet and health. Recent scientific evidence supports the role of probiotic LAB in mediating many positive health effects. Traditional probiotics dairy strains of lactic acid bacteria have a long history of safe use and most strains are considered commensal microorganisms with no pathogenic potential (Soomro et al., 2002).
Lactic acid bacteria (LAB) occur naturally as indigenous microbiota in raw milk and several strains associated with food systems, produce bacteriocins able to inhibit spoilage and pathogenic micro-organisms (Jack et al., 1995). Thus biotechnologists are becoming increasingly interested in characterizing them with a view to use in food preservation in dairy systems. Among LAB, members of the enterococci are widely distributed throughout nature as inhabitants of the gastrointestinal tract of humans and other animals and are also present in vegetables, plant material and foods (Giraffa 2002). Bacteria belonging to this genera are recognized by their bacteriocinogenic activity against Listeria monocytogenes and in recent years new enterocins have been characterized by many research groups (Galvez et al., 1989, 1998; Aymerich et al., 1996; Casaus et al., 1997; Cintas et al., 1997,1998, 2000;
Chapter 1
Introduction
Ennahar et al., 1998, 1999; Herranz et al., 1999, 2001; Du Toit et al., 2000; Ohmomo et al., 2000; Sabia et al., 2002, 2004; Marekova et al., 2003).
1.4. Use of Lactic acid bacteria (LAB) in food Industry

The beneficial role of LAB and their safety in food fermentation are well documented. They are extensively used in food processing, such as in dairy and meat fermented products for their contribution to shelf life, texture, and organoleptic properties (Cogan and Hill, 1993; Stiles 1996; Wood, 1997). These microorganisms have been used in food and feed preservation for centuries since they can produce a variety of antimicrobial agents, including organic acids like lactic and acetic acid, ethanol, carbon dioxide, diacetyl, and hydrogen peroxide (Gould 1992; De Vuyst and Vandamme, 1994; Holzapfel et al., 1995). Further more, some of them inhibit spoilage bacteria and foodborne pathogens including Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum, and thus may be of great interest to the food industry as natural food preservatives (Stiles, 1996).
Strains used as probiotics usually belong to species genera Lactobacillus, Enterococcus, and Bifidobacterium. These LAB are typically
chemoorganotrophic and ferment carbohydrates with lactic acid as a major end product. Some physiological characteristics are of interest for their function as probiotics, a pre-condition of which is survival in the gastrointestinal tract and low pH, bile and their temperature growth (Fuller, 1989). In the last decade, numerous strains of lactic acid bacteria have been isolated which show production of bacteriocins. Producers were also found among representatives of the genus Enterococcus (Klaenhammer 1988; Parente and Hill, 1992; Laukova et al., 1993; De Vuyst and Vandamme, 1994; Vlaemynck et al., 1996; Farias et al., 1996; Casaus et al., 1997; Cintas et al. 1997; Cintas et al., 2000; Ennahar et al., 2000; Marekova et al., 2007).
Chapter 1
Introduction
1.5. Enterococcus as an Agent for Control of Listeria and Clostridia

Enterococci are associated with several fermented foods, especially artisanal dairy products such as Mediterranean cheeses, where they are thought to play a role in flavor and aroma development (Centeno et al., 1999). Although some strains have been implicated as causal agents of antibiotic resistance and human infections, non-pathogenic enterococci seem to have promising commercial potential (Franz et al., 1999). Enterococci are already being used as starter cultures for certain artisanal food fermentations and are currently commercially available as probiotics (Giraffa, 1995; Stiles and Holzapfel, 1997). Moreover, many enterococci produce one or more bacteriocins (enterococcins and enterocins), and may therefore be considered as protective towards spoilage and pathogenic bacteria (De Vuyst, 1994; Giraffa, 1995; Cintas et al., 1997). The genus Enterococcus is the most controversial group of lactic acid bacteria. Studies on the microbiota of many traditional cheeses in the Mediterranean countries have indicated that enterococci play an important role in the ripening of these cheeses, probably through proteolysis, lipolysis, and citrate breakdown, hence contributing to their typical taste and flavor. Enterococci are also present in other fermented foods, such as sausages and olives. However, their role in these products has not been fully elucidated. Furthermore, the production of bacteriocins by enterococci is well
documented. Moreover, enterococci are nowadays used as probiotics. At the same time, however, enterococci have been associated with a number of human infections. Several virulence factors have been described and the number of vancomycin-resistant enterococci is increasing (Sarantinopoulos et al., 2001). Enterococci are widespread in nature and constitute essential part of the gut microbiota of human and animals. However their presence in food is controversial: enterococci are considered as indicators of fecal contamination and responsible for food spoilage, but also beneficial for their potential use as
Chapter 1
Introduction
starter cultures in food fermentation, their use as probiotics, and their ability to produce antimicrobial peptides generally referred as bacteriocins (Cintas et al., 2000; Eijsink et al., 2002) Fig. 1.1. Benefits Probiotics strains Food fermentation Feed fermentation culture Producer of bacteriocins Indicator Microorganism Enterococcus Fecal contamination Carrier of virulence factors Involvement in nosocomial infections Risks Donor and receptor of genes (e.g. antibiotic resistance properties
Fig.1.1. The position of enterococci among benefits and risks in medical and food microbiology.
The interest in natural preservatives is increasing, which is in accordance with the consumers demand for healthy, safe and fresh food. However, chemicals such as potassium nitrate are still used in cheese making to prevent late gas formation by Clostridium tyrobutyricum during cheese ripening. On the other hand, the risk of Listeria spp. contamination of milk and cheese must be minimized to achieve a zero tolerance policy (De Vuyst, 2000).
In general, bacteriocins produced by Enterococcus faecium and Enterococcus faecalis are small, hydrophobic and thermostable peptides with an interesting technological potential (Giraffa, 1995). Indeed, an increasing number of enterococcal bacteriocins against foodborne pathogens such as Listeria spp. and Clostridium spp. have been reported (Ben Embarek et al., 1994; Farias et al., 1994; Giraffa et al., 1994; Maisnier-Patin et al., 1996; Vlaemynck, 1996; Bennik et al., 1999; McAuliffe et al., 1999; Mendoza et al., 1999). Even activity towards Gram negative bacteria like Vibrio cholerae has been shown
Chapter 1
Introduction
(Simonetta et al., 1997). Wachsman et al., (1999) reported the antiviral activity of 3.5 KDa enterocin CRL 35 from Enterococcus faecium against Herpes simplex viruses type I and 11 (HSV-1 and HSV-2).
The use of bacteriocinogenic lactic acid bacterial strains as starter or cocultures is a promising alternative in food fermentation processes like cheese manufacture, both to prevent late blowing and to combat Listeria spp. (De Vuyst 2000).
Enterococci are also used as starter cultures in some cheeses and even as animal and human probiotics. Many food hygienists are still reluctant to accept the presence of enterococci in food because there is no consensus as to whether they can be generally recognized as safe micro-organisms, despite their long history in foods without causing health concerns (Achemchem et al., 2005).
1.6. Enterococci and Health Perspective

Enterococcus is a genus of lactic acid bacteria of the phylum Firmicutes. Members of this genus were classified as Group D Streptococcus until 1984 when genomic DNA analysis indicated that a separate genus classification was appropriate Schleifer and Kilpperbazz (1984). The genus Enterococcus is comprised of Gram-positive, microaerophilic cocci, which are not motile and occur in chains or pairs. These are difficult to distinguish from Streptococci on physical characteristics alone. The genus is defined by a combination of
antigenic, haemolytic, and physiological characteristics. Enterococci are facultative anaerobic organisms (Fischetti et al., 2000). They typically exhibit gamma-hemolysis on sheep's blood agar. Enterococcus is widely present in food products like sausage, evaporated milk, cheese, meat croquettes, meat pie, pudding, raw milk, and pasteurized milk. Entrance into the food chain is due to under-processing and/or poor and unsanitary food preparation.
Chapter 1
Introduction
Two species are common commensal organisms in the intestines of humans: Ent. faecalis (90-95%) and Ent. faecium (5-10%). Enterococcus are also a common cause of nosocomial diseases of which major cause are Ent. faecalis which accounts for 8090% and E. faecium for 510% of clinical enterococci. Enterococcus faecalis can cause endocarditis, as well as bladder, prostate, and testicle infections; nervous system infections are less common. The existence of enterococci in such a dual role is facilitated by its intrinsic and acquired resistance to virtually all antibiotics currently in use (Ryan and Ray 2004). Enterococcus faecalis is resistant to many commonly used antimicrobial agents (aminoglycosides, aztreonam, cephalosporins, clindamycin, penicillins, nafcillin, oxacillin and trimethoprim-sulfamethoxazole). Exposure to
cephalosporins is a particularly important risk factor for colonization and infection with enterococci. Resistance towards vancomycin and ampicillin has increased alarmingly over the past few years. In 1997, 52% and 83% of isolates sampled in the United States were vancomycin and ampicillin resistant, respectively.
1.7. Bacteriocins
Based on structural, physiochemical and molecular properties, bacteriocins from LAB can be subdivided into three major classes. Nonetheless, this classification is continuously being reviewed and it is evolving with the accumulation of knowledge and the appearance of new bacteriocins (Cleveland et al., 2001; Ennahar et al., 2000; Riley and Wertz, 2002; Eijsink et al., 2002; Rodriguez et al., 2003; Cotter et al., 2005; Fimland et al., 2005). Class I bacteriocins are modified bacteriocins, the lantibiotics, i.e. small, cationic, hydrophobic, and heat-stable peptides that contain unusual amino acids (e.g. the thioether amino acids lanthionine and/or 3-methyl-lanthionine). Class II bacteriocins are small, cationic, hydrophobic, heat-stable peptides that are not post-translationally modified, except for cleavage of a leader peptide from the prebacteriocin peptide. Within this class, three subclasses can be distinguished: subclass IIa or pediocin-like bacteriocins with a strong
Chapter 1
Introduction
antilisterial effect, possessing the consensus sequence YGNGV in their Nterminus; subclass IIb or bacteriocins that require two polypeptide chains for full activity; and subclass IIc are bacteriocins that do not belong to the other subgroups. Class III bacteriocins are a group of large, hydrophilic, and heatlabile proteins. Since 1955, when the first bacteriocin-like substance within the group D streptococci was reported by (Kjems, 1955), a large number of enterocins has been studied. Kramer and Brandis (1975) reported the antiListeria activity of the enterocin E1A produced by E. faecium E1. The first enterocin purified to homogeneity was the enterocin AS-48 produced by E. faecalis S-48 (Galvez et al., 1989; Martinez-Bueno et al., 1994), which was defined as a cyclic peptide antibiotic. Since then, the number of new enterocins characterized is increasing. However, many of these molecules have not been purified to homogeneity. Enterocins are found within the class I, class IIa, class IIc, and class III bacteriocins mentioned above.
Bacteriocin production is widespread among LAB present in milk or dairy products (Vaughan et al., 1994; Martinez et al., 1995; Coventry et al., 1997). The identification of LAB bacteriocins with broad inhibitory spectra against spoilage and pathogenic microorganisms has gained increasing interest towards their application as food preservatives in dairy systems. Bacteriocins are agents which are most often encoded in the genetic material carried by plasmids, with the purpose of killing or inhibiting closely related species or even different strains of the same species. This property is similar to that of the antibiotic resistance genes which are also encoded by plasmids, however bacteriocins are much more specific in their action. Bacteriocins arisen from the need for survival in over populated environment / culture, thus cells containing the plasmid encoding for a bacteriocin, have the capability of destroying surrounding cells without the bacteriocin plasmid. There are several inherent characteristics of bacteriocins that distinguish it from other microbial compounds, these have low molecular weight, narrow and specific antimicrobial spectrum, these are synthesized on ribosomes, undergo post transcriptional modifications, and these are non-toxic to
10
Chapter 1
Introduction
eukaryotes. The genetic determinants of most of the bacteriocins are located on the plasmids, apart from few which are chromosomally encoded and producer cells are generally immune to them (Nissen-Meyer and Nes, 1997).
Proteolytic enzymes rapidly inactivate the antagonistic activity whereas glycolytic and lipolytic enzymes had no effect. Bioactivity may remain stable in the presence of several organic solvents and detergents. They have potential application against a wide range of human and animal diseases (Cleveland et al., 2001). Due to their resistance to temperature and low pH, the bacteriocins are digested by human and animal peptidases, thus avoiding resistance and problems associated to the presence of residues in feed and food (Russel and Mantovani, 2002). Bacteriocins are of interest in medicine because they are made by nonpathogenic bacteria that normally colonize the human body. Loss of these harmless bacteria following antibiotics use may allow opportunistic pathogenic bacteria to invade the human body. Bacteriocins are named after the species that produces them e.g. colicins from E. coli, enterocins from Enterococcus spp., mutacins from S. mutans and epidermin from S. epidermidis etc. It should also be noted that a naturally occurring plasmid only encodes for one type of bacteriocin, if it encodes for one at all. A much studied bacteriocin is colicin, which is encoded by the Col plasmids, and as expected, originates from the bacterial species, Escherichia coli. Colicins function by disrupting the cell membrane of surrounding cells. The bacteriocins produced by the Ent. faecalis strains show a narrow spectrum of activity, mainly against other Enterococcus spp. compared with those from the E. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The foodborne pathogen Listeria monocytogenes has become the focus of bacteriocin investigators during the past decade. This has led to the description of a large number of anti-listerial bacteriocins. The best studied anti-listerial bacteriocin is nisin. One approach to reduce the prevalence of Listeria in milk products is the application of bacteriocin and/or
11
Chapter 1
Introduction
bacteriocin producing cultures. Enterococcal bacteriocins characterized at the biochemical level (enterocins A, B, I, and P), proved to be strong inhibitors of food borne pathogens such as Listeria monocytogenes, since they all belong to the class II of LAB bacteriocins (Aymerich et al. 1996; Casaus et al. 1997; Cintas et al., 1997; Floriano et al., 1998). These strains may be of great technological importance in cheese manufacture when used as starter and/or coculture, since Listeria monocytogenes has been shown to be able to survive the manufacture and ripening conditions of cheeses. Aymerich et al., (2000) experimented and recommended the application of enterocins as bio preservative against Listeria innocua in meat products. Before a bacteriocin is considered for application in food, information on its antibacterial spectrum, biochemical and genetic characteristics, effectiveness in food systems, and regulatory implications should be known. Most wanted one is their safety studies; they should not possess any virulence or antibiotics resistance. Another important factor to consider will be the economical aspects or cost of using a bacteriocin in foods. One way to reduce the cost is to determine the optimum parameters for the production of a bacteriocin. For economical use in food, the bacteriocins have to be produced in large amounts and preferably by growing the strains in media containing food grade ingredients. Production of a bacteriocin in a simple medium can be increased by growing the cells at optimum pH and supplementing with nutrients specific for a species/strain. Conditions that provide high cell density favor high bacteriocin production (Ennahar et al., 2000).
1.8. Identification and characterization of enterococci with modern molecular techniques

Modern taxonomical concepts are needed to validate the identification of enterococci upto species and strains level. Such concepts, based on molecular biology techniques for classification and species identification of enterococci, have become available and are already been used, especially in medical microbiology, e.g. polymerase chain reaction (PCR) amplifications of the intergenic spacer between the 16S and 23S rRNA (Tyrrell et al., 1997),
12
Chapter 1
Introduction
whole cell protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Descheemaeker et al., 1997) randomly amplified polymorphic DNA (Quednau et al., 1998), broad-range 16S rDNA PCR (Monstein et al., 1998) and determination of 16S rRNA sequences. (Frahm et al., 1998; Michiei et al., 2006) applied species specification 23S rDNA-targeted oligonucleotide probes for the detection of enterococci in water hygiene control. Enterococci isolated from forage crops have been identified on the basis of DNA-DNA homology by restriction analysis of PCR-amplified 16S rDNA, as well as whole-cell protein profiles by means of SDS-PAGE (Ulrich and Mueller 1998).
Martin et al., (2006) designed species specific PCR and partial sequencing of 16S rRNA and sodA genes to identify enterococcal population. Enterococcus faecium was the most dominant followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD-PCR) revealed
species specific clusters and allowed strain typing.
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Introduction
Aims and Objectives of the Research Project

Aim of this study was to isolate, identify and characterize enterococci from processed dairy products of Pakistan and to investigate their safety level and their potential technological properties. Following were the specific objectives of present research project. 1. Isolation of Lactic acid bacteria (LAB) especially Enterococcus spp. from dairy products. 2. Screening of isolated strains for potential bacteriocins by using Listeria spp. and Enterococcus spp. as indicator organisms. 3. Precise identification of the bacteria by molecular genetics techniques with 16S and 23S rRNA amplification and sequencing. 4. Optimization of growth and media conditions for optimum production of bacteriocin. 5. Locus identification for bacteriocins genes by plasmid curing and amplification for known bacteriocin genes from isolated enterococci by PCR. 6. Safety investigation of the isolated strains by determination of critical virulence factors. 7. Purification and characterization of bacteriocins produced by the selected isolates.
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Review of Literature
2.1. BACTERIOCINS
2.1.1. History Most of the bacteria are capable of producing a heterogeneous array of molecules that may be inhibitory either for themselves or for other bacteria. These molecules include toxins, bacteriolytic enzymes, bacteriophages, by products of primary metabolites, antibiotics, bacteriocin and bacteriocin like substances. The division within this spectrum is ill defined and in practice there is considerable overlap. The bacteriocin family is the most abandoned and diverse group of bacterial defense system (Riley and Wertz, 2002). Bacteriocins were first discovered by Gratia in 1925. He was involved in the process of searching for ways to kill bacteria, which also resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few years. He called his first discovery a colicin because it killed E. coli. Bacteriocins are antimicrobial, proteinaceous compounds with a bactericidal mode of action against bacteria closely related to the producer strain. They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. They are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches. These compounds are produced by both Gram-negative and Gram-positive organisms (Tagg et al., 1976). The bacteriocins from E. coli are called colicin. They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. In fact, one of the oldest known so-called colicins was called colicin V and is now known as microcin V. It is much smaller and produced and secreted in a different manner than the classic colicins (Riley and Wertz, 2002). Both Gram positive and Gram negative bacteria produce bacteriocins, which constitute a heterologous subgroup of ribosomally synthesize antimicrobial peptides. In general these substances are cationic peptides that produce 15
Chapter 2
hydrophobic and amphiphilic properties and the bacterial membrane is the target for their activity in most cases (Cleveland et al., 2001). Enterococcal bacteriocin CCM 4231 was characterized as a small, thermostable, hydrophobic substance with a broad antimicrobial spectrum against Gram-positive and Gram-negative organisms produced by the ruminal isolate Enterococcus faecium CCM 4231 (Laukova et al., 1999). Enterococcos faecalis INIA 4, isolated from raw ewes milk Manchego cheese, produced an enterocin with bactericidal activity against L. monocytogenes strains Ohio, CA, V7 and Scott A (Rodriguez et al., 1997). The structural gene encoding enterocin 4 (Joosten et al., 1996) was shown to be very similar to that encoding peptide AS-48, a cyclic peptide with antibiotic activity produced by Enterococcus faecalis S-48 (Martinez et al., 1994). Enterococcus faecium WHE 81, a strain isolated from cheese, has been reported to produce a bacteriocin designated enterocin 81, which exhibits several properties of interest with regards to its possible use as food preservative (Ennahar et al., 1998). These include protease sensitivity, a narrow spectrum of activity, with a strong inhibition of L. monocytogenes strains, an extremely rapid bactericidal effect inducing an important efflux of intracellular material, and a broad pHrange activity. Since the last decade the number of characterized bacteriocins produced by Enterococci (enterocins) is increasing. Interestingly, enterocins are particularly active against pathogenic bacteria, such as Listeria, Clostridium and Staphylococcus (Vlaemynch 1996; Bennik et al., 1999; Laukova et al., 1999; Aymerich et al., 2000; Moreno et al., 2002; 2003; Achemchem et al., 2005; Kang and Lee, 2005; Criado et al., 2006). The best characterized bacteriocins from Enterococci (enterocins) were described from strains originating from food sources. These include the class II enteroins A, B, P. CRL 35 and bacteriocin 31 (Aymerich et al., 1996; Farias et al., 1996; Tomita et al., 1996; Casaus et al., 1997; Cintas et al., 1997; Franz et al., 1999; Okeeffe et al., 1999). Enterocins L50A and L50B, also produced by an enterococcal food isolate, do not belong to any of the three classes of bacteriocins as defined by (Nes et al., 1996) but show homology to the staphylococcal peptide toxins
16
Chapter 2
(Cintas et al., 1998). Well characterized enterocins from the clinical sources are the cyclic antibiotic peptide, AS-48 (Galvez et al., 1989), and the class I bacteriocin, cytolysin (Gilmore et al., 1994). Interestingly, the bacteriocin phenotype is frequently associated with pheromone-responsive conjugative plasmids of Enterococcus faecalis, as is the case for bacteriocin 31, cytolysin and AS-48 bacteriocins. There are only a few reports of bacteriocins production by Enterococci from an intestinal source, and the amino acid sequences of these enterocins were generally not determined (Kramer and Brandis 1975; Laukova et al., 1993; Du Toit et al., 2000; Martin et al., 2006). Additionally, several strains of LAB produce bacteriocins, which are peptides or proteins with an antibacterial activity against bacteria closely related to the producer strain. Further more, some bacteriocins are also active against food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum, Clostridium perfringens, Bacillus subtilis and thus may be of great interest to the food industry as natural food preservatives (Stiles, 1996; wood 1997). For this reason, they have received much attention for use as natural, or so-called bio-preservatives in foods in recent years (Cleveland et al., 2001; Galvez, 2007).
2.1.2. Classification Bacteriocins of LAB have been classified on the basis of their biochemical characteristics in three groups (Nes et al., 1996). The previously proposed class IV, consisted of bacteriocins form large complexes with macromolecules (Klaenhammer 1993); was probably because of artifacts or partial purifications (Cleveland et al., 2001). Class I bacteriocins, termed lantibiotics, are small, membrane-active and heat-stable peptides that contain unusual thioether amino acids, like lanthionine and B-methyllanthione. The model of this group, nisin, which is bacteriocin isolated from Lactococcus lactic is probably the most well-known and studied bacteriocins produced by LAB (Cleveland et al., 2001; Ross et al., 2002; Eijsink et al., 2002).
17
Chapter 2
Class II bacteriocins of lactic acid bacteria (LAB) have emerged in recent years as the most promising bacteriocin candidates for food preservation, since they have been shown to display overall better performance, in terms of biological activity and physiochemical properties, than most bacteriocins from other classes (Klaenhammer 1993; Nes et al.,1996; Ennahar et al., 1999). Class II bacteriocins contain small (4-6 kDa), heat-stable, non-modified peptides (Nes and Holo 2000; Cleaveland et al., 2001) which are subdivided into Class IIa, Listeria-active or anti-Listerial peptides, which are pediocin-like and which are characterized by a well conserved YGNGVXaaC consensus motif at their N-terminal ends. These contain the recently described lactococcin MMFII (Ferchichi et al., 2001). and sakacin G (Simon et al., 2002) and Class IIb, twopeptide bacteriocins; consisting in the association of two different peptides for full activity, such as lactococcin G (Nissen-Meyer et al., 1992) or lacticin F (Muriana and Klaenhammer 1991). A third subgroup (IIc) or Class III, is suggested to contain sec-dependent secreted bacteriocins (Nes et al., 1996) though it was shown more recently that enterocin P, which contains the IIa consensus motif, is also secreted in a sec-dependent manner (Cintas et al., 1997). On the other hand, bacteriocins, like carnobacteriocin A and enterocins B and I (Worobo et al., 1994; Casaus et al., 1997; Floriano et al., 1998), which are sec-independent secreted one-peptide bacteriocin lacking the YGNGVXaaC motif, could neither be included in one of the subgroups described in the current classification, nor could they be clearly categorized within a particular subgroup. This classification scheme will therefore certainly be evolving based on new evidences, particularly the identification of new natural peptides. Class III bacteriocins are secreted by the bacterial preprotein translocase (sec-pathway) and are large (>30kDa), heat-labile proteins (Nes et al., 1996). This group is not well documented. Only few large bacteriocins produced by LAB are described at the molecular level, such as helveticin J produced by Lactobacillus helveticus 481 (Joerger and Klaenhammer 1990) or enterolysin A from Enterococcus faecalis LMG 2333. Nevertheless, some of the incompletely described antibacterial compounds could be suspected to be class III bacteriocins, although not yet classified, e.g. helveticin V1829 from L.
18
Chapter 2
helveticus1829 (Vaughan et al., 1992), the antimicrobial substance from L. helviticus CNRZ450 strain (Thompson et al., 1996) or the recently reported enterocin R69 from Enterococcus faecalis R69 (Elotmani et al., 2002), enterocin AS 48 (Galvez et al., 1989); and enterocins 1071A and 1071B from E. faecalis (Balla et al., 2000), enterocin B (Casaus et al., 1997), enterocins L50A and L50B (Cintas et al., 1998), and enterocin Q from Enterococcus faecium (Cintas et al., 2000). Many of the bacteriocins have been characterized at molecular level (Table 2.1.). Franz et al., (2007) reviewed the structural and genetic characteristics of enterocins, the bacteriocins produced by enterococci. Some of these can be grouped with typical bacteriocins produced by lactic acid bacteria according to traditional classification, whereas others are atypical and structurally distinct from the general classes of bacteriocins. These atypical enterocins recently played an important role in and prompted reclassification of the class II bacteriocins into a new scheme. In that review, a more simplified classification scheme for enterocins based on amino acid sequence homologies was proposed.
19
Chapter 2
Table. 2.1. List of characterized enterocin with their amino acid sequences Type
Enterocin AS-48 Enterocin A Enterocin CRL 35 Bacteriocin 31 Enterocin B Enterocin P Enterocin L50A Enterocin L50B Enterocin Q Enterocin EJ97
Producer strain
E. faecalis S-48 E. faecium CTC492 E. faecium CRL 35 E. faecalis YI717 E. faecium T136 E. faecium P13 E. faecium L50 E. faecium L50 E. faecium L50 E. faecalis EJ97
M.M
7167 4833
Amino acid sequence of the purified enterocin

MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVQGSGGLSLLAAAGR ESIKAYLKKEIKKKGKRAVIAW TTHSGKYYGNGVYCTKNKCTVDWAKATTQCIAGMSIGGFLGGAIPGKC KYYGNGVTLNKXGXSVNXXXA. . .
Reference Martinez et al., 1994 Aymerich et al., 1996 Farias et al., 1996 Tomita et al., 1996 Casaus et al., 1997 Cintas et al., 1997 Cintas et al., 1998 Cintas et al., 1998 Cintas et al., 2000 Galvez et al., 1998; Sanchez et al., 2003 Balla et al., 2000 Balla et al., 2000 Yamamoto et al., 2003 Marekova et al., 2007
5009 5465 4630 5190 5178 3952 5328
ATYYGNGLYCNKQKCWVDWNKASREIGKIIQVNGWVQHGPWAPR ENDHRMPNELNRPNNLSKGGAKCGAAIAGGLFGIPQKGPLAWAAGLA NVYSKCN ATRSYGNGVYCNNSKCWVNWGEAKENIAGIQVISGWASGLAGMGH MGAIAKLVAKFGWPIVKKYYKQIMQFIGEGWAINQKIIEWIKKHI MGAIAKLVTKFGWPLIKKFYKQIMQFIGQGWTIDQQIEKWLKRH MNFLKNGIAKWMTGAELQAYKKKYGCLPWEKISC MLAKIKAMIKKFPNPYTLAAKLTTYEINWYKQQQYGRYPWERPVA
Enterocin 1071B Enterocin 1071A Enterocin RJ-11 Enterocin M
E. faecalis BEF1071 E. faecalis BEF 1071 E. faecalis RJ-11 E. faecium AL40
3899 4286 5049 4628
GPGKWLPWLQPAYDFVTGLAKGIGKEGNKNKWKNV ESVFSKIGNAVGPAAYWILKGLGNMSDVNQAQDRINRKKH APAGLVAKFGRPIVKKYYKQIMQFIGEGSAINKIIPQWIARMWRT ATRSYGNGVYCNNSKCW-NVGEAKENIA GIVISGKASGL
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2.1.3. Production and characterization of bacteriocins There have been numerous reports on bacteriocin-producing Enterococci, primarily among strains of Ent. faecium associated dairy products, fermented sausages, fish, vegetables, silage, and the mammalian gastrointestinal tract. In fact, most of the bacteriocinogenic strains have been isolated from foods: dairy products (Torri-Tarelli et al., 1994; Vlaemynck et al.,1994; Farias et al. 1996; Maisnier-patin et al., 1996; Nunez et al., 1997; Okeeffe et al., 1999); sausages (Aymerich et al., 1996; Casaus et al., 1997; Cintas et al., 1997), fish (Ben Embarek et al., 1994); and vegetables (Mc Kay, 1990; Villani et al. 1993; Franz, et al., 1999; Bennik et al., 1999; Floriano et al. 1998). All enterocins share a number of common characteristics such as heat stability, stability over a wide range of pH values, and activity towards L. monocytogenes (G. Giraffa, 1995). Du Toit et al., (2000) isolated and screened 7 out of total 92 enterococcal strains from the faeces of minipigs of which four were identified as Enterococcus faecalis and three as Enterococcus faecium. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by protease enzymes but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 34 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B. Moreno et al., (2003) concluded from their study that the seven enterococci isolated from different sources were sensitive to the glycopeptide antibiotics
21
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vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. Park, et al., (2003) screened bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, with respect to bacteriocins. Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, a-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or -glucosidase. The inhibitory activity was stable at 100 C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-SDSPAGE. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. Achemchem et al., (2005) characterized Ent F-58 a broad spectrum bacteriocin produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goats cheese manufactured without starter cultures. The
22
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antimicrobial substance was produced during the growth phase, with maximum production after 1620 h of incubation at 30 C, and was stable over a wide pH range (48) and at high temperatures (5 min at 100 C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. They verified that it also holds the structural gene for P-like enterocin. Kang and Lee, (2005) reported the bacteriocin produced by Ent. faecium GM1, a broad spectrum bacteriocins against indicator strains of E. coli, S. aureus, Vibrio spp., S. typhimurium, L. monocytogenes, Lact. acidophilus, and Strept. thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from Ent. faecium GM-1 was obtained when the culture conditions were pH 6.06.5 and 35-40 C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of Ent. faecium GM-1 by PCR and direct sequencing methods. Gutierrez et al., (2005) studied cloning and expression of enterocin P (entP) produced by Enterococcus faecium P13, in Escherichia coli. PCR-amplified products of the pre enterocin P gene (entP) or entP plus the putative EntP immunity gene (entiP), were cloned in plasmid pETBlue-1 under the control of the inducible T7lac promoter. Functional EntP from the supernatants of E. coli Tuner(DE3)pLacI (pJG01) cultures grown in a complex medium was
23
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recovered, at a high efficiency, by immunoaffinity chromatography in a single step. A purification method based on hydrophobic adsorption and reversephase chromatographies also permitted the recovery of active EntP from the supernatants of the same cultures grown in a minimally defined medium. Cuozzo et al., (2006) have reported purification and heterologous expression of enterocin P (entP), the sec-dependent bacteriocin produced by Enterococcus faecium, in Escherichia coli. PCR-amplified product of the enterocin P structural gene entP was cloned into plasmid pET-32b under the control of the inducible T7lac promoter. The neo-synthesized EntP was genetically modified by an addition of 3 extra amino acids, leading to recombinant EntRP. Active EntRP was recovered from the cytoplasmic soluble fraction of E. coli harboring appropriate recombinant plasmid, characterized by ELISA and Western-blot analysis and purified by immunoaffinity chromatography. The use of E. coli as heterologous host and pET-32b as expressing vector offers promising tools for heterologous production of class IIa bacteriocin. Criado et al., (2006) reported the locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (entQ), and enterocin P (entP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occured at different locations; entL50AB (encoding EntL50A and EntL50B) were on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) was on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) was on the chromosome. Martin et al., (2006) isolated enterococci from samples of intestinal contents and carcasses of wood pigenos and evaluated their antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcus faecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme linked immunosorbent assay (NCI-ELISA), permitted characterization of enterococci coding that described
24
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bacteriocins and their expression. The ejaAfim determinant was the only virulence gene detected in Ent. faecium, whereas Ent. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Sparo et al., (2006) reported the new enterocin MR99 from Enterococcus faecalis MR99, the only strain with inhibitory activity out of 100 enterococcal isolates, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromoneresponsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 C. Enterocin MR99 was bactericidal on strains of L. monocytogenes, S. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. Martin, et al., (2007) done replacement of the leader sequence of enterocin A (entA), a bacteriocin produced by Enterococcus faecium PLBC21, by the signal peptide of enterocin P (entP), a sec dependent bacteriocin produced by Ent. faecium P13, permitted production of entA in Lactococcus lactis. Chimeras encoding the entP signal peptide (SPentP) fused to mature EntA (entA), with or without its immunity gene (entiA), were cloned into the expression vector pMG36c to generate the recombinant plasmids, pMPA15 (SPentP:entA) and pMPA10i (SPentP:entA+entiA). Transformation of competent L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000 with the recombinant plasmids permitted production of entA by the transformed cells, and the co-production of nisin A and entA by the L. lactis subsp. lactis DPC5598 transformants. Ghrairi et al., (2008) done the screening for bacteriocin production by strains of lactic acid bacteria from a Tunisian dairy product. They reported the
25
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detection of Ent. faecium bacteriocin producing strain. The anti-microbial compounds produced by this novel strain were heat stable and inactivated by trypsin, proteinase K and pronase E. Bacteriocins produced inhibited not only the closely related LAB, but also Listeria monocytogenes and Staphylococcus aureus. Mass spectrometry analysis and RP-HPLC showed that Ent. faecium MMT21 produced two bacteriocins enterocin A (4828.67 Da) and enterocin B (5463.8 Da). That result was confirmed by amplification by PCR of enterocins A and B genes. Soomro and Tariq (2008) reported that only one out of thirty strains, Lactobacillus acidophilus J1, caused inhibition of E. coli and E. faecalis. The antibacterial effect of producer organism was highest when MRS broth was supplemented with tryptone and glucose with initial medium pH of 5.5 and the incubation was carried out at 37 C between late logarithmic and early stationary phases of growth. The antibacterial substance in cell-free culture supernatant was sensitive to proteolytic enzymes and was heat-stable. The putative bacteriocin was purified by using ammonium sulphate precipitation (60% saturation) and by solvent extraction method with chloroform. Further purification to homogeneity was performed by HPLC. It displayed a bacteriocin with an estimated molecular mass of approximately 5-6 kDa.
2.1.4. Applications of bacteriocins Enterococci are natural inhabitants of gastrointestinal tract and are thus considered as indicator bacteria for faecal pollution of water and foods. They are also promising for the biological preservation of cheeses and vegetables as additional starter cultures (Bennik et al., 1999; Moreno et al., 2002; Sarantinopoulos et al., 2002). Further, Enterococci are used as Probiotic in some countries (Holzapfel et al., 1998). For instance, the strain Ent. faecium SF68 has been studied in detail as a probiotic in the treatment of antibioticassociated diarrhea (Franz et al., 1999; Marteau et al., 2001). However there is a potential health risk in the use of Enterococci (Libertin et al., 1992; Booth et al., 1996; McDonald et al., 1997). Some Enterococcus faecalis strains have been associated with a number of human infections (e.g. endocarditis), and
26
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also Ent. faecium has been implicated as the causative agent in 20% of enterococcal infections (Stiles and Holzapfel 1997). In addition, there is a clear increasing antibiotic resistance development among Enterococci (McDonald et al., 1997). Nisin, the bacteriocin commercially available as a bio-preservative, was traditionally used in the prevention of late blowing mainly gas blowing defect in cheese (Hirsch et al., 1951). Nisin is produced by numerous Lactococcus lactis strains and has been approved in over 50 countries for many applications in a large range of food products, particularly in cheese manufacturing (Ross et al., 2002). Two naturally occurring nisin variants namely nisin A and nisin Z that differ in a single amino acid residue but showing similar activities have been described. Nisin A possesses a histidine and nisin Z, an asparagine at position 27 (Mulders et al., 1991). Nisin kills sensitive strains by pore formation in the membrane of sensitive bacteria and is active at the nano-molar level by targeting the lipid-bound cell wall precursor lipid II as a docking molecule (Breukink et al., 1999; Wiedemann et al., 2001). Generally, bacteriocins exhibit inhibitory activity towards bacteria closely related to their producing strain (Nes et al., 1996). Consequently, some Lactococcus lactis starter strains are inhibited by nisin. Nevertheless, producer strains are protected from their own bacteriocin by expressing a specific immunity protein. Thus, starter blends designed with nisin-producers associated to insensitive strains with appropriate technological properties could be of interest, particularly in cheese manufacturing (Roberts and Zottola 1993). Its application has been extended to the inhibition of Listeria monocytogenes in Camembert (Maisnier-Patin et al., 1992) or ricotta-type cheese (Davies et al., 1997) and of L. innocua in Manchego cheese (Nunez, et al., 1997). Lacticin 3147 produced by a Lactococcal strain has been successfully used as inhibitor of L. monocytogenes in cottage cheese (McAuliffe et al., 1999) and to control the growth of non-starter LAB in Cheddar cheese (Ryan et al., 1996) Also, pediocin PA-1 produced by pediococci and lactobacilli strains reduced the levels of L. monocytogenes on the surface of Munster cheese
27
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(Ennahar et al., 1998). Enterococcal bacteriocins have shown a strong antiListerial activity in milk and cheese (Giraffa et al., 1995; Nunez et al., 1997). Raw milk represents a source of new strains of LAB with the potential to inhibit undesirable microflora for use in bio-preservation of dairy products. A well-studied Enterococcus strain used as probiotic is Ent. faecium SF 68, which is produced in Switzerland (CylactinLBC SF68 ME10, F. Hoffmann-La Roche Ltd., Basel, Switzerland). It has been proposed to be clinically effective in the prevention of antibiotic-associated diarrhea. In Denmark, a fermented milk product that contains Ent. faecium SF 68 (GAIO; MD Foods, Aarhus, Denmark) has been sold for several years because of its hypocholesterolemic effect on individuals. In a recent work, the same fermented milk product GAIO was used to study the relationship of the Ent. faecium SF 68-containing probiotic and a vancomycin treatment. It was concluded that Ent. faecium SF 68 from the GAIO product was not found to acquire vancomycin resistance during the study period (Lund et al., 2000). Rossi et al., (1999) used the strain Ent. faecium CRL 183 for the development of a novel fermented soymilk product with potential probiotic properties. Ent. faecium CRL 183 in combination with Lactobacillus jugurti resulted in a 43% decrease in cholesterol in vitro. Also, the probiotic strain Ent. faecium PR88 (Ent. faecium Fargo 688 from Quest International, Naarden, Netherlands) was studied in a human clinical trial. The consumption of that strain led to alleviation of the symptoms of irritable bowl syndrome in humans (Moreno et al., 2006). In addition to the available commercial preparations of nisin and pediocin AP1/AcH, other bacteriocins like lacticin 3147, enterocin AS-48 or variacin, enterocin A, B and P offer promising perspectives. Broad spectrum bacteriocins present potential wider uses, while narrow spectrum bacteriocins can be used more specifically to selectively inhibit certain high risk bacteria in foods like Listeria monocytogenes with out affecting harmless microbiota. Bacteriocins can be added to foods in the form of concentrated preparations as food preservatives, shelf-life extenders, additives or ingredients, or they
28
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can be produced by bacteriocinogenic starters, adjunct or protective cultures. Immobilized bacteriocins can also find application for development of bioactive food packaging. In recent years, application of bacteriocins as part of hurdle technology has gained great attention. Several bacteriocins show additive or synergistic effects when used in combination with other antimicrobial agents, including chemical preservatives, natural phenolic compounds, as well as other antimicrobial proteins (Galvez et al., 2007). In case of Greek Feta cheese the presence of Listeria spp. has never been reported. This could be attributed to the Feta cheese ecosystem, where Enterococci frequently comprise a significant part of the microflora in the mature product. Enterococcus faecium FAIR-E 198, a strain isolated from Greek Feta cheese, was previously found to exhibit antilisterial activity (Sarantinopoulos, 2002). The use of enterococci as probiotics remains a controversial issue. While the probiotic benefits of some strains are well established, the emergence and the increased association of enterococci with human disease and multiple antibiotic resistances (see below) have raised concern regarding their use as probiotics. The fear that antimicrobial resistance genes or genes encoding virulence factors can be transferred to other bacteria in the gastrointestinal tract contributes to this controversy (Franz et al., 2007).
Enterocins are of interest for their diversity and potential for use as food biopreservatives. The emergence of multiple antibiotic-resistant enterococci among agents of nosocomial disease and the presence of virulence factors among food isolates requires a careful safety evaluation of isolates intended for potential biotechnical use. Nevertheless, enterococcal bacteriocins produced by heterologous hosts or added as cell-free preparations may still be attractive for application in food preservation (Moreno et al., 2003).
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2.2. Food safety issues

Listeria monocytogenes is a ubiquitous microorganism which is widely distributed in nature and has been isolated from a variety of foods, including dairy products. Contamination of food with L. monocytogenes continues to be a threat to public health (Gaya et al., 1996; Wan et al., 1997). The Food and Drug Administration (FDA) reported that 5% of raw milk samples tested were positive of Listeria, and 18% of the milk of lactating Holstein cows contained L. monocytogenes serotype 4b (Ryser, 1999). The first outbreak of listeriosis was demonstrated by Schlech et al., (1983), coleslaw as a vehicle of infection in an outbreak of listeriosis in Nova Scotia, Canada, in 1981. Listeria monocytogenes was assumed to be of animal origin since the cabbages used in coleslaw were fertilized with sheep manure originating from a herd with cases of ovine listeriosis. After the coleslaw outbreak, four epidemics, two in USA, one in Switzerland and one in Denmark, were linked to dairy products, thus generating concern about presence of L. monocytogenes in these products (Fleming et al., 1985; Linnan et al., 1988; Bille 1989). Several epidemics have subsequently been described and various kinds of food products linked with the outbreaks. In Finland, Junttila and Brander (1989) described a Listeria septicemia in an 80 year old man to be associated with consumption of home-made salted mushrooms. In 1997, five persons suffered from a febrile gastroenteritis caused by L. monocytogenes originating from cold-smoked rainbow trout (Miettinen et al., 1999). Moharem et al., (2007) investigated the incidence of Listeria species in seafood products of mysore, India. A total of 164 fresh and dry fish samples collected from retail outlet shops of Mysore, South India, during the period August 2005 through August 2006 were examined for the presence of Listeria species by using ISO 11290 protocols. The incidence of Listeria species was positive in 62 samples (37.8%), and L. monocytogenes was isolated from only three (1.83%) fresh fish samples. Listeria species in seafood were
30
Chapter 2
predominant in the order of Listeria innocua (50) (30.49%), Listeria grayi (eight) (4.9%), L. monocytogenes (three) (1.83%) and Listeria seeligeri (one) (0.6%). There are several reasons for the late establishment of the food-borne nature of listeriosis. The symptoms of invasive listeriosis are severe and differ from classical food-borne diseases. Moreover, listeriosis is a rare disease, occurring mainly among a high-risk group of people, and majority of cases are believed to be sporadic. In addition, the long incubation time hinders tracing of vehicles of infections and often no food is left for microbiological analysis. As a consequence of recurring and serious listeriolysis outbreaks (Gahan and Collins 1991), the food borne pathogen Listeria monocytogenes became the focus of bacteriocin investigators during the past two decade. The best studied anti-listerial bacteriocin is nisin, a class I bacteriocin, which is produced by strains of Lactococcus lactis and is used as a food preservative on a commercial scale in several countries. Recent reports however clearly indicate that bacteriocins of class IIa are more interesting anti-listerial agents as they do not have as broad an inhibitory spectrum as nisin and are more effective in killing Listeria strains (Ennahar et al., 1999). Class IIa regroups 14 bacteriocins of the pediocin family, i.e. peptides identified on the basis of their strong amino acid sequence similarity, particularly their distinctive N-terminal part and their strong anti-Listerial activity (Klaenhammer 1993; Ennahar et al., 1999). Class IIa bacteriocins are believed to be next in line if more bacteriocins are to be approved in the future. However, application studies have shown that there are limitations to the usefulness of class IIa bacteriocins as anti listerial agents, as full suppression of the pathogen is rarely achieved in food systems (Ennahar et al., 1999). This is due the difference between bacteriocin production media and food ecosystem, pH and competition from diversified microflora. Also the interference form food macromolecules mainly lipid, casein, lactose ect. reduces exposure of target organism to the potent bacteriocin.
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Enterococci, and especially the vancomycin-resistant enterococci (VRE), are recognized as one of the most important causes of nosocomial infections in seriously ill and immunocompromised patients (Emori and Gaynes, 1993; Patel, 2003). They are intrinsically resistant or poorly susceptible to several antibiotics, and under antimicrobial pressure can develop resistance to other important drugs, such as aminoglycosides and glycopeptides. VRE have been frequently isolated from farm animals and food of animal origin in Europe and several reports have suggested that their presence could be associated with the use of the vancomycin-related glycopeptide avoparcin as growth promoter in animal production. Some strains of enterococci are opportunistic human pathogens and can cause nosocomial infections such as bacteraemia, endocarditis, urinary tract and other infections (Morrison et al., 1997; Landman and Quale 1997; Leclercq 1997). This, in combination with the production of virulence factors such as aggregation substance (as), gelatinase (gel), cytolysin (cyl), enterococcal surface protein (esp), adhesin to collagen from Ent. faecalis (ace) and Enterococcus endocarditis antigen (efafs or efafm from Ent. faecalis or Ent. faecium respectively), determines that enterococci are not considered as generally recognised as safe (GRAS) organisms (Wegener et al., 1997). However, antibiotic resistance as such cannot explain the virulence of enterococci. Although enterococci possess subtle virulence traits (Shankar, 1999) considerable progress has recently been made in determining these. For example, studies have shown that phenotypes such as -hemolysin/bacteriocin (also called cytolysin) and aggregation substance (as), which are encoded by E. faecalis pheromone-responsive plasmids, are related to pathogenicity and enhance the virulence of enterococci in animal models. Majority of Enterococcus spp. isolated from food origins did not show these virulence traits (Singh et al., 1998; Abriouel, 2005; Choho et al. 2008).
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Materials and Methods
3.1. ISOLATION AND CHARACTERIZATION OF LACTIC ACID BACTERIA 3.1.1. Isolation and Growth Characteristics Lactic acid bacteria (LAB) were isolated from different food commodities like local processed cheese, fresh milk, yoghurt, butter, spoiled dairy products and chicken sausages. Isolation of lactic acid bacteria was done by serial 10-fold dilution technique. Simply one gram of food sample was mixed in 9 mL of quarter strength ringer solution (Oxoid, Basingstoke, UK) and further serially diluted, 0.1 ml sample of suitable dilution was plated onto non selective medium de Man Rogosa sharpe (MRS) agar (Oxoid, Basingstoke, UK). These plates were incubated for 24-48 hours for checking initial growth at 37 C. Purified isolated colonies were screened for the enumeration of Enterococcus spp. by growth on modified MRS (6.5% NaCl, 9.6 pH), at 10 and 45 C and growth on selective media (kanamycin esculin azide & Slantz and Bartely medium (Oxoid, Basingstoke, UK). Enterococcus faecium LMG 11423T provided by Gent Culture Collection Centre, Belgium was used as bacteriocin producing control strain and Enterococcus faecium ATCC 27273 was used as control strains for molecular characterization. Seven bacterial isolates of Enterococcus spp. selected and named as IJ-03, IJ-06, IJ07, IJ-11, IJ-12, IJ-21 and IJ-31. Listeria monocytogenes, Bacillus subtlis and Bacillus cereus were used as indicator strains and were propagated at 35 C in brain heart infusion (BHI) broth (Oxoid, Basingstoke, UK). Stock cultures of all strains were kept in the appropriate medium with 15% (v/v) glycerol, and stored at -20 C. Strains were propagated twice in the appropriate broth before use as fresh bacteriocin producing or indicator culture. Agar medium were prepared by addition of 1.5% bacteriological agar (Oxoid, Basingstoke, UK) to the broth medium and overlay agar medium contained 0.7% bacteriological agar all sterilization were carried out in situ at 121 C for 15 min with steam autoclave.
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3.1.2. Morphological and Biochemical Characterization Simple, Negative and Gram staining was performed for the morphology and Gram reaction of the isolates. The sugar fermentation profile of these isolates was checked by using phenol red broth medium with glucose, lactose, salicin and manitol as sole carbon source in the medium. The antibiotic sensitivity test of the isolates was performed by using teicoplanin and vancomycin disks (Oxoid, Basingstoke, UK) and the growth inhibition zones were measured in mm according to National Committee for Clinical Laboratory Standards (NCCLS 2002). The haemolysis was checked by culturing of the isolates on blood agar medium (Fluka). Catalase test and methylene blue reductase test were also performed. Identification was done with the help of Analytical Profile Index (API 20) Strep System (bioMrieux Germany, Nrtingen, Germany) according to users instructions manual. Biochemical tests in API 20 Strep are voges proskauer (VP), hydrolysis of hippuric acid (HIP), esculin hydrolysis (ESC), pyrrolidonyl arylamidase (PYRA), -galactosidase ( GAL), -glucuronidase ( GUR), -galactosidase ( GAL), alkaline phosphatase (PAL), leucine amino peptidase (LAP), arginine dihydrolase (ADH), along with sugar fermentation tests like ribose (RIB), arabinose (ARA), mantiol (MAN), sorbitol (SOR), lactose (LAC), trehalose (TRE), inulin (INU), raffinose (RAF); amidon acidification (AMD) and glycogen (GLY). The biochemical and sugar fermentation tests of this system allowed presumptive identification of Enterococcus up to species level. Results from biochemical and sugar fermentation test were recorded after 5 and 24 hours. The 8 digit numerical code was derived and identification was done with the users profile.
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3.1.3. Molecular Characterization

3.1.3.1. Extraction of Genomic DNA Overnight culture from de Man Ragosa Sharpe broth (Fisher Scientific, USA) was used to harvest the cell pellet from 1.5 ml culture of the representative Enterococcus faecium and Enterococcus faecalis strains. DNA extraction was done with DNeasy Blood and Tissue Kit (Qiagen, USA) as per users instruction manual. Isolated Genomic DNA from these Enterococcus strains was quantified on Nano-Drop spectrophotometer at 230, 260 and 280nm and analyzed on 0.8% agarose gel.
3.1.3.2. PCR amplification for 16S rRNA of Ent. faecium and Ent. faecalis For molecular identification of isolated Enterococcus faecium and Enterococcus faecalis primers were designed to amplify almost complete region of 16S rRNA. Primers for 16S rRNA were designed from the Eneterocccus faecium strain LMG 11423T. The primer with higher melting temperature (Tm) and GC contents (G+C%) were designed to avoid low temp anneling and non specific amplification. Preferably Tm was selected in range of 65-70 C and GC contents 50-60%. All of the oligos were purchased from Sigma Genoys (Sigma Alrdrich, USA). The amplification primers used for 16S and 23S rRNA are given in the Table 3.1. PCR Reaction Following PCR conditions were used to amplify 16S rRNA from Enterococcus strains. Ingredients Template (0.2ng/ L) Primers (10 M) dNTPs Mix (10mM) PCR Standard Buffer Taq DNA Polymerase ddH2O Concentration 1ng 1 L each 1 L per reaction 5 L per reaction 1 Unit 36.75 L Volume 5 L 2 L 1 L 5 L 0.25 L 36.75 L
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Total reaction volume was 50 L. Amplification was carried out in Eppendroff thermocycler (Eppendroff, USA) with following conditions. Denaturation temperature Time Annealing and extension temperature Time Final extension temperature Time Hold 95 C 30 sec. 72 C 2 min. 72 C 5 min. 4 C until removed 30 cycles
The PCR products were analyzed on 0.9% agarose gel. The size of 16S rRNA fragment was 1.5 kb. 1kb DNA marker (Trackit, Invitrogen) was used to compare the size of PCR amplified fragments. The primer designed for amplification of Enterococcus faecium also amplified 16S rRNA fragement from Enterococcus faecalis strains as the target sites were same for both Ent. faecium and Ent. faecalis.
3.1.3.3. PCR amplification for 23S rRNA of Ent. faecium and Ent. faecalis The 23S rRNA fragment from these isolated Enterococcus faecium and Enterococcus faecalis were amplified with the primers designed to amplify almost complete region of 23S rRNA. Primers for 23S rRNA were designed from the Enterococcus faecium strain LMG 11423T and Enterococcus faecalis strain V583 sequences published at National Commission for Biotechnology Information (NCBI) Gene Bank. PCR reaction and amplification conditions were same as used previously for amplification of 16S rRNA with 35 cycles instead of 30. The PCR products were analyzed on 0.9% agarose gel with 1X TAE buffer at constant voltage of 80V. 1kb DNA marker (Trackit, Invitrogen) were used to compare the size of PCR amplified fragments. The size of 23S rRNA fragment was 2.9 kb. Separate primers were designed for amplification of Enterococcus faecium and Enterococcus faecalis.
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Table 3.1 Primers for amplification of 16S and 23S rRNA from isolated Enterococcus spp. Target gene Primer Name Orientiation Position Oligoneculeotide Product size bp
16S rRNA Entrococcus spp.
LMG-16S-S20
Sense, Forward
20-41
5 GACGAACGCTGGCGGCGTGCC 3 1500
LMG-16S-AS1520
Antisense, Reverse Sense, Forward Antisense, Reverse
1496-1520
5 CGGCTACCTTGTTACGACTTCACC 3
LMG-23S-S1 23S rRNA Enterococcus faecium
1-24
5 GGTTAAGT AATAAGGGCGCACGG 3 2904
LMG-23S-AS2904
2877-2904
5 CCTCGATCGATTAGTATC AGTCCGCTC 3
V583-23S-S250246 23S rRNA Enterococcus faecalis
Sense, Forward
250246250273
5 GTTAAGTGAATAAGGGCGCACGGTGG 3 2905
V583-23SAS253151
Antisense, Reverse
253124253151
5 GTCCTCGACCGATTAGTATTGGTCCGC 3
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3.1.3.4. PCR product purification After PCR amplification purification of amplicons was done by using QIA quick-spin PCR purification kit (Qiagen) as per users instruction manual. This column removes primers and other reagents from the PCR product. The PCR products were run of either 0.9% or 2.0% for smaller fragments for analytical purpose. The quantification of the PCR purified fragments was carried out by Nano Drop Spectropotometer by measuring OD at 230, 260 and 280nm. The concentration of 15ng/100bp was made as required for DNA sequencing of these PCR purified amplicons.
3.1.3.5. DNA sequencing and identification of bacterial strains The almost complete 16S and 23S rRNA nucleotide sequencing was done form representative enterococci strains occurring in two genomic subgroups namely Ent. faecalis IJ-03, IJ-07, IJ-11 and IJ-12 along with Ent. faecium IJ-06, IJ-21, IJ-31 and ATCC27273. Determination of nucleotide sequence of the PCR purified fragments was performed using the ABI PRISM BigDye Terminator cycle sequencing kit and the automatic DNA sequencer ABA PRISM at the DNA sequencing service of University of Texas at Austin. The 16S rRNA and 23S rRNA sequences were compared with other strains by using NCBI BLAST analysis for identification purpose and comparison of homologies of isolated strains with previous characterized strains. The sequencing primers for 16S and 23S rRNA are given in Table 3.2.
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Table 3.2. Sequencing primers used for 16S and 23S rRNA nucleotide sequencing of isolated Enterococcus strains
Published sequence strain Enterococcus faecium LMG 11423
T
Nomenclature LMG-16S-S20 LMG-16S-AS232 LMG-16S-S420 LMG-16S-AS656 LMG-16S-S852 LMG-16S-AS1065 LMG-16S-S1294 LMG-16S-AS1520
Oligodeoxynucleotise primer 5 GACGAACGCTGGCGGCGTGCC 3 5 ACCGCGGGTCCATCCATCA 3 5 GAAGAAGGTTTTCGGATCGTAAA 3 5 CCTCTTCTGCACTCAAGTCTC 3 5 TCC GCC CTT CAGTGCTGC 3 5 CACGAGCTGACGACAACCAT 3 5 TTAAAGCTTCTCTCAGTTCGGATT 3 5 CGGCTACCTTGTTACGACTTCACC 3 5 GGTTAAGTAATAAGGGCGCACGG 3 5 GCCGCTACTCAGGGAATCG 3 5 TTCCCATGACCGCGCAGCCGAC 3 5 CACCTTCAACCTGGACATGGG 3 5 ATATCCGGGAGTCAGACTG 3 5 AAGTTCCTGATGTCACACTGCC 3 5 TTGATAAACAGTCGCTTGGGCC 3 5 AAAGGCGTAACGATTTGGGCA 3 5 CACTGTCTCCCACCACGATTA 3 5 CAAGAGTCCACATCGACGGG 3 5 GCGGTAAGTCCATCCCGGTC 3
Position 20-41 213-232 420-443 635-656 852-870 1045-1065 1294-1318 1496-1520 1-24 232-251 492-512 728-749 970-999 1588-1318 1777-1799 1982-2004 2244-2223 2480-2500 2677-2697
Enterococcus faecium LMG 11423
LMG-23S-S1 LMG-23S-AS251 LMG-23S-S497 LMG-23S-AS749 LMG-23S-S970 LMG-23S-S1588 LMG-23S-AS1799 LMG-23S-S1982 LMG-23S-AS2244 LMG-23S-S2480 LMG-23S-AS2697
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3.1.4. Safety Assessment of Isolated Enterococcus spp.

3.1.4.1. Antibiotic susceptibility of the isolates The isolated Enterococcus strains were checked for their susceptibility against commonly used antibiotics with Kirbby and Bauer disc diffusion assay (Monica Cheesbrough, 2002). Standard antibiotics namely methicillin, kanamycin, vancomycin, teicoplanin, ampicillin, erythromycin, ciprofloxacin, tetracycline and gentamycin were used for this assay. Further resistance to glycopeptides antibiotics namely vancomycin and teicoplanin was checked by determining minimum inhibitory concentration (MIC) of these drugs using standard antibiotics with serial dilution broth technique (NCCLS, 2002).
3.1.4.2. Qualitative assessment of virulence factors Haemolysis of these isolates was checked by plating onto Blood Agar Base (Fluka, UK) supplemented with 5-7% of sterile sheep blood. The results were noted for clear haemolysis as -haemolysis, slight haemolysis as -haemolysis and with no haemoysis as -haemolysis. Catalse and coagulase test were perforemed to confirm the absence of virulence characteristics.
3.1.4.3. Detection of virulence genes form isolated strains Absence of potential virulence determinants was confirmed by PCR amplification of reported haemolysin/cytolysin and antibiotics resistance genes in Enterococcus genra. The PCR was carried out for glycopeptides antibiotics resistance mainly vancomycin resistance namely vanA, and vanB. The absence of hamolysis was confirmed with haemolysin primers. The PCR reaction conditions were same as described previously and 2.0% agarose gel was run for qualitative purpose. The primers used for amplification of virulence determinats genes were from Sigma Genoys (Sigma Alrdrich, USA) and are listed in Table 3.3.
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Table 3.3. Primers for amplification of virulence determinants from Enterococcus spp.
Target Gene
Primer Name
E.f Ent.cyl-S754
Oligoneculeotide
5 GGGTTGGTGGCGGTATTTTTACTGGA 3 5 AATAATGCACCTACTCCTAAGCCTATGGT 3 5 GCATGGCAAGTCAGGTGAAGATGGAT 3 5 GGTCCACCTCGCCAACAACTAACG 3 5 CGGAATGGGAAGCCGATAGTCTCC 3 5 TTCCCATGACCGCGCAGCCGAC 3
Position
754-780
Product Size bp
cyl E.f Ent.cyl-AS1019 E.f Ent.vanA-S227 vanA E.f Ent.vanA-AS631 E.f Ent.vanB-S230 vanB E.f Ent.vanB-AS722 700-722 607-631 230-254 990-1019 227-253
265
404
492
cyl; Cytolsin, vanA; vancomycin resistance gene type A, vanB; vancomycin resistance gene type B
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3.2. BACTERIOCIN PRODUCTION BY BACTERIAL ISOLATES

3.2.1. Screening of Isolates for Bacteriocin Production Isolated strains were screened for their antimicrobial potential with stab overlay method and agar well diffusion assay. Listeria monocytogenes strains obtained from Nestle Milk Products Ltd. (Pakistan) and Enterococcus faecalis isolated previously were used as indicator strains. As per definition bacteriocins have potential antimicrobial activity against closely related genera and strains. The main focus for screening was selection of strains with good antilisterial activity. The antimicrobial activity of isolated strains was performed with crude bacteriocin against several Gram-positive and Gram-negative strains was tested using spot-onlawn method and the modified deferred antagonism method (Kwon et al., 2002) as follows: an overnight culture of the isolated strain was stabbed onto the surface of an MRS agar plate, and then incubated at 37 C for 24 h to allow for colony development. Indicator strain (100 l at OD600=0.45) was inoculated into 7 ml of a soft (0.7%) MRS agar and poured over the plate in which the bacteriocin-producing bacteria was grown. After 24 h of incubation, the plates were checked for the appearance of an inhibition zone. Indicator strains were subcultured in the appropriate medium and then inoculated into soft agar medium of the same composition. 3.2.2. Batch fermentation for bacteriocin production by selected isolates The isolated strains namely Ent. faecium IJ-21, IJ-31 and Ent. faecalis IJ-11 along with control strain were used for the production of bacteriocin. Each experiment was done in triplicate. Batch fermentation experiments using MRS broth (100 ml) were performed under static conditions. Incubation time was optimized by performing the fermentation experiment for 72 hrs at 37 C with 1% inoculum and the samples were collected at 8, 12, 16, 24, 36, 48 and 72 hrs of incubation. Analysis of growth (CFU/ml) protein concentration and bacteriocin activity (AU/ml) were performed at each step.
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3.2.3. Optimization of physical and chemical parameters for bacteriocin production by selected Enterococcus spp. Parameters such as optimum incubation time, media pH, incubation temperature, inoculum size and media composition were optimized. Further the sets of experiments were performed at different temperatures 25, 30, 35, 37, 40 and 45 C. The flasks were incubated for 96 hrs and samples were collected at 8, 12, 16, 24, 36, 48, 72 and 96 hrs and analyzed. Optimization of media pH was done with MRS broth adjusted to pH 4, 5, 6, 7, 8, 9 and 10, inoculated with 1% of bacteriocin producing culture grown overnight at 37C. Inoculum size was optimized for 1, 2.5 and 5.0% of inoculum at 37 C and constant pH 7.0. Addition of different sodium chloride concentrations (0, 10, 20, 40 and 60 g/l) and glucose concentrations (5, 10, 20, 40 and 80 g/l) were done in original MRS broth medium for their effect on bacteriocin production. The composition of MRS broth is given in Table 3.4.
Table 3.4. Composition of MRS broth for 1 liter medium Peptone Lab Lemco Powder Yeast Extract Glucose Tween 80 Di Potasium Hydrogen Phosphate Sod. Acetate.3 H2O Tri ammonium citrate Magnesium sulphate.7 H2O Maganese Sulphate. 4 H2O 10 gms 8 gms 4 gms 20 gms 1ml 2 gms 5 gms 2 gms 0.2gms 0.05gm
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The effect of peptone, yeast extract, meat extract and tween 80 on the bacteriocin production was checked by modifying the concentrations of individual components in the media used in fermentation experiments. Different media compositions M1-M7 were made by varying the concentration of peptone, yeast extract, meat extract and tween 80 in MRS broth, remaining ingredients were same as reported by de Man et al. 1960. The modification and composition of MRS broth is given in Table 3.5.
3.3. ANALYTICAL METHODS

3.3.1. Bacteriocin Quantification Bacteriocin activity in the liquid was determined by an adaptation of the Agar well diffusion assay and critical dilution method, currently used for the assay of bacteriocins De Vuyst et al., (1996). Serial twofold dilutions of cell-free culture supernatant containing bacteriocin were spotted (10 l) on agar plates containing overlay of fresh cultures of a sensitive strain or the cell free culture supernatant (50 l) was loaded into the well. These overlay cultures were prepared by propagating fresh cultures to an optical density at 600 nm of 0.45, and adding 100 l of the cell suspension to 3.5 ml of overlay agar. Overlaid agar plates were incubated for 18 h at the appropriate temperature. The bacteriocin activity was defined as the reciprocal of the highest dilution that demonstrated complete inhibition of the indicator lawn, and was expressed in activity units (AU) per milliliter of culture medium. The units were derived by multiplication with factor 20 for agar well diffusion assay and with 100 for spot on lawn assay to make activity unit per milliliter (AU/ml). 3.3.2. Study of growth pattern Growth pattern of potential strains for bacteriocin were studied by 1% inoculation of fresh culture into 500ml of MRS broth. The growth OD was monitored at 600nm and protein estimation was done with Bradford protein assay at every 30min interval and viable cell count was derived for colony forming units (CFU) after every one hour by serial dilution and plating method.
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Table 3.5. Modified MRS media with different levels of peptone, yeast extract, meat extract and tween 80
Modified Media M1 M2 M3 M4 M5 M6 M7
Peptone (g/l) 10 10 10 10 5 5 2.5
Yeast extract(g/l) 5 5 5 0 0 2.5 7.5
Meat extract (g/l) 2 2 0 0 0 0 0
Tween 80(g/l) 1 0 0 0 0 0 0
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3.3.3. Protein Quantification Protein concentrations were determined by Bradford method (Bradford, 1976) using a protein assay kit from BioRad Laboratories (California, USA). Bovine serum albumin (BSA) was used as standard. To 0.9 ml of Bradford reagent, 100l of protein sample was added and OD was taken at 595nm on Agilent spectrophotometer (Agilent Technologies) after 15 min of incubation at room temperature. Bradford reagent (0.9 ml) mixed with 100l of distilled water was used as blank. The standard solution was prepared by adding 100 l of BSA stock solution in 200 l of distilled water. 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 l of the standard were added in each test tube containing 0.9 ml of Bradford reagent. The absorbance of the tubes was determined at 595nm within 15-16 min and readings were plotted for quantification of total protein.
3.4. CHARACTERIZATION OF BACTERIOCIN

3.4.1. Locus identification of bacteriocin genes Plasmid curing was done to check the location of bacteriocin genes on plasmid with different concentrations of ethidium bromide according to the method of Trevors (1986). Over night culture of isolated strains of Enterococcus faecium were grown in different dilutions of ethidium bromide. Highest dilution showing the visible growth was cultured on the MRS media. Resulted colonies were subcultured and residual bacteriocin activity was checked with stab overlay assay for bacteriocin production.
3.4.2. Characterization of bacteriocins genes with Secondary PCR The potential bacteriocin producing strains Ent. faecium IJ-06, IJ-21, IJ-31, ATCC2773 along with Ent. faecalis IJ-11 were characterized by amplification of bacteriocin genes from total genome of representative strains. Specific sets of primers were used for the amplification of fragment of bacteriocins reported until now with the aim to hunt any novel bacteriocin. The bacteriocin genes reported so far are bac A, bac B, bac P, bac L 50 A, bac L 50B, bac 1071 A, bac 1071 B, bac 31 and bac AS 48. Primers were
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designed from the reported genes sequence deposited with Gene Bank at National center for biotechnology information (NCBI). PCR Reaction Following PCR conditions were used for amplification of bacteriocin genes from Enterococcus spp. Ingredients Template (0.2ng/L) Primers (10 M) Go Taq master mix (2X conc.) ddH2O Total reaction volume was 50 L. Amplification was carried out in Eppendroff thermocycler with following conditions. Denaturation temperature Time Annealing and extension temperature Time Final extension temperature Time Hold 95 C 30 sec. 72 C 2 min. 72 C 5 min. 4 C until removed 30 cycles Concentration 1 ng 1 L each 25 L 18 L Volume 5 L 2 L 25 L 18 L
The PCR products were analyzed on 2.0 % agarose gel with 1X TAE buffer at constant voltage of 80 V. 50 bp and 100 bp DNA marker (TrackIt, Invitrogen) were used to compare the PCR amplicons. The secondary PCR was done with the same protocol by taking 2 L of template from primary PCR reaction. The primers used for amplification of bacteriocin genes were obtained from Sigma Genoys (Sigma Aldrich, USA) and are listed in Table 3.6.
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Table 3.6. Primers for amplification of bacteriocins genes from isolated strains of enterococci
Target gene bacA
Primername
Oligodeoxynucleotiseprimer
Position
Product sizebp 155
AM746970EntAS571 AM746970EntAAS726
5CACAACTTATCTATGGGGGTACCACTC3 5CCCTGGAATTGCTCCACCTAAAAAACC3 5ATGCAAAATGTAAAAGAATTAAGTACGAAAGAGATG3
571598 699726 12541290 14411478 236264 314344 42664300 44284450 5489 532565 459493 11841215
bacB
AF076604EntBS1254
224
AF076604EntBAS1478 5CTTCTTTTTTAGTTGCATTTAGAGTATACATTTGCTA3
bacP
AF005726EntPS236 AF005726EntPAS344
5GTACAAAAGTTGATGCAGCTACGCGTTC3 5CGATTCCTGCAATATTCTCTTTAGCTTCTC3 5AATTAGTTATTTGTGGCATTATTGGGATTGGTT3 5GCCCAAGGGCCATGTTGTACCC3 5TTGAGGAGGAGTATCATGGTTAAAGAAAATAAATT3 5GGAAAATACTCTTTTAACCAAACTACTCTCTTG3 5CATTCTTCCACTTATTTTTATTTCCTTCTTTTCC3 5CTTCATTATCAGCTTGAATAGCAGGAGAATC3
108
bac31
E.fEnt31S4266 E.fEnt31AS4450
184
bacAS48
E.fEntAS48S54 E.fEntAS48AS532
511
bac1071 A,B
E.fEnt1071A&BS459 E.fEnt1071A&B AS1215
756
Bac. L50A, L50B
AJ223633EntL50A S1364 AJ223633EntL50A AS1473 AJ223633EntL50B AS1650
5ATGGGAGCAATCGCAAAATTAGTAGC3
13641390
286
5AATGATTTTGTTAATTGCCCATCCTTC3
14731500
5.CATTAATGTCTTTTTAGCCATTTTTCAATTT.3
16191650
177
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3.4.3. Cloning and transformation of bacteriocin genes in E. coli Cloning of amplified bacteriocin genes was done in competent E. coli by using TOPO cloning vector (Invitrogen, USA) according to the manufacturers instruction guide. Briefly 1 l of PCR reaction mixture was mixed with 1 l of TOPO cloning vector. This mixture was incubated for 5 min at room temperature for liagation. TOPO cloning vector are linear with 3-T overhang and topoisomerase I which is activated to readily accept PCR product with 3-A overhang. This enables fast, 5min TOPO cloning and yield upto 90% recombinants. The cloned vector was transformed in New England Biolabs (NEB) competent cells of NEB Turbo E. coli according to the users protocol and selection was done on nutrient agar plates supplemented with ampicillin. Plasmid extraction was done with overnight culture of transformed E. coli with Plasmid Mini Kit (Qiagen, USA) as per users instruction manual. Isolated plasmid DNA from these E. coli clones subsequently sequences with primers M13 universal primer (-20) forward
5GTAAAACGACGGCCAGT3
and
M13
reverse
primer
(-24)
5GGAAACAGCTATGACCATG3. Resulted sequences were checked for their homologies
with already reported one at NCBI using BLAST. 3.4.4. Partial purification of bacteriocins Enterocins were isolated from culture of the Ent. faecium IJ-06, IJ-21, IJ-31 and
ATCC27273 grown in MRS broth medium at 37C for 16 and 24 hrs. The pH of culture supernatant was adjusted to 65 by addition of 0.1N NaOH. The cells were removed by centrifugation (5500 rpm, 4C 20 min). This cell-free culture supernatant was brought to a final ammonium sulphate concentration of 20 and 40% saturation by slow addition of the salt, and was stirred overnight at 4C. Then, the mixture was centrifuged (5500 rpm, 4C, 30 min) and the surface pellicles and bottom pellets were harvested and resuspended in 10 ml of sodium phosphate buffer (pH 65). To one volume of the resuspended product, 10 volumes of a methanol-chloroform mixture (1: 2, v/v) were added, and the mixture was extracted at 4C for 1 h. The sample was centrifuged (5500 rpm, 4C, 30 min), the supernatant fraction decanted and the pellet was air-dried. The pellet was resuspended in 2 ml of ultrapure water (Gibco). This partially purified
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enterocin was stored at
20C. Protein concentrations were determined by a
modification of the method of Bradford (1976) using a Protein Assay Kit from BioRad Laboratories (California, USA). Bovine serum albumin was used as a standard. 3.4.5. Characterization of partially purified bacteriocins 3.4.5.1. Effect of enzymes Sensitivity of the bacteriocin to proteolytic enzyme trypsin, lipase, lysozyme, and catalase was tested using partially purified enterocin samples. Partially purified bacteriocin was treated with the following enzymes (Sigma, St Louis, MO, USA): trypsin (1000 units mg/ml), lipase (724 units mg/ml), lysozyme (47 000 units mg/ml), and catalase (29 900 units mg/ml). Each enzyme was dissolved in 10 mM sodium phosphate buffer (pH 7.0), and the solutions were added to the bacteriocin solution for a final concentration of 1 mg/ml. Following incubation at 37 C for 2 h, the mixture was heated at 100 C for 10 min to denature the enzymes. The residual bacteriocin activity (AU/ml) was measured by bioassay, using Ent. faecium ATCC 27273 as indicator strain. 3.4.5.2. Thermal stability Thermal stability of the partially purified enterocins was determined by incubation of partially purified enterocin solutions at 60 C for 20min, at 100 C for 15 min, and at 121 C for 15 min. Residual activity was determined for bacteriocin using sensitive strain as indicator.
3.4.5.3. pH stability To evaluate the effect of pH on bacteriocin activity, the supernatant pH levels were adjusted between 4.0 and 9.0 using 1 N HCl and 1 N NaOH. The pH stability was assayed at pH 40, 50, 60, 70, 80, and 90, at room temperature (25C) after 1 and 24 h of incubation of partially purified enterocin solutions. The remaining activity (AU/ml) was measured by bioassay. Untreated samples were used as the control.
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3.4.6. Antilisterial activity The antibacterial effect of the enterocins from Ent. faecium IJ-06, IJ-21, IJ-31 and control was investigated by adding partially purified enterocin to a log-phase culture (OD600=0.6) of L. monocytogenes in BHI (Oxoid Ltd.) at 35 C. Ten milliliter of the culture was distributed aseptically in different tubes. Each tube contained 05 ml of the partially purified enterocin, except for the control that only contained sterile distilled water. The OD600 and cell counts (cfu/ml) were measured after 10 min, 1 h, 2 h, and 4 h of incubation. Cell counts were determined on both BHI agar and Listeria selective agar (Oxoid, UK) plates.
3.4.7. Purification by chromatography Bacteriocin from Ent. faecium IJ-31 was purified further by using gel filtration chromatography Sephadex G-50 and 50 mM phosphate buffer (pH 6.5). Five grams of Sephadex-G-50 was soaked in 200 ml of 50 mM phosphate buffer (pH 6.5) containing 0.1 gm of sodium azide and incubated for 72 hours at room temperature. After soaking the gel was deaerated and poured in a 0.9 x 60 cm column. Void volume was determined by passing blue dextran (2000 kDa) through the column. The sample was loaded to the column 2.0 ml at a time. The above mentioned buffer was used to elute the sample fractions each of 3.0 ml were collected at a flow rate of 0.2 ml/min and read at 280nm using spectrophotometer. Antimicrobial assay was performed against indicator organism. The active fractions were pooled and subjected to freeze drying (lyophilization). The lyophilized sample was used for further analysis.
3.4.8. Molecular weight characterization of Bacteriocins Tricine-SDS- Polyacrylamide Gel Electrophoresis To estimate the molecular mass of the bioactive peptides, tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) was carried out as described by Schagger and Jagow (1987). Polyacrylamide concentrations in the
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stacking and the separating gel were 75 and 120%, respectively. Electrophoresis was conducted at a constant voltage of 60 V for 1 h and 90 V for 12 h. standard weight protein marker was run for size estimation. After electrophoresis, the gel was cut and the part that contained the standard proteins was stained with Coomassie brilliant blue R 250 (Sigma-Aldrich N.V., Bornem, Belgium) and gel was visualized using Chemidoc (Bio-Rad Laboratories, USA). Gel was then extensively washed for 5-6 hours by continuously replacing ultra pure water (milliQ) and antimicrobial assay was done to check the zone of inhibition by using bacteriocin sensitive strains as indicator.
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4.1. Isolation and Characterization of Lactic Acid Bacteria

4.1.1. Isolation and growth characteristics Fermented dairy products namely imported processed cheese, local processed cheese, fresh milk, yoghurt, butter, spoiled dairy products and chicken sausages were used for isolation of lactic acid bacteria. Predominant colony types were selected and purified by continuous streaking on MRS media. Thirty four strains of lactic acid bacteria (LAB) were isolated on this non selective media (MRS) from dairy products of Islamabad, Pakistan. Further selective media Kanamycin Aesculin Azide (KAA) and Slanetz and Bartley (SB) were used for selective enumeration of Enterococcus spp. from these LAB isolates. By selective enumeration, 7 out of 34 strains were found to be species belonging to genus Enterococcus. These isolates were characterized for their morphology by microscopy and Gram staining. The colony morphology on MRS agar was off white, round, with smooth edges and raised from center. All of the isolated Enterococcus spp. were Gram positive cocci with arrangement of singles cells to clusters for Enterococcus spp. IJ-03, IJ-06, IJ-11, IJ-31 along with control strain, while Enterococcus spp. IJ-12 and IJ-21 were found in single or in pairs. Enterococcus spp. IJ-03, IJ-06, IJ-07, IJ-11 and IJ-12 were isolated from local processed cheese, Enterococcus spp. IJ-21 and IJ-31 were isolated from yoghurt and butter samples respectively (Table 4.1). Enterococcus faecium LMG 11423T was used as the control strain.
4.1.2. Morphological and Biochemical Characterization Screening of these isolates was done by culturing on Kanamycin Aesculin Azide Agar (KAA) and Slantz and Bartely (SB) medium. After 24 h incubation at 42 C, typical colonies of presumptive enterococci (colonies surrounded by black zones) were selected and subcultured in order to obtain pure isolates. All of the isolates were able to ferment ribose, manitol and lactose. Enterococcus spp. IJ-03, IJ-07, IJ-11 and IJ-12 were unable to fermented raffinose but fermented sorbitol, that was reverse for Entrococcus spp. IJ-06, IJ-21, IJ-31 and control which fermented raffinose and could not ferment sorbitol. All the isolates showed positive growth at 10 and 45 C, and MRS broth with pH 9.6, 6.5% NaCl (Table 4.2).
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Table 4.1. Morphological characterization of bacterial isolates
Bacterial Isolate
Source
Shape
Arrangement
Gram Staining
IJ-03 IJ-06 IJ-07 IJ-11 IJ-12 IJ-21 IJ-31 LMG 11423 T
Local cheese Yoghurt Butter Gent University Culture
coccobacilli Cocci Cocci Cocci Cocci Cocci Cocci Cocci
Single to clusters Single to clusters Tetrade to clusters Clusters Singles to pairs Single to pairs Single to clusters Single to clusters
G +ive G +ive G +ive G +ive G +ive G +ive G +ive G +ive
Collection Center (Belgium)
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Table 4.2. Growth characteristic and screening of bacterial isolates Growth Characteristic IJ-03 Growth at 10 C 45 C 9.6 pH 6.5% NaCl SB media Kaa media Fermentation of sugars ribose Manitol Lactose Raffinose Sorbitol +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive +ive IJ-06 +ive +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive IJ-07 +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive +ive Bacterial Isolates IJ-11 +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive +ive IJ-12 +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive +ive IJ-21 +ive +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive IJ-31 +ive +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive LMG 11423T +ive +ive +ive +ive +ive +ive +ive +ive +ive +ive -ive
SB; Slantz and Bartely medium, Kaa; kanamycin aesculin azide Agar, +ive; positive growth, -ive; no growth
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4.1.2.1. Preliminary identification of isolated strains Preliminary identification of isolated strains IJ-03, IJ-06, IJ-07, IJ-11, IJ-12, IJ-21, IJ-31 and control strains was carried out upto species level by standard biochemical and morphological tests as well as with API 20 Strep. system (bioMrieux, Germany). All of the isolated strains showed positive results for voges proskauer (VP), hydrolysis of hippuric acid (HIP), esculin hydrolysis (ESC), pyrrolidonyl arylamidase (PYRA), leucine amino peptidase (LAP), arginine dihydrolase (ADH), ribose (RIB), and were negative for -galactosidase ( GL), glucuronidase ( GUR) and alkaline phosphatase (PAL). Isolated strains IJ-06, IJ21, IJ-31 and control were positive for -galactosidase ( GAL) but IJ-03, IJ- 07, IJ-11 and IJ-12 showed negative results. All of the isolated Enterococcus strains were able to ferment ribose (RIB), mantiol (MAN), lactose (LAC) and trehalose (TRE) but none was able to ferment inulin (INU), raffinose (RAF) and glycogen (GLYG). IJ-03, IJ-07, IJ-11 and IJ-12 were unable to ferment arabinose (ARA) but IJ-06, IJ-21 and IJ-31 did. It was reverse for sorbitol fermentation results where IJ-03, IJ-07, IJ-11 and IJ-12 were positive but IJ-06, IJ-21, IJ-31 and control strain were unable to ferment (Table 1A). Results were recorded at 4 hrs and confirmed after 24 hrs of incubation at 37 C and 8 digit numeric codes were generated from these test results. Identification of strains was done with API analytical profile index along with control strains (Table 4.3). Preliminary identification showed that isolated Enterococcus spp. IJ-06, IJ-21, IJ31 were identified as Enterococcus faecium and strains IJ-03, IJ-07, IJ-11 and IJ12 were identified as Enterococcus faecalis. Isolated Enterococcus spp. were 21% of the normal microbial population of lactic acid bacteria (LAB) in indigenous dairy products of Islamabad. The two genomic subgroups were in relative proportion being Enterococcus faecalis (n=4, 12%) and Enterococcus faecium (n=3, 9%).
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Table 4.3. Identification of isolated enterococci with API 20 Strep system (bioMrieux, Germany) Bacterial isolates IJ-03 IJ-06 IJ-07 IJ-11 IJ-12 04 hrs 24 hrs 04 hrs 24 hrs 04 hrs 24 hrs 04 hrs 24 hrs 04 hrs 24 hrs IJ-21 IJ-31 04 hrs 24 hrs 04 hrs 24 hrs Enterococcus 04 hrs faecium LMG 11423
T
Time
API Code 7143511 7143711 7157510 7157511 7143511 7143711 7143511 7143711 7143511 7143711 7157510 7157511 7157510 7157511 7157510 7157511
Cumulative code 7143511/7143711 7157510/7157511 7143511/7143711 7143511/7143711 7143511/7143711
Species identification with API 20 Strep System Enterococcus faecalis
Enterococcus faecium
Enterococcus faecalis
7157510/7157511 7157510/7157511 7157510/7157511
24 hrs
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4.1.3. Molecular Characterization

4.1.3.1. Extraction of genomic DNA Extraction of Genomic DNA was done from overnight culture of isolated Enterococcus faecium and Enterococcus faecalis strains with Qiagen DNeasy Blood and Tissue Kit (Qiagen Sciences, Maryland, USA) according to the manufacturers instruction manual. The size of purified genomic DNA of these bacterial isolates was in range of 15-20kb when visualized on 0.8% agarose gel with 1X Tris acetate EDTA buffer at constant voltage of 80V (Fig 4.1). Absorbance ratios (A 260/280) of these preparations were in range of 1.80-1.90 as measured by Nano Drop Spectrophotometer (Nano Drop Technologies, USA).
4.1.3.2. PCR amplification for 16S rRNA of isolated Enterococcus spp. Genus specific primers designed for the amplification of 16S rRNA were able to amplify the region giving ~1.5kb size fragment in all of the isolated strains of Ent. faecium IJ-06, IJ-21, IJ-31 control and Ent. faecalis IJ-03, IJ-07, IJ-11 and IJ-12 (Fig. 4.2). Amplicons of 16S rRNA were column purified and subsequently sequenced for ~1.5 kb fragment using a set of primers designed from Ent. faecium LMG 11423T and Ent. faecalis V583 the sequences of which are known and available with Gene Bank at National Center for Biotechnology Information (NCBI). After sequencing the resulted nucleotide sequences were checked for their homologies with the known ones using BLAST tool at National Centre for Biotechnology Information (NCBI) to identify these strains on molecular level. Genotypic analysis confirmed four of the isolated strains IJ-03, IJ-07, IJ-11 and IJ12 were Enterococcus faecalis while IJ-06, IJ-21 and IJ-31 were found to be member of Enterococcus faecium. These 16S rRNA sequences of Ent. faecalis IJ-03, Ent. faecium IJ-06, Ent. faecalis IJ-07, Ent. faecalis IJ-11, Ent. faecalis IJ12, Ent. faecium IJ-21, Ent. faecium IJ-31 and control strain Ent. faecium ATCC 27273 were deposited in NCBI Gene Bank with accession Nos. EU547773, EU547774, EU547775, EU547776, EU547777, EU547778, EU547779 and EU547780 respectively.
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M 2 3 4 5 6 7 8 9
1636 bp
Fig 4.2. PCR amplification for 16S rRNA from isolated Enterococcus strains on 0.9% agarose gel M: 1kb TrackIt DNA marker (Invitrogen), L2: Ent. faecalis IJ- 03, L3: Ent. faecium IJ- 06, L4: Ent. faecalis IJ- 07, L5: Ent. faecalis IJ- 11, L6: Ent. faecium IJ- 12, L7: Ent. faecium IJ- 21, L8: Ent. faecium IJ- 31, L9: Ent. faecium ATCC 27273
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4.1.3.3. PCR amplification for 23S rRNA of isolated Enterococcus spp. The species specific primer designed form Ent. faecium LMG 11423T and Ent. faecalis V583 were able to amplify 23S rRNA region of their respective genomic subgroup of Ent. faecium and Ent. faecalis. Amplicons of 23S rRNA were column purified and subsequently sequenced for ~2.8 kb. These sequences were deposited in NCBI gene bank with accession nos. EU547781, EU547782, EU547783, EU547784, EU547785, EU547786, EU547787 and EU547788 for Ent. faecalis IJ-03, Ent. faecium IJ-06, Ent. faecalis IJ-07, Ent. faecalis IJ-11, Ent. faecalis IJ-12, Ent. faecium IJ-21, Ent. faecium IJ-31 and Ent. faecium ATCC 27273 respecitvely. The PCR product of ~2.8 kb size of all of the isolated strains were visualized on 0.9% agarose gel with 1X TAE buffer under constant voltage(110V) and are shown in Fig 4.3 a & b. Amplification was observed with primer set 1 in lane 2, 8 and 11 for Ent. faecalis IJ-03, Ent. faecalis IJ-07 and Ent. faecalis IJ-11. Ent. faecium IJ-06 was amplified with primer set 2 in lane 6. There was no amplification in Lanes 3, 4, 5, 7, 9, 10, 12 and 13 (Fig. 4.3a). In Fig 4.3b amplification was observed with primer set 1 in lane 2 representing Ent. faecalis IJ-12, while Ent. faecium IJ-21, IJ-31 and ATCC27273 were amplified with primer set 2 in lane 6, 9 and 12. There was no amplification in Lanes 3, 4, 5, 7, 8, 10, 11 and 13. The PCR product of ~2.8 kb size was column purified with Qiagen PCR purification column and were visualized on 0.9% agarose gel with 1X TAE buffer under constant voltage (110V) and are shown in the Fig 4.4. Nucleotide BLAST results for 16S and 23S rRNA of isolated enterococci are presented in Table 4.4. Phylogenetic relationship of isolated strains of Ent. faecalis IJ-03, IJ-07, IJ-11, IJ12 showed that these strains are clonely related and share homologies in their conserved regions. Same was the case for Ent. faecium IJ-06, IJ-21 and IJ-31. Results are shown in Fig 4.1a. & 4.1b. respectively.
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M 2 3 4 5 6 7 8 9 10 11 12 13 M
3054 bp
Fig 4.2. PCR amplification for 23S rRNA from isolated Enterococcus spp. on 0.9% agarose gel Primer set 1: Ent. faecalis V583, Primer set 2 & 3: Ent. faecium LMG 11423T M: 1kb DNA marker (Invitrogen), L2: Ent. faecalis IJ-03 (Primer set 1), L3: Ent. faecalis IJ-03 (Primer set 2), L4: Ent. faecalis IJ-03 (Primer set 3), L5: Ent. faecium IJ-06 (Primer set 1), L6: Ent. faecium IJ-06 (Primer set 2), L7: Ent. faecium IJ-06 (Primer set 3), L8: Ent. faecalis IJ-07 (Primer set 1), L9: Ent. faecalis IJ-07 (Primer set 2), L10: Ent. faecalis IJ-07 (Primer set 3), L11: Ent. faecalis IJ-11 (Primer set 1), L12: Ent. faecalis IJ-11 (Primer set 2), L13: Ent. faecalis IJ-11 (Primer set 3).
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.M 2 3 4 5 6 7 8 9 10 11 12 13 M
3054 bp
Fig 4.3. PCR amplification for 23S rRNA from isolated Enterococcus spp. on 0.9% agarose gel Primer set 1: Ent. faecalis V583, Primer set 2 & 3: Ent. faecium LMG 11423T M: 1kb DNA marker (Invitrogen), L2: Ent. faecium IJ-12 (Primer set 1), L3: Ent. faecium IJ-12 (Primer set 2), L4: Ent. faecalis IJ-12 (Primer set 3), L5: Ent. faecalis IJ-21 (Primer set 1), L6: Ent. faecium IJ-21 (Primer set 2), L7: Ent. faecium IJ-21 (Primer set 3), L8: Ent. faecium IJ-31 (Primer set 1), L9: Ent. faecium IJ-31 (Primer set 2), L10: Ent. faecium IJ-31 (Primer set 3), L11: Ent. faecium ATCC 27273 (Set 1), L12: Ent. faecium ATCC 27273 (Set 2), L13: Ent. faecium ATCC 27273 (Set 3)
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M 2 3 4 5 6 7 8 9 M
3054 bp
Fig 4.4. PCR column purified amplicons for 23S rRNA from isolated Enterococcus spp. on 0.9% agarose gel M: 1kb TrackIt DNA marker (Invitrogen), L2: Ent. faecalis IJ-03, L3: Ent. faecium IJ-06, L4: Ent. faecalis IJ-07, L5: Ent. faecalis IJ-11, L6: Ent. faecium IJ-12, L7: Ent. faecium IJ-21, L8: Ent. faecium IJ-31, L9: Ent. faecium ATCC 27273
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Table 4.4: 16S rRNA nucleotide BLAST results and homologies of isolated strains of enterococci and control Strain and Gene Bank Accession Nos.
Ent. faecalis IJ-03 (EU547773) Ent. faecium IJ-06 (EU547774) Ent. faecalis IJ-07 (EU547775) Ent. faecalis IJ-11 (EU547776) Ent. faecalis IJ-12 (EU547777) Ent. faecium IJ-21 (EU547778) Ent. faecium IJ-31 (EU547779) Ent. faecium ATCC27273 (EU547780) AB291625.1, EF653454.1, DQ411814.1, DQ239694.1, AY850358.1, AY692453.1, AY395018.1, AB036835.1, AF515223.1 AJ276355.1, EF510748.1, AB326300.1, EU547780.1, AB246407.1, EU722747.1, EF510434.1, EU547779.1, AY172570.1 EF653454.1, AM697463.1, AM697450.1, DQ239694.1, AY850358.1, AY692453.1, AB036835.1, AE016830.1, AB362602.1 DQ411814.1, DQ239694.1, AY850358.1, AY692453.1, AY395018.1, AB036835.1, AF515223.1, AB154827.1, AE016830.1 EU768717.1, EU460312.1, EU460273.1, EU547777.1, AB291625.1, AB362592.1, EU459207.1, AM697463.1, EF653454.1 EU768717.1, EU460312.1, EU460273.1, EU459207.1, AB291625.1, EU547777.1, AB362592.1, EF653454.1, AM697463.1 AB246407.1, AY172570.1, EU547780.1, EU722747.1, AB326300.1, EF510748.1, EF510434.1, AJ276355.1, EF510794.1 EU547779.1, AB246407.1, AY172570.1, EU722747.1, AB326300.1, EF510748.1, EF510434.1, AJ276355.1, DQ411813.1 2706-2717 99-100% 0.0 2725 99-100% 0.0 99100% 99100% 2152 100% 0.0 100% 2302 100% 0.0 100% 2704 99% 0.0 99% 2772 99% 0.0 2699-2710 99-100% 0.0 99100% 99% 2725 99% 0.0 99%
Closely related accession Nos.
Score
Query coverage
F value
Max. ident
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4.1.4. Safety Assessment of Isolated Enterococcus Strains

4.1.4.1. Antibiotic susceptibility of the isolates The safety investigation revealed that all the strains of the Ent. faecium and Ent. faecalis were sensitive to most of the commonly used antibiotics namely vancomycin, teicoplanin, ampicillin, erythromycin, ciprofloxacin, tetracycline and gentamycin. They were highly sensitive to tetracycline and least sensitive to streptomycin but were resistant to methicillin and kanamycin, expressing natural resistance phenotype (Fig. 4.5). Further vancomycin and teicoplanin MIC values were in the range of 1-2 g/ml, indicating sensitivity of the isolated strains of Enterococci for these antibiotics. MIC limit for these glycopeptide antibiotics is 4 g/ml (NCCLS, 2002).
4.1.4.2. Assessment of virulence factors by qualitative tests All of the indigenously isolated Enterococci were catalase negative and non haemolytic on blood agar base media (Oxoid, UK) supplemented with 5-7% of sterile sheep blood. Gelatinase and DNAse test revealed the absence of Gel and DNAse activity in all isolated strains. None of the isolated enterococcus spp. showed hemolysis on 5-7% sterile sheep blood (Table 4.5).
4.1.4.3. Detection of virulence genes form isolated strains PCR amplification was done with different sets of primers confirmed the absence of glycopeptides resistance genes van-A and van-B. Moreover no antibiotic resistance plasmid was detected in any of the strains. Absence of known virulence determinants was confirmed by amplification of critical virulence genes mainly cytolysin (cyl), haemolysin, enterococcal surface protein (esp) and gelatinase (gel) with PCR. None of the enterococci tested contained the structural genes for the production of cytolysin (Table 4.5).
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Table 4.5. Detection of virulence determinants and antibiotics resistance of bacteriocin producing isolates.
Potential Strain
Antibotic resistance
Van. MIC Types of g/ml Haemolysis
Gelatinase DNAse Catalase and
PCR amplification of
Coagulase test virulence genes
vanA vanB cyl Ent. faecium IJ-06 Kanamycin 2 & methicillin Ent. faecalis IJ-11 Ent. faecium IJ-21 Ent. faecium IJ-31 Control 4 2 1 2 -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive -ive
esp gel -ive -ive
-ive -ive -ive -ive
-ive -ive -ive -ive
van; Vancomycin, MIC; Minimum inhibitory concentration, ; Gamma haemolysis, vanA; vancomycin type A, vanB; vancomycin type B, cyl; cytolysin, esp-1 enterococcal surface protein, gel ; gelatinase, -ive; negative
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40 Zone of Inhibition(mm)
IJ E 03 IJ 06 IJ 07
30
IJ 11 IJ 12 IJ 21
20
IJ 31 C ontrol
10
0 Vancomycin Teicoplanin Ampicillin Erythromycin Ciprofloxacin Methicilin Tetracyclin Gentamycin Kanamycin
A ntibiotic s
Fig 4.5. Antibiotics suscptibility of isolated enterococci to commonly used antibiotics.
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4.2. Bacteriocin Production by Bacterial Isolates

4.2.1. Screening of Isolates for Bacteriocin Production Isolated strains showed good antimicrobial potential against closely related strains. Ent. faecium IJ-06, Ent. faecalis IJ-11 and control strain showed good inhibition of tested strains of Enterococcus spp. along with Listeria spp. Ent. faecium IJ-21 and Ent. faecium IJ-31 were active against Listeria spp. Enteroccous spp. Bacillus cereus and Bacillus subtlis. Ent. faecium IJ-31 also showed week inhibition against S. aureus (Table 4.6).
4.2.2. Study for growth pattern of potential strains Almost same pattern of bacteriocin production was observed by the strains Ent. faecium IJ-21, Ent. faecium JI-31 along with Ent. faecium LMG 11423T. Bacteriocin production was first detected 6 hrs after inoculation. Bacteriocin production then increased, reaching a maximum of 3200 AU/ml after 12 hrs. Bacteriocin activity remained stable at this level for the next 40 hrs, before declining to a level of 800 AU/ml after 120 h incubation (Fig. 4.6, 4.7, 4.8).
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Table 4.6. Antimicrobial activity of isolated Enterococcus strains Producer strain IJ-06 IJ-11 IJ-21 Listeria spp. Listeria spp. Listeria spp. Indicator strain Tested strains a 5 5 5 3 4 5 3 4 4 5 Enterococcus spp. 11 Enterococcus spp. 11 Enterococcus spp. 11 Bacillus cereus Bacillus subtlis IJ-31 Listeria spp. Inhibited strains b 4 11 2 8 4 11 2 2 4 11 2 3 2 4 11 6400 6400 800 1600 6400 6400 3200 6400 6400 6400 3200 6400 1600 6400 6400 Activity c Average Zone of inhibition d (mm) 25 28 21 23 25 28 24 26 25 28 15 20 19 23 25
Enterococcus spp. 11 Bacillus cereus Bacillus subtlis Stap. Aureus Control Listeria spp.
Enterococcus spp. 11
a b c d
The counts of strains tested The count of inhibited strains Activity in Arbitrary units per ml (AU/mL) Activity expressed as average zone of inhibition in mm
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2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
3500 3000 2500 AU/ml 2000 1500 1000 500 0 OD at 600nm Protein mg/ml AU/ml
O.D. at 600nm
Fig. 4.6. Growth pattern and bacteriocin production by Ent. faecium IJ-21
1.8 1.6 O.D. at 600nm 1.4 1.2 1 0.8 0.6 0.4 0.2 0
0 2 4 6 8 10 12 24 48 72 96 12 0
Time (hrs)
Fig. 4.7. Growth pattern and bacteriocin production by Ent. faecium IJ-31
0 2 4 6 8 10 12 24 48 72 96 12 0
Times (hrs)
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0.9 0.8 O.D. at 600nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Fig. 4.8. Growth pattern and bacteriocin production by Ent. faecium LMG 11423T
0 2 4 6 8 10 12 24 48 72 96 12 0
Time (hrs)
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4.2.3. Optimization of growth parameters and media composition 4.2.3.1. Optimization of temperature and incubation time Optimum production of bacteriocin was observed at 24hrs of incubation at 30 C and pH 7.0 form cultures of Ent. faecalim IJ-31 and control strain, antimicrobial activity started decreasing after 48 hrs. In case of Ent. faecium IJ-21 best activity and bacteriocin production was observed at 36 hrs. Enterococcus faecalis IJ-11 showed activity at 48 hrs during later growth stage. Bacteriocin production and antimicrobial activity started decreasing after that and very low activity was detected from all the samples at 96 hrs (Fig. 4.9). Listeria monocytogenes was used as indicator strain for antimicrobial assay. When production of bacteriocin by selected strains was performed at 35 C optimum production for Ent. faecium IJ-21, IJ-31 and control strain was observed at 24 hrs. After that antimicrobial activity started diminishing for Ent. faecium IJ21, IJ-31 as well as control strain. On the other hand Ent. faecalis IJ-11 sowed best activity at 48 hrs at later growth stage and activity then started decreasing (Fig. 4.10). At 40 C and constant pH 7.0 maximum production of bacteriocin and antimicrobial activity for Ent. faecium IJ-21 and IJ-31 was observed at 36 hrs of incubation, while control strain showed maximum antimicrobial activity at 48 hrs then the bacteriocin production and antimicrobial activity started decreasing. On contrary Ent. faecalis IJ-11 have not shown inhibitory activity until 96 hours (Fig. 4.11). Optimization of incubation temperature and time revealed the optimum inhibitory activity and bacteriocin production was at 35 C at constant pH 7.0 after 24 hrs of incubation for Ent. faecium IJ-31 and control. While in case of Ent. faecium IJ-21 optimum bacteriocin was observed in range of 24-36 hrs and Ent. faecalis IJ-11 showed maximum bacteriocin production after 48 hrs during the later growth stage after. Bacteriocin production was not observed at 25 and 45 C.
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25
E. faecalis IJ-11
Z on e of In h ibition (m m )
20
E. faecium IJ-21
15 10 5 0 0 20 40 60 80 Incubation Time(hrs) 100 120

E. faecium IJ-31
Control
Fig. 4.9. Time course of bacteriocin production by selected bacterial isolates at pH 7.0 and 30 C
E. faecalis IJ-11
30 Zone of Inhibition (mm) 25 20 15 10 5 0 0 20 40 60 80 100 120

Control E. faecium IJ-31 E. faecium IJ-21
Incubation Time (hrs)

Fig. 4.10. Time course of bacteriocin production by selected bacterial isolates at pH 7.0 and 35 C.
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25 20 Zone of Inhibition (mm) 15

E. faecium IJ-31
E. faecium IJ-21
10 5 0 0 20 40
Incubation Time(hrs)
Control
60
80
Fig 4.11. Time course of bacteriocin production by selected bacterial isolates at pH 7.0 and 40 C
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4.2.3.2. Optimization of media pH Optimization of pH revealed the optimum inhibitory activity and bacteriocin production of Ent. faecalis IJ-11 was observed at pH 7 and 8 followed by pH 6 during the later growth stage after 48 hours of incubation (Fig. 4.12). In case of Ent. faecium IJ-21 optimum bacteriocin production was observed at pH 7 at 24 hrs followed by pH 6 and 8 and 48 hrs. So the bacteriocin production could be carried out in range 5-9 but optimally around neutral. The bacteriocin production and antimicrobial activity started decreasing after 48 hrs during the later growth stage (Fig. 4.13). Optimum bacteriocin production was observed at pH 7 and 8 at 24 hrs followed by pH 6 in case of Ent. faecium IJ-31. The bacteriocin production and antimicrobial activity started decreasing after 48 hrs during the later growth stage (Fig. 4.14). The control strain showed optimum bacteriocin production at pH 7 followed by pH 8 at 24 hrs. Bacteriocin production was observed in wide pH range 5-8. The bacteriocin production and antimicrobial activity started decreasing after 48 hrs during the later growth stage (Fig. 4.15).
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Zone of Inhibition (mm)
30 25 20 15 10 5 0 0
pH5
24
pH6
48 72 Time(hrs)
pH7 pH8
96
pH9
Fig. 4.12. Effect of media pH for bacteriocin production form Ent. faecalis IJ-11 at 35 C
30 25 Zone of Inhibition (mm) 20 15 10 5 0 0

pH5
35 C
24
pH6
48 Time(hrs)
pH7
72
pH8
96
pH9
Fig. 4.13. Effect of media pH for bacteriocin production from Ent. faecium IJ-21 at
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30 25 Zone of Inhibition (mm) 20 15 10 5 0 0

pH5
24
pH6
48 Time(hrs)
pH7
72
pH8
96
pH9
Fig. 4.14. Effect of media pH for bacteriocin production from Ent. faecium IJ-31 at 35 C
30 Zone of Inhibition (mm) 25 20 15 10 5 0 0

pH5
C
24
pH6
48 Time(hrs)
pH7
72
pH8
96
pH9
Fig. 4.15. Effect of media pH for bacteriocin production from control strain at 35
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4.2.3.3. Optimization of inoculum size Optimization of inoculum size revealed the maximum production of bacteriocin and inhibition of sensitive strain was found to be 1% for all of the isolated strains of Ent. faecium IJ-21, IJ-31, Ent. faecalis IJ-11 along with control. Maximum inhibitory activity was observed by the strain Ent. faecium IJ-21 followed by IJ-31, control and Ent. faecalis IJ-11. Bacteriocin production decreased by either increasing or decreasing the size of inoculum (Fig. 4.16).
4.2.3.4. Optimization of glucose and NaCl concentrations At constant pH 7 and temperature moderate level of sodium chloride (10-20 g/l) improves bacteriocin activity levels in the supernatant by all of these strains and activity decreased at concentration of 40 and 60 g/l. All Ent. faecium IJ-06 showed lower level of activity than Ent. faecium IJ-21 and IJ-31 but increased to same fold was observed in all of the strains with increasing NaCl concentration (Table 4.7). Addition of different concentrations of glucose in the media showed enhancing effect, as concentration increased from 5 to 40 g/l. While further increase has inhibitory effect and bacteriocin actively decreased at concentration of 80 g/l. However, bacteriocin stability decreased or increased at low levels of sugar or complex nutrients respectively (Table 4.7). Pattern was same for all the three potential bacteriocin producing isolates. Level of activity was little low (1600 AU/ml at 5 g/l) for Ent. faecium IJ-06 as compared to others Ent. faecium IJ-21 and IJ-31 (3200AU/ml at 5 g/l)
4.2.3.5. Influence of medium pH and components on bacteriocin production The effect of medium pH on production of bacteriocins is shown in Table 4.8. Ent. faecium strains IJ-06, IJ-21 and IJ-31 were not capable of growth and bacteriocin production at pH 3.0. Although their growth occurred at pH 4.0 and 5.0, no bacteriocin was produced within the 24 h incubation period except for Ent. faecium IJ-31. Clearly, therefore, low medium pH inhibited bacteriocin production. Bacteriocins production occurred to optimum levels (6400 AU/ml) at pH 6.0-8.0, while at pH 9.0 bacteriocin production was reduced (3200 AU/ml) for IJ-06 and IJ-31 but IJ-21
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sowed 1600 AU/ml at that pH. Bacteriocin production started declining at pH 10.0. Growth and bacteriocin production, therefore, occurred at optimum levels in the neutral or slightly alkaline range. Hence, for optimizing production of enterocins in vitro, Ent. faecium strains should be grown at pH values between 6.0 and 9.0. The effect of medium composition on bacteriocins production is shown in Table 4.9. All MRS-based broth media, bacteriocin production was clearly dependant on medium components. The presence and concentrations of peptone, yeast extract and Tween 80, but not that of meat extract influenced bacteriocin production. Bacteriocin production was highest in MRS medium (commercial formulation, Merck, Darmstadt, Germany) containing all the medium components tested at their normal concentrations (Table 4.9). Omitting Tween 80 from the MRS medium formulation decreased bacteriocin production from 1600 to 3200 AU/ml (Table 4.9). When the yeast extract concentration in MRS-based medium was increased (M 7), bacteriocin could be recovered at 1600 AU/ml, even though peptone concentration was reduced to one quarter that of commercial MRS. Bacteriocin production occurred to a similar level (1600 AU/ml) in MRS-based media in which peptone concentration was halved, while that of yeast extract remained the same or was reduced to half that of commercial MRS (M 5 and M 6, respectively, Table 4.9). Decreasing the peptone concentration to one quarter while increasing yeast extract concentration may be of advantage in bacteriocin purification trials.
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30 Zone of inhibition (mm) 25 20 15 10 5

Control E. faecalis IJ-11 E. faecium IJ-21 E. faecium IJ-31
0 0.00%
1.00%
2.00%
3.00%
4.00%
5.00%
6.00%
Size of inoculum (%)
Fig. 4.16. Effect of size of inoculum on bacteriocin production by selected bacterial isolates in batch fermentation
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Table 4.7. Effect of different concentrations of glucose and NaCl on bacteriocin production Strain IJ-06 IJ-21 IJ-31 IJ-06 IJ-21 IJ-31 IJ-06 IJ-21 IJ-31 IJ-06 IJ-21 IJ-31 IJ-06 IJ-21 IJ-31 Glucose (g/l) 5 5 5 10 10 10 20 20 20 40 40 40 80 80 80 Bacteriocin activity AU/ml 1600 3200 3200 3200 6400 6400 6400 6400 6400 3200 6200 6400 1600 3200 3200 0 0 0 10 10 10 20 20 20 40 40 40 60 60 60 800 1600 1600 3200 6400 6400 1600 1600 3200 800 1600 1600 200 400 400 NaCl (g/l) Bacteriocin activity AU/ml
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Table 4.8. Influence of initial pH of MRS medium on numbers (log CFU/ml) and bacteriocin production (AU/ml) after growth for 24 h at 37C pH 3 Strain Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 4 Ent. faecium IJ-06 Ent. faecium IJ-31 5 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 6 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 7 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 8 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 9 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 10 Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 Log CFU/ml 0 0 0 5.71 5.90 8.05 7.90 8.14 8.50 8.16 8.81 8.23 8.41 8.77 8.80 8.61 9.11 9.02 8.90 9.26 8.95 9.10 9.28 Bacteriocin activity (AU/ml) 0 0 0 0 0 0 400 800 1600 3200 3200 6400 6400 6400 6400 6400 3200 6400 3200 1600 3200 1600 800 1600
Ent. faecium IJ-21 4.97
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Table 4.9. Influence of medium composition [modified MRS medium Ml-M7 on numbers (log CFU/ml) and bacteriocin production (AU/ml) of isolated strains Media M1 Strain IJ-06 IJ-21 IJ-31 M2 IJ-06 IJ-21 IJ-31 M3 IJ-06 IJ-21 IJ-31 M4 IJ-06 IJ-21 IJ-31 M5 IJ-06 IJ-21 IJ-31 M6 IJ-06 IJ-21 IJ-31 M7 IJ-06 IJ-21 IJ-31 2.5 7.5 0 0 5 2.5 0 0 5 0 0 0 10 0 0 0 10 5 0 0 10 5 2 0 Peptone (g/l) 10 Yeast extract (g/l) 5 Meat 2 Tween 1 CFU 8.23 8.50 8.8 1 8.12 8.45 8.79 8.29 8.55 8.62 8.81 8.08 7.90 8.67 8.02 8.19 8.69 8.50 8.88 8.79 8.65 8.70 Bacteriocin activity AU/ ml 3200 6400 6400 1600 3200 3200 1600 1600 1600 1600 800 800 1600 800 800 1600 1600 1600 1600 1600 1600
extract (g/l) 80 (g/l)
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4.3. Characterization of Bacteriocins

4.3.1. Plasmid curing The plasmid curing done with different concentrations of ethidium bromide confirmed the absence of plasmid encoding gene for bacteriocins of all the isolated Enterococcus strains. Further there was no indication of plasmid during plasmid extraction done with Qiagen Plasmid Mini Prep. (Qiagen Sciences, Maryland, USA) (Fig. 4.17). It confirms the presence of bacteriocins gene on chromosomal DNA.
4.3.2. Characterization of bacteriocins genes with Secondary PCR After agarose gel electrophoresis, a PCR fragment with a size of 109 bp was amplified from genomic DNA of Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium IJ-31, which corresponds to the enterocin P. A PCR fragment for enterocin A (155 bp) was detected in the case of Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium ATCC 27273 (Fig. 4.18). No signals were obtained that corresponded with the enterocins 31, AS-48, L50A, and L50B. This indicated that Ent. faecium IJ-06, Ent. faecium IJ-21 have the potential to produce both enterocin A and P, while Ent. faecium ATCC 27273 and Ent. faecium IJ-31 were able to produce enterocin A and P respectively. No signal was obtained for DNA of Ent. faecalis IJ-11 (lane 5 in Fig. 4.18), indicating that the antimicrobial activity was because of an enterocin different from those checked by PCR (Table 4.10).
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2 3
Fig 4.17. Plasmid isolation of isolated strains with Qiagen plasmid mini prep. Lanes: 1: 1kb TrackIt DNA marker (Invitrogen), L2: Ent. faecalis IJ- 03, L3: Ent. faecium IJ- 06, L4: Ent. faecalis IJ- 07, L5: Ent. faecalis IJ- 11, L6: Ent. faecium IJ- 12, L7: Ent. faecium IJ- 21, L8: Ent. faecium IJ- 31, L9: Ent. faecium ATCC 27273, L10: +ive control E. coli transformed with Phyg
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Table 4.10. Potential strains for bacteriocins and PCR amplification of bacteriocin gene
Potential Strain
Bacteriocin activity against Presence of bacteriocin genes indicator strains L. monocytogenes and Ent. faecalis Ent A + + + Ent B Ent P + + + Ent AS 48 Ent 31 Ent L50A & B Ent 1071A & 1071 B -
Ent. faecium IJ-06 Ent. faecalis IJ-11 Ent. faecium IJ-21 Ent. faecium IJ-31 Ent. faecium ATCC 27273
3200AU/ml 1600AU/ml 6400AU/ml 6400AU/ml 3200AU/ml
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M 2 3 4 5
6 7
8 9 M
Fig. 4.18. Apmplification of bacteriocin genes from isolates on 2.0% Agarose gel M: 100bp DNA marker, L2: Ent. faecium IJ-06 entA, L3: Ent. faecium IJ-06 entP, L4: Ent. faecalis IJ-12 entA, L5: Ent. faecium IJ-21 entA, L6: Ent. faecium IJ-21 entP, L7: Ent. faecium IJ-31 entA, L8: Ent. faecium IJ-31 entP, faecium ATCC 27273 entA L9: Ent.
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4.3.3. PCR product purification, Cloning and transformation of bacteriocin genes in E. coli Amplicons from secondary PCR were column purified for representative enterocin genes namely enterocin A (ent A) and enterocin P (ent P) from Ent. faecium IJ-06, IJ-21, IJ-31 along with ATCC 27273. Cloning of amplified bacteriocin genes was done into TOPO cloning vector (Invitrogen, USA) and cloned vectors were transformed into competent E. coli cells. Plasmid extraction was done from overnight culture of transformed E. coli and these clones were subsequently sequenced with universal primers M13 forward and reverse. Column purified amplicons were also sequenced with the same set of primers used for PCR amplification. Following sequences were obtained for enterocin A and enterocin P. Enterocin A (ent A) TCATTTAGCCACTTCCCTGGAATTGCTCCACCTAAAAAACCACCTATAGACATTCCTGGA ATACAAGTAGTTGCCTTGGCCCAATCGACCGTACATTTATTTTTAGTGCAATACACTCCA TTTCCATAATATTTTCCACTATGAGTGGTACCCCCA Enterocin P (ent P) GTACAAAAGTTGATGCAGCTACGCGTTCATATGATAATGGTATTTATTGTAATAATAGTA AGTGCTGGGTTAATTGGGGAGAAGCTAAAGAGAATATTGCAGGAATCG These partial sequences of enterocins showed homologies (90-100%) with the previously reported enterocin A and P genes. Transformed E. coli clones were checked for their expression of bacteriocin genes with stab-overlay assay using Ent. faecalis IJ-11 as indicator strain. Bacteriocin production was not observed with any of the clone from qualitative assay.
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4.3.4. Isolation and partial purification of enterocins Activity of bacteriocin-producing enterococci against different indicator strains during the partial purification process is shown in Table 4.11. For the enterocins produced by the Ent. Faecium IJ-06 no activity was detected in the cell-free culture supernatant. However, after ammonium sulphate precipitation and chloroform/methanol extraction, titres of 3200 AU/ml was measured. The antimicrobial activity towards L. monocytogenes (AU/ml), protein concentration (mg/ml), and increase in specific activty SA (AU/mg) of all the enterocins upon isolation and partial purification are shown in Table 4.12. For the enterocins produced by Ent. faecium IJ-21 and Ent. faecium IJ-31, the degree of purification expressed as the increase in SA (AU/mg) was less than 100.
4.3.5. Sensitivity of partially purified enterocins to pH, enzymes and temperature treatment Partially purified enterocin from Ent. faecium IJ-06, IJ-21, IJ-31 and control strain were stable in a wide range of pH. It appeared stable to adjustment of pH of 4.0, 5.0, 6.0, 7.0 and 8.0. The temperature stability of the enterocins depended on the pH. At acidic pH values the enterocins studied were more heat-stable than at neutral or alkaline pH. The activity was higher at low pH. Antibacterial activity of the partially purified enterocins studied was completely destroyed showing no residual activity upon treatment with trypsin. Enterocin activities were not affected by lipase, lysozyme, and catalase (Fig 4.19). Partially purified bacteriocin form Ent. faecium IJ-06, IJ-21, IJ-31 and control retained their activity after the temperature treatment of 60, 80 100 and 121 C for 15 min (Fig. 4.20).
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Table 4.11. Activity of bacteriocin-producing enterococci against different indicator strains
Indicator Strain
Producer Strains Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 Sup C/M + + + + + Sup + + + + + C/M + + + + + + + + Sup + + + + + + + + C/M + + + + + + + + Ent. faecium LMG 11423T Sup + + + + + C/M + + + + + -
Ent. faecalis IJ-03 Ent. faecalis IJ-07 Ent. faecalis IJ-11 Ent. faecalis IJ-12 Ent. faecium LMG 11423T Listeria spp. ARL-03 Listeria spp. ARL 05 Bacillus subtllis Bacillus cereus
Sup = cell free culture supernatant C/M = chloroform-methanol extract + = inhibitory activity, - = no inhibitory activity
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Table 4.12. Antilisterial activity of selected bacteriocin-producing strains using L. monocytogenes as indicator strains Producer strain Cell free culture supernatant AU/ml Ent. faecium IJ-06 Ent. faecium IJ-21 Ent. faecium IJ-31 Ent. faecium LMG 11423T 0 400 1600 3200 mg/ml 13.2 15.4 16.1 14.6 AU/mg 0 25.97 99.38 219.17 Ammonium sulphate precipitation AU/ml 400 3200 16200 16200 mg/ml 7.0 5.9 6.2 5.4 AU/mg 57.15 542.37 2612.9 3000 Chloroform methanol extract AU/ml 3200 6400 16200 16200 mg/ml 2.2 1.8 1.7 1.3 AU/mg 1454 3555 9529 12461
Antilisterial activity (AU/ml) = Reciprocal of the highest dilution x 1000 / volume of the sample used. Protein conc. (mg/ml) = measured by Bradford assay taking BSA as a standard. Specific activity (AU/mg) = Total activity of the sample / Total protein of the sample.
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25 Zone of inhibition (mm) 20 15 10 5 0 Supernatant + Protease Supernatant + Catalase Supernatant + lipase Supernatant + Lysozyme
E. faecium IJ-06 E. faecium IJ-21 E. faecium IJ-31 Control
Enzymes
Fig 4.19. Effect of different enzymes on the residual activity of partially purified bacteriocin
25 Zone of inhibition (mm) 20

E. faecium IJ-06
15
E. faecium IJ-21
10
E. faecium IJ-31
5
Control
0 60 80 100 121
Temperature (C)
Fig. 4.20. Thermo-stability assay of partially purified bacteriocin
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4.3.6. Mode of inhibition Under the experimental conditions used in this study, L. monocytogenes was very sensitive to the different enterocin preparations investigated. Ten minutes after the addition of the enterocins from Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium IJ-31, the decrease observed was between 3.56 and 3.16 log, compared with the control; after 4 h of incubation the differences were still the same. A bacteriolytic effect was noted for the enterocins produced by Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium IJ-31.
4.3.7. Inhibitory spectrum The cell-free culture supernatant and the partially purified enterocins were tested against a broad range of Gram positive bacteria (45 strains) and a Gram-negative control (E. coli). The results are shown in Table 4.5. A narrow inhibitory spectrum was found for both cell-free culture supernatant and partially purified enterocins when only lactic acid bacteria other than enterococci were considered as indicator strains. An increase of the inhibitory spectrum was observed with the partially purified enterocin preparation, but this increase was mainly observed for spoilage or pathogenic bacteria, such as other enterococci or Listeria spp. as indicator strains.
4.3.8. Tricine-SDS-PAGE A single inhibitory zone was observed for all enterocins after electrophoresis followed by a bioassay. Zone of inhibition was observed in range of ~4.5 kDa when checked Ent. faecalis IJ-11 as indicator strain. Single inhibitory zone were observed in lanes 1, 3, 5 and 7 representing enterocin purified from 16hrs cultures of Ent. faecium IJ-06, IJ-21, IJ-31 and ATCC 27273 respectively. Increase in antimicrobial activity was observed in lanes 2, 4, 6 and 8 for the enterocin samples of 24hrs from cultures of Ent. faecium IJ-06, IJ-21, IJ-31 and ATCC 27273 (Fig. 4.21). Single inhibition zone was observed when checked L. monocytogenes as indicator strain in lanes 1, 3, 5 and 7 representing 20% ammonium sulphate saturation of enterocin from Ent. faecium IJ-06, IJ-21, IJ-31 and ATCC 27273
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respectively. No zone was observed in lanes 2, 4, 6 and 8, having activity in 40% saturation (Fig.4.22).
4.3.9. Gel filtration using sephadex G-50 Total 30 fractions each of 3.0 ml were collected at a flow rate of 0.2 ml/min and monitored at 280nm with the spectrophotometer. Antimicrobial assay was performed using B. cereus as indicator strain. The separation profile resulted in two well-separated peaks. The bacteriocin activity was detected in five fractions corresponding to the second peak i.e. in fractions No. 11 to 15. The active fractions were collected and subjected to lyophilization (Fig 4.23). The specific activity of lyophilized sample was increased to 12075 AU/mg resulting in 60 fold purification (Table 4.13).
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Fig. 4.21. Antimicrobial assay SDS-PAGE Gel on Ent. faecalis IJ-11 as indicator strain Conc. of stacking gel 7.5%, resolving gel 15% washed with ultra pure water for 56 hours M: Precision plus protein marker (Bio-Rad), L1: Ent. faecium IJ-06 Enterocin 16 hrs, L2: Ent. faecium IJE 06 Enterocin 24hrs, L3: Ent. faecium IJ-21 Enterocin 16 hrs, L4: Ent. faecium IJ-21 Enterocin 24hrs, L5: Ent. faecium IJ-31 Enterocin 16 hrs, L6: Ent. faecium IJ-31 Enterocin 24hrs, L7: Ent. faecium ATCC 27273 Enterocin 16hrs, L8: Ent. faecium ATCC 27273 Enterocin 24hrs
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Fig 4.22. Antimicrobial assay SDS-PAGE Gel (20 and 40% of ammonium sulphate saturation) on L. monocytogenes as indicator strain M: Precision plus protein marker (Bio-Rad), L1: Ent. faecium IJ-06 Enterocin 20% saturation, L2: Ent. faecium IJ-06 Enterocin 40% saturation, L3: Ent. faecium IJ21 Enterocin 20% saturation, L4: Ent. faecium IJ-21 Enterocin 40% saturation L5: Ent. faecium IJ-31 Enterocin 20% saturation, L6: Ent. faecium IJ-31 Enterocin 40% saturation, L7: Ent. faecium ATCC 27273 Enterocin 20% saturation, L8: Ent. faecium ATCC 27273 Enterocin 40% saturation
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2.5
Absorbance at 280nm
14 Zone of inhibition (m m )
Bacteriocin activity (mm)
Absorbance at 280nm
2 1.5 1 0.5 0
12 10 8 6 4 2 0
1 2 3 4 5 6 7 8 9 10 1112131415 1617181920 2122232425 2627282930
Fractions
Figure 4.23: Purification of bacteriocin by Sephadex G-50 column. Column was equilibrated and eluted with sodium phosphate buffer, pH 6.5 at a flow rate of 0.2 ml/min.
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Table 4.13. Summary of purification of bacteriocin from crude culture filtrate of Enterococcus faecium IJ-31.
Treatment
Total volume (ml)
Activity (AU/ml) 1 3200 51200
Protein (mg/ml) 2 15.9 8.3
Specific activity Fold (AU/mg) 3 201 6168 purification4 1 31
Cell free crude filtrate 200 Ammonium precipitation Methanol-chloroform extraction Gel filtration with 1 G-50 2 sulfate 2
12800
1.6
8000
40
6400
0.53
12075
60
sephadex (Lyophilized)
1) Activity (AU/ml) = Expressed in terms of arbitrary units defined as reciprocal of the highest dilution x 1000 / volume of the sample used. 2) Protein (mg/ml) = measured by Bradfords method taking BSA as a standard. 3) Specific activity (AU/mg) = Total activity of the sample / Total protein of the sample. 4) Fold purification = Specific activity of the subsequent step / Specific activity of crude sample.
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CONCLUSION 1. Enterococcus spp. could be isolated from dairy products mainly cheese, butter and yoghurt. 2. Isolated strains Enterococcus faecium IJ-06, IJ-21, IJ-31 along with Enterococcus faecalis IJ-11 have good potential for bacteriocin production. 3. Molecular characterization of isolated bacterial strains with 16S rRNA and 23S rRNA region amplification showed 4 out of 7 seven strains were Ent. faecalis and other to be Ent. faecium. 4. Optimization of growth parameters revealed the maximum bacteriocin was produced at pH 7 and 8 at 35 C after 24hrs of incubation with 1% inoculum. 5. Moderate level of NaCl increase bacteriocin production but glucose has not significant effect. 6. Safety investigation and antibiotics susceptibility of isolated strains showed non pathogenic characteristics. 7. Absence of known virulence determinants was confirmed by PCR. 8. The resistance to vancomycin, which is a common trait for enterococci of nosocomial origin, is not wide spread among dairy enterococci. 9. Amplification of bacteriocin genes revealed entA and entP genes were present in Ent. faecium IJ-06 and IJ-21 along with the control strain Ent. faecium ATCC 27273 have entA. In case of Ent. faecium IJ-31 there was evidence of entP. 10. No expression of bacteriocin was found in transformed E. coli 11. These bacteriocins were found to be highly thermostable upto 121 C. 12. Specific activity was increased after partial purification. 13. Characterization of bacteriocin that a single protein with mol weight of ~4.5 and have very good antilisterial activity. Future Prospects Purification of bacteriocin to homogeneity and molecular mass determination with Mass Spectrometry (MALDI-TOF). N-Terminal amino acid sequencing of purified bacteriocin. Heterologus expression of bacteriocin in different hosts especially in L. lactic and E. coli. Application of these potential bacteria and their bacteriocins in dairy products for control of undesirable microorganism.
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Discussion
The present study was carried out with the objective of isolation, screening and characterization of indigenous bacterial strains for potential bacteriocin production. Lactic acid bacteria were isolated from a variety of the food products, namely cheese, yoghurt, butter, fresh milk, spoiled food products along with chicken sausages. Enterococcus spp. were only identified from cheese (5), yoghurt (1) and butter (1). So these seven strains were identified on the basis of morphological and biochemical characterization. Isolated Enterococcus spp. were a significant part (21%) of the normal microbial population of lactic acid bacteria (LAB) in indigenous dairy products of Islamabad. Many other studies also reported isolation of Enterococcus species, Franzetti et al., (2004) isolated 64 Enterococcus spp. from different sources. Du Toit et al., (2000) isolated and screened 7 out of total 92 enterococcal strains from the faeces of minipigs out of which four were identified as Enterococcus faecalis and three as Enterococcus faecium. Cheese was the best source for isolation of enterococci; five isolates IJ-03, 06, 07, 11 and 12 were isolated from local processed cheese while IJ-21 and IJ-31 were isolated from yoghurt and butter samples respectively. Gupta and Malik (2007) isolated enterococci from a total number of 68 dairy products and were screened for bacteriocin production. The incidence of the bacteriocinproduction trait was highest among the cream samples (33.51%), followed by yoghurt samples (15.55%), raw milk samples (9.51%) and cheddar cheese (9.2%) samples. Genotypic analysis on the basis of 16S and 23S rRNA homology identified 4 (12%) of the isolated strains IJ-03, IJ-07, IJ-11 and IJ-12 as Enterococcus faecalis while other 3 (9%) IJ-06, IJ-21 and IJ-31 were found to be Enterococcus faecium. Morandi et al., (2006) isolated a total of 68 strains of enterococci from different North West Italian areas. The isolates were identified as belonging to Ent. faecalis, Ent. faecium and Ent. durans. They concluded from their study that Ent. faecalis and Ent. faecium were the 100
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dominant enterococcal species present in different dairy products, this is also reported by many authors for different cheese varieties Vaughan et al., 1994; Martinez et al., 1994; Coventry et al., 1997; Laukova et al., 1999; Sarantinopoulos, 2002; Achemchem et al., 2005). One of the most important characteristic for the evaluation of enterococci is the susceptibility for different antibiotics. In our studies, all of the isolated strains of enterococci IJ-03, IJ-06, IJ-07, IJ-11, IJ-12, IJ-21 and IJ-31 were susceptible to commonly used antibiotics namely ampicillin, erythromycin, ciprofloxacin, tertracycin, gentamycin along with vancomycin and teicoplanin. These strains were resistant to methicllin and kanamycin. Abriouel et al., (2006) had almost same findings for their isolated bacteriocin producing Ent. faecium strain RJ16 which was resistant to ciprofloxacin and levofloxacin and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, teicoplanin. Further safety aspects tested showed negative test results for catalase, gelatinase, DNAse activity along with absence of hemolysis. There was no detection of any of the virulence genes, vancomycin resistance (van A, van B), cytolysin (cyl), haemolysin (hm), enterococcal surface protein (esp) and gelatinase (gel) by PCR amplification. Results for detection of virulence determinants were in accordance with the findings of Morandi et al., (2006) they found none of the isolates carried van A or van B gene of enterococci isolated from Italian cheese. Sanchez et al., (2007) studied antagonistic enterococci for potential virulent genes and reported that none of Ent. faecium strain harbored potential virulence factors, except the cell wall adhesion (efaAfm), which agrees with other results of Eaton and Gasson (2001) and Martin et al., (2006). The novel bacteriocin producing strain Ent. faecium MMT21 was non haemolytic, sensitive to vancomycin and other clinically relevant antibiotics and was an antilisterial strain (Ghrairi et al., 2008). None of the isolated enterococcus spp. showed hemolysis on 5-7% sterile human blood. These findings are in accordance with the findings of Franzetti et al., 101 tetracycline, quinupristin/dalfopristin, vancomycin and
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(2004) they inferred that none of the isolated enterococci was hemolytic out of 64 strains of different food origins. Vancomycin minimum inhibitory concentration (MIC) values of isolated strains of enterococci from dairy products in the present study ranged from 2-4 g/ml. The threshold of susceptibility of enterococci to vancomycin is 1-4 g/ml (Leclercq et al. 1989). So all of the isolated strains can be considered susceptible to the glycopeptide antibiotic vancomycin. Giraffa and Sisto (1997) found out that twenty isolated dairy enterococci were susceptible to glycopeptide antibiotics mainly van A, and minimum inhibitory concentration values ranged from 1 to 4 g/ml. Induction of van B resistance phenotype had been reported in vancomycin resistance but not in vancomycin susceptible enterococci (Woodford et al., 1995). The van C type resistance, which is a constitutive low level resistance to vancomycin, is an intrinsic property of motile enterococci such as Ent. Gallinarumand Ent. Casseliflavus (Dutka Malen et al., 1995; Woodford et al., 1995). However the vancomycin resistant enterococci (VRE) were found in nosocomial and of meat isolate (Woodfords et al., 1995; Wegener et al., 1997)
The bacterial isolates were then screened for their potential for bacteriocin production. Four isolates namely Ent. faecalis IJ-11, Ent. faecium IJ-06, IJ-21 and IJ-31 showed good antimicrobial activities against closely related species. Ent. faecalis IJ-11 and Ent. faecium IJ-06 were capable of inhibition of other Enterococcus spp. and Listeria spp. but no antimicrobial activity was detected from the supernatant of Ent. faecium IJ-06, however activity was found both with stab overlay assay and after partial purification. In case of Ent. faecium IJ-21 and IJ-31 potent bacteriocin with good activities against Enterococcus spp., Listeria spp. Bacillus cereus, Bacillus subtlis, Staphlococcus aureus and other lactic acid bacteria (Table 4.6). Du Toit et al., (2000) conferred that bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and 102
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Propionibacterium spp. Sanchez et al., (2007) revealed from their study that Ent. faecalis comprised the largest enterococcal species with antagonistic activity followed by Ent. faecium, Ent. hirae. Optimization of incubation temperature and time revealed the optimum inhibitory activity and bacteriocin production was observed at 35 C at constant pH 7.0 after 24 hrs of incubation for Ent. faecium IJ-31 and control. Enterococcus faecium IJ-21 gave maximum activity at 36 hrs, before and after which a less activity was observed and Ent. faecalis IJ-11 showed maximum bacteriocin production after 48 hrs during the later growth stage after. Low levels of activities were seen at 30 and 40 C by the selected strains and bacteriocin production was not observed at 25 and 45 C. Maximum bacteriocin production occurred in exponential growth phase in the case of Ent. faecium IJ-21, IJ-31 as is typical for bacteriocin production by most LAB (De Vuyst and Vandamme 1994) therefore, displayed primary metabolite kinetics. Decreasing bacteriocin activity after 48 hrs incubation may be explained by bacteriocin degradation due to culture proteases, or low culture pH (Parente and Hill, 1992; Torri Tarelli et al., 1994). In addition, readsorption of bacteriocin to the producer cell surface at low pH may contribute to the observed bacteriocin decrease in the culture medium. We also observed that maximum activity was observed at 35 C, while 25 and 45C did not supported bacteriocin production. Criado et al., (2006) concluded from their study that temperature has a strong influence on bacteriocin production by their strain, Maximum EntL50A and EntL50B production was detected at 25 C, while EntP and EntQ are maximally produced at 37 and 47 C, respectively. Shin et al., (2008) reported 37 C and pH 5-7 as optimum for the bacteriocin production from Pediococcus pentosaceus K23-2. Hu et al., (2008) reported enterocin from Enterococcus duran over a temperature range 20-43 C. The antimicrobial activities of Ent. faecium IJ-06, IJ-21 and IJ-31 in prolonged fermentation dramatically decreased peaking at 3648 h. That pattern had been observed for other LAB bacteriocins (Aasen et al., 2000; Mataragas et 103
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al., 2003). Bacteriocins are often produced during the growth phase and then decrease owing to proteolytic degradation, protein aggregation and adsorption by the cells (Parente and Ricciardi 1994; Parente et al., 1994; De Vuyst et al., 1996; Aasen et al. 2000). Initial media pH showed profound effect on the bacteriocin production by the selected isolates. Ent. faecalis IJ-1, optimum activities were observed at pH 5 and 6 after 48 hrs with higher pH 7, 8 and 9 maximum production was observed at 72 hrs of incubation time (Fig 4.11). In case of Ent. faecium IJ-21 all pH 5-9 favour bacteriocin production but at 48 hrs with higher levels as pH 6 and 7 than 8 and 9 (Fig. 4.12). Ent. faecium higher level of bacteriocin activities was seen in culture supernatant of pH 8.0 after 48 hrs compared to other pH values (Fig. 4.13), control strain also showed same level. Effect of pH for the bacteriocin production by the selected strains in the present study revealed that no growth and production was observed at pH 3.0, although their growth occurred at pH 4.0 and 5.0, no bacteriocin was produced within the 24 h incubation period except for Ent. faecium IJ-31. According to Van de Berghe et al., (2006) bacteriocin production demonstrated primary metabolite kinetics but was limited to the early growth phase. The critical biomass for switching off bacteriocin production was dependent on medium pH and incubation temperature, and was inversely correlated with the specific bacteriocin production. According to Leroy and De Vuyst (2002) At pH 6.5, high enterocin activity was obtained in the temperature range 2035 C and at 35 C, enterocin activity could only be detected between pH 5.5 and 8.0. Leroy et al., (2003) also reported in their study at constant pH 6.5, high enterocin activity was obtained in the temperature range of 20-30 C. Enterocin activity was only found between pH 5.5 and 8.0. Kang and Lee (2005) done the optimization trail for bacteriocin produced by Ent. faecium GM-1. They reported the optimal production of bacteriocin was obtained when the culture conditions were pH 6.0-6.5 and 35-40 C. Enterococcus faecalis IJ-11 showed different behavior, that is bacteriocin production in late stage of growth peaking after 48hrs (Fig. 4.14). In batch 104
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fermentation of Ent. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase (Yoon et al., (2005). Further for Ent. faecalis IJ-11 no signal was obtained for amplification of bacteriocin genes form genomic DNA, indicating that the antimicrobial activity was because of an enterocin different from those checked by PCR or because of some other metabolite. Moreno et al., (2003) were unable to amplify bacteriocin genes from Ent. faecium SF 68. Optimization of inoculum size revealed the maximum production of bacteriocin and inhibition of sensitive strain was found to be 1% for all of the isolated strains of Ent. faecium IJ-21, IJ-31, Ent. faecalis IJ-11 along with control (Fig. 4.18). One percent (1%, v/v) inoculum was used in bacteriocin production experiments by many authors (Leroy et al., 2003; Moreno et al., 2003; Achemchem et al., 2005). At constant pH 7 and temperature moderate level of sodium chloride (2040g/l) improved bacteriocin activity levels in the supernatant by all of these strains and activity started decreasing by further increasing its concentration. All Ent. faecium IJ-06 showed lower level of activity than Ent. faecium IJ-21 and IJ-31 but increased to same fold was observed in all of the strains with increasing NaCl concentration (Table 4.7). Neysens et al., (2003) reported maximum bacteriocin activities after the addition of 10 g/l of NaCl. On the other hand, high sodium chloride concentrations have a negative influence on the bacteriocin production (Leroy and De Vuyst, 1999). Addition of different concentrations of glucose in the media showed enhancing effect, as concentration increased from 5g/l to 40g/l. While further increase has inhibitory effect and bacteriocin actively decreased at concentration of 80g/l (Table 4.7). Increasing the glucose concentration of MRS broth leads to an increase of the specific bacteriocin production by Lb. amylovorus DCE 471, but, on the other hand, decreases the biomass level at which bacteriocin production stops (Lejeune et al., 1998).
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Plasmid curing revealed that the bacteriocin production genes are mediated by chromosomal DNA. Our findings are in accordance with Franz et al., (1996) and Du Toit et al., 2000 they reported that no plasmid could be isolated from Enterococcus faecium indicating that the gene for bacteriocin activities are located on the chromosome DNA. Moreno et al., (2003) reported the presence of entA, entB and entP genes on the genome of the previously isolated Enterococcus faecium and faecalis strains. Kang and Lee (2005) also found a bacteriocin gene similar to enterocin P from the total genomic DNA of Ent. faecium GM-1 by PCR and direct sequencing methods. Abriouel et al., (2005) findings are in contrast to our findings, they reported the presence of structural genes for enterocin P (entP) by hybridization studies on plasmid. Achemchem et al., (2005) also found from PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harbored by that strain. PCR fragment with a size of 109 bp was amplified from genomic DNA of Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium IJ-31, which corresponds to the enterocin P. A PCR fragment for enterocin A (155 bp) was detected in the case of Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium ATCC 27273. No signals were obtained that corresponded with the enterocins 31, AS-48, L50A, and L50B. Our findings are in accordance with the results of Moreno et al., (2003) they reported enterocin A in Ent. faecium CCM 4231, Ent. faecium 306 I.2.20, Ent. faecium LMG 11423T, Ent. faecium RZS C5, Ent. faecium RZS C13, and Ent. faecalis Y. Cocolin et al., 2007 also detected ent A and ent B from Ent. faecium M241 and M249 strain isolated from Italian goat milk. These strains were capable of producing enterocin active against L. monocytogenes and C. butyricum. Direct inhibitory activity was observed from cell free supernatant (CFS) of Ent. faecium IJ-21, IJ-31 and control strain. For the enterocins produced by the Ent. Faecium IJ-06 no activity was detected in the cell-free culture supernatant. However, after ammonium sulphate precipitation and chloroform/methanol extraction, titer of 3200 AU/ml was measured. For the enterocins produced by Ent. faecium IJ-21 and Ent. faecium IJ-31, the degree 106
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of purification expressed as the increase in specific activity (SA; AU/mg) was less than 100. Moreno et al., (2003) reported increase in SA (AU/mg) of all the enterocins upon isolation and partial purification. The activity of bacteriocins produced by Entrococcus faecium IJ-06, IJ-21 and IJ-31 were thermo-stable and retained their activity even after the heat treatment at 121 C for 15 minutes (Fig. 4.22). These results are consistent with the stability of bacteriocins by the other findings, Franz et al., (1996) reported that the activity of enterocin BFE 900 retained its activity after heating at 100 C and 121 C. Sparo et al., 2006 also reported a stable enterocin MR 99 from Ent. faecium. Marekova et al., (2007) exposed their bacteriocin termed as enterocin M with 60, 80 and 100 C for 1 hour and long term storage at 4 C or -20 C had no effect on its residual activity. Partially purified bacteriocin appeared stable to adjustment of pH range of 4-10. results are supported by findings of (Moreno et al., 2003; Sparo et al., 2006; Abriouel et al., 2006; Marekova et al., 2007; Shin et al., 2008; Ghrairi et al., 2008). Antibacterial activity of the partially purified enterocins studied was completely destroyed upon treatment with trypsin (residual activity of 0 AU/ml). Enterocin activities were not affected by lipase, lysozyme, and catalase results are in accordance with the findings of Park et al., 2003; Abriouel et al., 2006; Cocolin et al., 2007 and Ghrairi et al., 2008 they identified and characterized enterocin produced by Ent. faecium and reported their inactivated by proteinase K, trypsin, -chymotrypsin and papain, but not by lysozyme, lipase, catalase or glucosidase. A bacteriolytic effect was noted against Listeria monocytogenes for the enterocins produced by Ent. faecium IJ-06, Ent. faecium IJ-21, and Ent. faecium IJ-31 as reported by Moreno et al., (2003) for the enterocins produced by Ent. faecium 306 I.2.20, Ent. faecium LMG 11423T, and Ent. faecium SF 68. The size of bacteriocins produced by Ent. faecium IJ-21, IJ-31 and ATCC 27273 was ~4.5 kDa when checked as a zone of inhibition after 107
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electrophoresis. A single inhibition zone was observed from the partially purified enterocin preparations from the strains of Ent. faecium IJ-06, IJ-21, IJ31 and ATCC 27273 showing the production of only one potent bacteriocin. Bacteriocin from Enterococcus faecium JCM 5804T was also reported to be 4.5 kDa as measured by Tricine SDS-PAGE (Park et al., 2003). Moreno et al., (2003) also reported single inhibitory zone for all enterocins after electrophoresis followed by a bioassay. The molecular mass of the enterocins studied averaged between 39 and 40 kDa, except for the enterocin from Ent. faecium SF 68, which was slightly smaller (38 kDa). Yoon et al., (2005) reported the apparent molecular mass of enterocin HJ35 was to be approximately 4 similar to 4.5 kDa on detection of its bactericidal activity after SDS-PAGE. Shin et al., (2008) reported 5 KDa pediocin from the Pediococcus pentosaceus.
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Test performe d IJ-03 API Code
Time 04:0 0 hrs 24:0 0 hrs
VP
HIP
ESC
PY RA
GL
GUR
GAL
PAL
LAP
AD H
RIB
AR A
MA N
SO R
LA C
TR E
IMU
RA F
AMD
GL YG
Hem
+ 1 7 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7
+ 2 + 2 + 2 + 2 + 2 + 2 + 2
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 4 0 4 + 1 5 + 1 5 0 4 0 4 0 4
0 0 0 0 0 0 0
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 3 + 1 3 + 1 7 + 1 7 + 1 3 + 1 3 + 1 3
+ 2 + 2 + 2 + 2 + 2 + 2 + 2
0 0 + 4 + 4 0 0 0
+ 1 5 + 1 7 + 1 5 + 1 5 + 1 5 + 1 7 + 1 5
0 + 2 0 0 0 + 2 0
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
+ 1 1 + 1 1 0 0 + 1 1 + 1 1 + 1 1 + 1 1
0 0 0 0 0 0 0
NC 0 0 NC 0 0 NC 0 0 NC 0
IJ-06
04:0 0 hrs 24:0 0 hrs
IJ-07
04:0 0 hrs 24:0 0 hrs
IJ-11
04:0 0 hrs
24:0 0 hrs IJ-21 04:0 0 hrs 24:0 0 hrs IJ-31 04:0 0 hrs 24:0 0 hrs LMG 11423T 04:0 0 hrs 24:0 0 hrs
+ 1 7 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7
+ 2 + 2 + 2 + 2 + 2 + 2 + 2
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 4 + 1 5 + 1 5 + 1 5 + 1 5 + 1 5 + 1 5
0 0 0 0 0 0 0
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 3 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7 + 1 7
+ 2 + 2 + 2 + 2 + 2 + 2 + 2
0 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 7 + 1 5 + 1 5 + 1 5 + 1 5 + 1 5 + 1 5
+ 2 0 0 0 0 0 0
+ 4 + 4 + 4 + 4 + 4 + 4 + 4
+ 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1 + 1 1
0 0 0 0 0 0 0
0 0 0 0 0 0 0
+ 1 1 + 0 0 + 1 1 0 0 + 1 1 0 0 + 1 1
0 0 0 0 0 0 0
0 NC 0 0 NC 0 0 NC 0 0
Table 4.1a. Test results for API Sterp system of isolated strains of enteroccocci
16S and 23S rRNA sequence of Enterococcus faecalis IJ-03 LOCUS EU547773 1473 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecalis strain IJ-03 16S ribosomal RNA gene, partial sequence. ACCESSION EU547773 VERSION EU547773.1 GI:183673460 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1473) AUTHORS Javed,I., Ali,M.I., Ahmad,B., Ghumro,P.B., Hameed,A., Chaudry,G.J. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1473 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-03" /db_xref="taxon:1351" <1..>1473 rRNA /product="16S ribosomal RNA" ORIGIN 1 cgcggcgtgc ctaatacatg caagtcgaac gcttctttcc tcccgagtgc ttgcactcaa 61 ttggaaagag gagtggcgga cgggtgagta acacgtgggt aacctaccca tcagaggggg 121 ataacacttg gaaacaggtg ctaataccgc ataacagttt atgccgcatg gcataagagt 181 gaaaggcgct ttcgggtgtc gctgatggat ggacccgcgg tgcattagct agttggtgag 241 gtaacggctc accaaggccr cgatgcatag ccgacctgag agggtgatcg gccacactgg 301 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc ggcaatggac 361 gaaagtctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaaactctgt 421 tgttagagaa gaacaaggac gttagtaact gaacgtcccc tgacggtatc taaccagaaa 481 gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 541 tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 601 caaccgggga gggtcattgg aaactgggag acttgagtgc agaagaggag agtggaattc 661 catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cagtggcgaa ggcggctctc 721 tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 781 gtagtccacg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc ttcagtgctg 841 cagcaaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 901 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 961 ccttaccagg tcttgacatc ctttgaccac tctagagata gagctttccc ttcggggaca 1021 aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1081 gcaacgagcg caacccttat tgttagttgc catcatttag ttgggcactc tagcgagact 1141 gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1201 gggctacaca cgtgctacaa tgggaagtac aacgagtcgc tagaccgcga ggtcatgcaa 1261 atctcttaaa gcttctctca gttcggattg caggctgcaa ctcgcctgca tgaagccgga 1321 atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggcc ttgtacacac 1381 cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttttggagc 1441 cagccgccta aggtgggata gatgattggg gtg LOCUS EU547781 2922 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecalis strain IJ-03 23S ribosomal RNA gene, partial sequence. ACCESSION EU547781 VERSION EU547781.1 GI:183673568 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 2922)
AUTHORS TITLE JOURNAL
FEATURES source
rRNA ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881
Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA Location/Qualifiers 1..2922 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-03" /db_xref="taxon:1351" <1..>2922 /product="23S ribosomal RNA" tggtnaagtg ggcactagga agctatgatc tcagtgaata aggaagagaa ccaacaagct aaggatttgg cctaggagga tcccgcaagg gaaaagcacc aagctcgtta tgcgaggtta gtatgtagtc aaacgcactg cggagaaatt gcctcggaat taccgaattc gataagatcc tgttaagtgg caccatttaa ggggctaaac ttctaagggc ggtatgagta gaaaggctcg atggacaaca gtaggctaag ttaaatgctt tcctgatgtc ccgacacagg tcggcaaaat ataggcccaa taggggctga tcagaattga tccttgtcgg gagagactcg agaccccatg ggtaggagcc ttgtgttatg cagtttgact aatggttgga aagtcgagca cgctcaacgg cgacggggag caagggttgg acagttcggt agaggaccgg tagctatgta gagatttccc gaagtggaag aataagggcg gccgatgaag cagagatttc catagctgat agaaaattcg tgcttgttgg aaaattccgc tcctgagtac ctaaatactc ccggaagggg atgagtgatg agtcgaagag gtagacccga gaggaccgaa ccaaacgaac tgagaatgat agataaactc gtagtcgaaa aaaaggatgt agagtgcgta atattaccga gttgaaggtc gcgaaagaca tccgccccag ggttgatatt gaatgcatgc tactctttaa acactgccaa tagtcgagga gaccccgtaa gcgactgttt cgcctgcccg agccccagta gtaagttccg gtgaaatttt gagctttact gatgagaccg aaccctctaa ggggcggtcg aatcattcga gggacgaaag ataaaagcta gtttggcacc gctgttcgcc ccctatccgt gatggactta gggaagggat atttctttaa gctagtgata cannnnnntt gacgggacta cgaatggggg tagaggtaga attccctgag ggttgtagga taaagagggt ggcggaacac cctagtgacc agtgaaatag gcgtgccttt acggagccgc aaccatgtga cccacgtacg ttggagatag ggaggtagag cgaatgccat gggaaacagc ggggttgcac atagctcact agctgtggac gatcgtgagg ggtgagaatc ggttagtcgg cctgtaccag gattggaagt ggacaagttg gaaaagcttc gagtatccta cttcgggaga atcaaaaaca gtgctggaag aacggcggcc acccgcacga agtacctgtg gtagtttgat gaacgctagt cccgcaccac cctcctaaaa agagtgtaaa tcgggcttag ccctggggat tcgatgtcgg cattaaagcg cgcgggcgtt ccgctggtgt aaacgctgaa gaaagtaaga gttggagcgg aagtnnntaa acaccgatat aacccaatat cgcagagaac tagcggcgag ctccaatatg gaaagccccg gagaaattcc gatagtgaac atcctgaaac tgtagaatga agcgaaagcg tctacccatg ttgaaaagtg ctggttctct cactgtttgg tcatttatat ccagaccacc agacaactag agtcgagtga tacaccatta acggctggag ctgtccaccg gacctaagcc ttgtttttgt gcatgtccaa tgacggggag tagtgagaaa aggtgagcga aggggtgctg caggtctctg gttaagagga gtaactataa aaggcgtaac aagatgcagg attgagtgtt ttcggaggag taatcgtggt ggtaacggag ggcagaaggg tgatccggtg aacaggctta ctcgtcgcat gcacgcgagc ggaaatttga accagttgtt agcatctaag cccctgagag accaatacta gggcgcacgg gctttgggga cttttatagg tgaaacatct cgaaacggga gtagtctgtt tagacgaaat gtcggaatcc cagtaccgtg cgtgtgccta accggcgagt agtctgaata tccaggttga cggggatgag ccgaaatagc actaggggcc ccgggagtca agctaaggtc gatgttggct ccctgcgccg ggtgtagtgg cgcttagaag tatgactaag gaggccgata ttgaacaatg gcaatgagtc cgaaataata acaactgccc gcgaactctc acttcggtca caaaatcgta tgggttagct cggtcctaag gatttgggca ttacccgcga tgtaccacat gcgctggtgg gggagacagt gcgcccaaag agcttgactg gttccgcatg tctcccccaa cctggggctg tgggttcaga gaggagctgt ctgccaaggg tgtgaagccc atgatcaggt at tggatgcctt gctgtaagta atattacttt tagtacctgc agagcccaaa agtatagttg gctaacaaca gcggggacca agggaaaggt caacaagtca tacgattgca gggcgaatga aggtgcggta gtgtgggtag tttagggcta catctcgggt gactgcgagt ccaaaatata tagaagcagc aaaatgtacc taggagagcg tgagaatgcc gtttcctggg ggcgtaggcg gagggacgca ttgagtagag gtagcgaagt gtaccgtaaa gttaaggaac gccgcagtga agatgaagta tcggcgaagc gtagcgaaat ctgtctcaac caggacggaa gtacaggata gatactaccc gtcagatggg gttccctcag cgagacctac gaagggccat gagtccacat tagtcggtcc acgtcgtgag ccttagtacg cattgctggg acctcaagat agataggttg
16S and 23S rRNA sequence of Enterococcus faecium IJ-06 LOCUS EU547774 1472 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain IJ-06 16S ribosomal RNA gene, partial sequence. ACCESSION EU547774 VERSION EU547774.1 GI:183673475 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1472) AUTHORS Javed,I., Ahmad,B., Ali,M.I., Chaudry,G.J. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA COMMENT Name: Enterococcus faecium. FEATURES Location/Qualifiers source 1..1472 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-06" /db_xref="taxon:1352" <1..>1472 rRNA /product="16S ribosomal RNA" ORIGIN 1 tgcctaatac atgcaagtcg aacgcttctt tttccaccgg agcttgctcc accggaaaaa 61 gaggagtggc gaacgggtga gtaacacgtg ggtaacctgc ccatcagaag gggataacac 121 ttggaaacag gtgctaatac cgtataacaa tcaaaaccgc atggttttga tttgaaaggc 181 gctttcgggt gtcgctgatg gatggacccg cggtgcatta gctagttggt gaggtaacgg 241 ctcaccaagg ccacgatgca tagccgacct gagagggtga tcggccacat tgggactgag 301 acacggccca aactcctacg ggaggcagca gtagggaatc ttcggcaatg gacgaaagtc 361 tgaccgagca acgccgcgtg agtgaagaag gttttcggat cgtaaaactc tgttgttaga 421 gaagaacaag gatgagagta actgttcatc ccttgacggt atctaaccag aaagccacgg 481 ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg 541 ggcgtaaagc gagcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 601 ggagggtcat tggaaactgg gagacttgag tgcagaagag gagagtggaa ttccatgtgt 661 agcggtgaaa tgcgtagata tatggaggaa caccagtggc gaaggcggct ctctggtctg 721 taactgacgc tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 781 acgccgtaaa cgatgagtgc taagtgttgg agggtttccg cccttcagtg ctgcagctaa 841 cgcattaaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 901 gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 961 caggtcttga catcctttga ccactctaga gatagagctt ccccttcggg ggcaaagtga 1021 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1081 agcgcaaccc ttattgttag ttgccatcat tcagttgggc actctagcaa gactgccggt 1141 gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1201 cacacgtgct acaatgggaa gtacaacgag ttgcgaagtc gcgaggctaa gctaatctct 1261 taaagcttct ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct 1321 agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1381 tcacaccacg agagtttgta acacccgaag tcggtgaggt aaccttttgg agccagccgc 1441 ctaaggtggg atagatgatt ggggtgaagt ct LOCUS EU547782 2873 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain IJ-06 23S ribosomal RNA gene, partial sequence. ACCESSION EU547782 VERSION EU547782.1 GI:183673583 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 2873)
Javed,I., Ali,M.I., Ahmad,B., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..2873 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-06" /db_xref="taxon:1352" <1..>2873 rRNA /product="23S ribosomal RNA" ORIGIN 1 gcacggtgga tgccttggca ctaggagccg atgaaggacg ggactaacac cgatatgctt 61 tggggagctg tacgtaagct atgatccaga gatttccgaa tgggggaacc caacatcttt 121 aataggatgt tacgattgtg tgaatacata gcacattcga ggtagacgca gagaactgaa 181 acatctaagt acctgcagga agagaaagaa aattcgattc cctgagtagc ggcgagcgaa 241 acgggaaaag cccaaaccag caagcttgct tgttggggtt gtaggactcc aatatggtag 301 ttctttcaga tagtcgaatg acttggaaaa gtcagtcaaa gagggtaaaa accccgtaga 361 cgaaatgtgg aagacaccta ggaggatcct gagtacggcg gaacacgaga aattccgtcg 421 gaatccggga ggaccatctc ccaaggctaa atactcccta gtgaccgata gtgaaccagt 481 accgtgaggg aaaggtgaaa agcaccccgg aaggggagtg aaatagaacc tgaaaccgtg 541 tgcctacaac aagtcaaagc ccgttaatgg gtgatggcgt gccttttgta gaatgaaccg 601 gcgagttacg attgcatgcg aggttaagtt gaagagacgg agccgcagcg aaagcgagtc 661 tgaatagggc gtttgagtat gtagtcgtag acccgaaacc atgtgatcta cccatgtcca 721 ggttgaaggt gcggtaaaac gcactggagg accgaaccca cgtacgttga aaagtgcggg 781 gatgaggtgt gggtagcgga gaaattccaa acgaacttgg agatagctgg ttctctccga 841 aatagcttta gggctagcct cggaattgag aatgatggag gtagagcact gtttggacta 901 ggggcccatc tcgggttacc gaattcagat aaactccgaa tgccattcat tcatatccgg 961 gagtcagact gtgagtgata agatccatag tcgaaaggga aacagcccag accaccagct 1021 aaggtcccaa aatatatgtt aagtggaaaa ggatgtgggg ttgcacagac aactaggatg 1081 ttggcttaga agcagccacc atttaaagag tgcgtaatag ctcactagtc gagtgaccct 1141 gcgccgaaaa tgtaccgggg ctaaacatat taccgaagct gtggagtaca cctttaggtg 1201 tattggtagg agagcgttct aagggcgtcg aaggcagatc gtgaggactg ctggagcgct 1261 tagaagtgag aatgccggta tgagtagcga aagacaggtg agaatcctgt ccaccgaatg 1321 actaaggttt cctggggaag gctcgtccgc ccagggttag tcgggaccta agccgaggcc 1381 gacaggcgta ggcgatggat aacaggttga tattcctgta cccgttgttt ttgtttgagc 1441 aatggaggga cgcaggaggc taaggaatgc agacgatcgg aaatgtctgt ccaagcagta 1501 agtctgaaga ggagtcaaat gcttcttttc ttaaggacaa gctgtgatgg ggagggaaat 1561 aatagtaccg aagttcctga tgtcacactg ccgagaaaag cttctagtga gaaaacagcg 1621 gcccgtaccg caaaccgaca caggtagtcg aggagagaat cctaaggtga gcgagagaac 1681 tctcgttaag gaactcggca aaatgacccc gtaacttcgg gagaaggggt gctgatcata 1741 cgatcagccg cagtgaatag gcccaagcga ctgtttatca aaaacacagg tctctgcaaa 1801 atcgtaagat gaagtatagg ggctgacgcc tgcccggtgc tggaaggtta agaggagtgc 1861 ttagcgcaag cgaaggtacg aattgaagcc ccagtaaacg gcggccgtaa ctataacggt 1921 cctaaggtag cgaaattcct tgtcgggtaa gttccgaccc gcacgaaagg cgtaacgatt 1981 tgggcactgt ctcaacgaga gactcggtga aattttagta cctgtgaaga tgcaggttac 2041 ccgcgacagg acggaaagac cccatggagc tttactgtag tttgatattg agtgtctgta 2101 ccgcatgtac aggataggta ggagccgtag aaatcggaac gctagtttcg atggaggcgc 2161 tggtgggata ctacccctgc gttatggcca ctctaacccg caccactaat cgtggtggga 2221 gacagtgtca gatgggcagt ttgactgggg cggtcgcctc ctaaaaggta acggaggcgc 2281 ccaaaggttc cctcagaatg gttggaaatc attcgaagag tgtaaaggca gaagggagct 2341 tgactgcgag accaacaagt cgagcaggga cgaaagtcgg gcttagtgat ccggtggttc 2401 cgcatggaaa gggccatcgc tcaacggata aaagctaccc tggggataac aggcttatct 2461 cccccaagag tccacatcga cggggaggtt tggcacctcg atgtcggctc gtcgcatcct 2521 ggggctgtag tcggtcccaa gggttgggct gttcgcccat taaagcggca cgcgagctgg 2581 gttcagaacg tcgtgagaca gttcggtccc tatccgtcgc gggcgttgga aatttgagag 2641 gagctgtcct tagtacgaga ggaccgggat ggacttaccg ctggtgtacc agttgttctg 2701 ccaagggcat tgctgggtag ctatgtaggg aagggataaa cgctgaaagc atctaagtgt 2761 gaagcccacc tcaagatgag atttcccatt tctttaagaa agtaagatcc ctgagagatg 2821 atcaggtaga taggtcagga gtggaagtac agtgatgtat ggagcggact gat
16S and 23S rRNA sequence of Enterococcus faecalis IJ-07 LOCUS EU547775 1517 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecalis strain IJ-07 16S ribosomal RNA gene, partial sequence. ACCESSION EU547775 VERSION EU547775.1 GI:183673491 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1517) AUTHORS Javed,I., Ali,M.I., Ahmad,B., Ghumro,P.B., Hameed,A., Chaudry,G.J. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1517 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-07" /db_xref="taxon:1351" <1..>1517/product="16S ribosomal RNA" rRNA ORIGIN 1 catgcaatga cgaacgctgg cggcgtgcct aatacatgca agtcgaacgc ttctttcctc 61 ccgagtgctt gcactcaatt ggaaagagga gtggcggacg ggtgagtaac acgtgggtaa 121 cctacccatc agagggggat aacacttgga aacaggtgct aataccgcat aacagtttat 181 gccgcatggc ataagagtga aaggcgcttt cgggtgtcgc tgatggatgg acccgcggtg 241 cattagctag ttggtgaggt aacggctcac caaggccacg atgcatagcc gacctgagag 301 ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 361 gaatcttcgg caatggacga aagtctgacc gagcaacgcc gcgtgagtga agaaggtttt 421 cggatcgtaa aactctgttg ttagagaaga acaaggacgt tagtaactga acgtcccctg 481 acggtatcta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 541 tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg caggcggttt cttaagtctg 601 atgtgaaagc ccccggctca accggggagg gtcattggaa actgggagac ttgagtgcag 661 aagaggagag tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg aggaacacca 721 gtggcgaagg cggctctctg gtctgtaact gacgctgagg ctcgaaagcg tggggagcaa 781 acaggattag ataccctggt agtccacgcc gtaaacgatg agtgmtaagt gttggagggt 841 ttccgccctt cagtgctgca gcaaacgcat taagcactcc gcctggggag tacgaccgca 901 aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 961 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaccactc tagagataga 1021 gctttccctt cggggacaaa gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1081 gatgttgggt taagtcccgc aacgagcgca acccttattg ttagttgcca tcatttagtt 1141 gggcactcta gcgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1201 tcatgcccct tatgacctgg gctacacacg tgctacaatg ggaagtacaa cgagtcgcta 1261 gaccgcgagg tcatgcaaat ctcttaaagc ttctctcagt tcggattgca ggctgcaact 1321 cgcctgcatg aagccggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1381 cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg 1441 aggtaacctt tttggagcca gccgcctaag gtgggataga tgattggggt gaagtcgtaa 1501 caaggtagcc gaatttg LOCUS EU547783 2899 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecalis strain IJ-07 23S ribosomal RNA gene, partial sequence. ACCESSION EU547783 VERSION EU547783.1 GI:183673603 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 2899)
Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..2899 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-07" /db_xref="taxon:1351" <1..>2899 rRNA /product="23S ribosomal RNA" ORIGIN 1 aagtgaataa gggcgcacgg tggatgcctt ggcactagga gccgatgaag gacgggacta 61 acaccgatat gctttgggga gctgtaagta agctatgatc cagagatttc cgaatggggg 121 aacccaatat cttttatagg atattacttt tcagtgaata catagctgat tagaggtaga 181 cgcagagaac tgaaacatct tagtacctgc aggaagagaa agaaaattcg attccctgag 241 tagcggcgag cgaaacggga agagcccaaa ccaacaagct tgcttgttgg ggttgtagga 301 ctccaatatg gtagtctgtt agtatagttg aaggatttgg aaaattccgc taaagagggt 361 gaaagccccg tagacgaaat gctaacaaca cctaggagga tcctgagtac ggcggaacac 421 gagaaattcc gtcggaatcc gcggggacca tcccgcaagg ctaaatactc cctagtgacc 481 gatagtgaac cagtaccgtg agggaaaggt gaaaagcacc ccggaagggg agtgaaatag 541 atcctgaaac cgtgtgccta caacaagtca aagctcgtta atgagtgatg gcgtgccttt 601 tgtagaatga accggcgagt tacgattgca tgcgaggtta agtcgaagag acggagccgc 661 agcgaaagcg agtctgaata gggcgaatga gtatgtagtc gtagacccga aaccatgtga 721 tctacccatg tccaggttga aggtgcggta aaacgcactg gaggaccgaa cccacgtacg 781 ttgaaaagtg cggggatgag gtgtgggtag cggagaaatt ccaaacgaac ttggagatag 841 ctggttctct ccgaaatagc tttagggcta gcctcggaat tgagaatgat ggaggtagag 901 cactgtttgg actaggggcc catctcgggt taccgaattc agataaactc cgaatgccat 961 tcatttatat ccgggagtca gactgcgagt gataagatcc gtagtcgaaa gggaaacagc 1021 ccagaccacc agctaaggtc ccaaaatata tgttaagtgg aaaaggatgt ggggttgcac 1081 agacaactag gatgttggct tagaagcagc caccatttaa agagtgcgta atagctcact 1141 agtcgagtga ccctgcgccg aaaatgtacc ggggctaaac atattaccga agctgtggac 1201 tacaccatta ggtgtagtgg taggagagcg ttctaagggc gttgaaggtc gatcgtgagg 1261 acggctggag cgcttagaag tgagaatgcc ggtatgagta gcgaaagaca ggtgagaatc 1321 ctgtccaccg tatgactaag gtttcctggg gaaggctcgt ccgcccaggg ttagtcggga 1381 cctaagccga ggccgatagg cgtaggcgat ggacaacagg ttgatattcc tgtaccagtt 1441 gtttttgttt gaacaatgga gggacgcagt aggctaagga atgcatgcga ttggaagtgc 1501 atgtccaagc aatgagtctt gagtagagtt aaatgcttta ctctttaagg acaagttgtg 1561 acggggagcg aaataatagt agcgaagttc ctgatgtcac actgccaaga aaagcttcta 1621 gtgagaaaac aactgcccgt accgtaaacc gacacaggta gtcgaggaga gtatcctaag 1681 gtgagcgagc gaactctcgt taaggaactc ggcaaaatga ccccgtaact tcgggagaag 1741 gggtgctgac ttcggtcagc cgcagtgaat aggcccaagc gactgtttat caaaaacaca 1801 ggtctctgca aaatcgtaag atgaagtata ggggctgacg cctgcccggt gctggaaggt 1861 taagaggatg ggttagcttc ggcgaagctc agaattgaag ccccagtaaa cggcggccgt 1921 aactataacg gtcctaaggt agcgaaattc cttgtcgggt aagttccgac ccgcacgaaa 1981 ggcgtaacga tttgggcact gtctcaacga gagactcggt gaaattttag tacctgtgaa 2041 gatgcaggtt acccgcgaca ggacggaaag accccatgga gctttactgt agtttgatat 2101 tgagtgtttg taccacatgt acaggatagg taggagccga tgagaccgga acgctagttt 2161 cggaggaggc gctggtggga tactaccctt gtgttatgaa ccctctaacc cgcaccacta 2221 atcgtggtgg gagacagtgt cagatgggca gtttgactgg ggcggtcgcc tcctaaaagg 2281 taacggaggc gcccaaaggt tccctcagaa tggttggaaa tcattcgaag agtgtaaagg 2341 cagaagggag cttgactgcg agacctacaa gtcgagcagg gacgaaagtc gggcttagtg 2401 atccggtggt tccgcatgga agggccatcg ctcaacggat aaaagctacc ctggggataa 2461 caggcttatc tcccccaaga gtccacatcg acggggaggt ttggcacctc gatgtcggct 2521 cgtcgcatcc tggggctgta gtcggtccca agggttgggc tgttcgccca ttaaagcggc 2581 acgcgagctg ggttcagaac gtcgtgagac agttcggtcc ctatccgtcg cgggcgttgg 2641 aaatttgaga ggagctgtcc ttagtacgag aggaccggga tggacttacc gctggtgtac 2701 cagttgttct gccaagggca ttgctgggta gctatgtagg gaagggataa acgctgaaag 2761 catctaagtg tgaagcccac ctcaagatga gatttcccat ttctttaaga aagtaagacc 2821 cctgagagat gatcaggtag ataggttgga agtggaaggc tagtgatagt tggagcggac 2881 caatactaat cggntcgag
16S and 23S rRNA sequence of Enterococcus faecalis IJ-11 LOCUS DEFINITION EU547776 1470 bp DNA linear BCT 23-APR-2008 Enterococcus faecalis strain IJ-11 16S ribosomal RNA gene, partial sequence. ACCESSION EU547776 VERSION EU547776.1 GI:183673505 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1470) AUTHORS Javed,I., Ali,M.I., Ahmad,B., Chaudry,G.J. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1470 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-11" /db_xref="taxon:1351" <1..>1470/product="16S ribosomal RNA" rRNA ORIGIN 1 gcgtgcctaa tacatgcaag tcgaacgctt ctttcctccc gagtgcttgc actcaattgg 61 aaagaggagt ggcggacggg tgagtaacac gtgggtaacc tacccatcag agggggataa 121 cacttggaaa caggtgctaa taccgcataa cagtttatgc cgcatggcat aagagtgaaa 181 ggcgctttcg ggtgtcgctg atggatggac ccgcggtgca ttagctagtt ggtgaggtaa 241 cggctcacca aggccagcga tgcatagccg acctgagagg gtgatcggcc acactgggac 301 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 361 agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 421 tagagaagaa caaggacgtt agtaactgaa cgtcccctga cggtatctaa ccagaaagcc 481 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 541 attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 601 ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 661 gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 721 tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 781 gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 841 caaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 901 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 961 taccaggtct tgacatcctt tgaccactct agagatagag ctttcccttc ggggacaaag 1021 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1081 acgagcgcaa cccttattgt tagttgccat catttagttg ggcactctag cgagactgcc 1141 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1201 ctacacacgt gctacaatgg gaagtacaac gagtcgctag accgcgaggt catgcaaatc 1261 tcttaaagct tctctcagtt cggattgcag gctgcaactc gcctgcatga agccggaatc 1321 gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc 1381 ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt ttggagccag 1441 ccgcctaagg tgggatagat gattgggggg LOCUS DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM EU547784 2884 bp DNA linear BCT 23-APR-2008 Enterococcus faecalis strain IJ-11 23S ribosomal RNA gene, partial sequence. EU547784 EU547784.1 GI:183673616 . Enterococcus faecalis Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. 1 (bases 1 to 2884)
REFERENCE
Javed,I., Ali,M.I., Ahmad,B., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..2884 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-11" /db_xref="taxon:1351" <1..>2884 rRNA /product="23S ribosomal RNA" ORIGIN 1 gggcgcacgg tggatgcctt ggcactagga gccgatgaag gacgggacta acaccgatat 61 gctttgggga gctgtaagta agctatgatc cagagatttc cgaatggggg aacccaatat 121 cttttatagg atattacttt tcagtgaata catagctgat tagaggtaga cgcagagaac 181 tgaaacatct tagtacctgc aggaagagaa agaaaattcg attccctgag tagcggcgag 241 cgaaacggga agagcccaaa ccaacaagct tgcttgttgg ggttgtagga ctccaatatg 301 gtagtctgtt agtatagttg aaggatttgg aaaattccgc taaagagggt gaaagccccg 361 tagacgaaat gctaacaaca cctaggagga tcctgagtac ggcggaacac gagaaattcc 421 gtcggaatcc gcggggacca tcccgcaagg ctaaatactc cctagtgacc gatagtgaac 481 cagtaccgtg agggaaaggt gaaaagcacc ccggaagggg agtgaaatag atcctgaaac 541 cgtgtgccta caacaagtca aagctcgtta atgagtgatg gcgtgccttt tgtagaatga 601 accggcgagt tacgattgca tgcgaggtta agtcgaagag acggagccgc agcgaaagcg 661 agtctgaata gggcgaatga gtatgtagtc gtagacccga aaccatgtga tctacccatg 721 tccaggttga aggtgcggta aaacgcactg gaggaccgaa cccacgtacg ttgaaaagtg 781 cggggatgag gtgtgggtag cggagaaatt ccaaacgaac ttggagatag ctggttctct 841 ccgaaatagc tttagggcta gcctcggaat tgagaatgat ggaggtagag cactgtttgg 901 actaggggcc catctcgggt taccgaattc agataaactc cgaatgccat tcatttatat 961 ccgggagtca gactgcgagt gataagatcc gtagtcgaaa gggaaacagc ccagaccacc 1021 agctaaggtc ccaaaatata tgttaagtgg aaaaggatgt ggggttgcac agacaactag 1081 gatgttggct tagaagcagc caccatttaa agagtgcgta atagctcact agtcgagtga 1141 ccctgcgccg aaaatgtacc ggggctaaac atattaccga agctgtggac tacaccatta 1201 ggtgtagtgg taggagagcg ttctaagggc gttgaaggtc gatcgtgagg acggctggag 1261 cgcttagaag tgagaatgcc ggtatgagta gcgaaagaca ggtgagaatc ctgtccaccg 1321 tatgactaag gtttcctggg gaaggctcgt ccgcccaggg ttagtcggga cctaagccga 1381 ggccgatagg cgtaggcgat ggacaacagg ttgatattcc tgtaccagtt gtttttgttt 1441 gaacaatgga gggacgcagt aggctaagga atgcatgcga ttggaagtgc atgtccaagc 1501 aatgagtctt gagtagagtt aaatgcttta ctctttaagg acaagttgtg acggggagcg 1561 aaataatagt agcgaagttc ctgatgtcac actgccaaga aaagcttcta gtgagaaaac 1621 aactgcccgt accgtaaacc gacacaggta gtcgaggaga gtatcctaag gtgagcgagc 1681 gaactctcgt taaggaactc ggcaaaatga ccccgtaact tcgggagaag gggtgctgac 1741 ttcggtcagc cgcagtgaat aggcccaagc gactgtttat caaaaacaca ggtctctgca 1801 aaatcgtaag atgaagtata ggggctgacg cctgcccggt gctggaaggt taagaggatg 1861 ggttagcttc ggcgaagctc agaattgaag ccccagtaaa cggcggccgt aactataacg 1921 gtcctaaggt agcgaaattc cttgtcgggt aagttccgac ccgcacgaaa ggcgtaacga 1981 tttgggcact gtctcaacga gagactcggt gaaattttag tacctgtgaa gatgcaggtt 2041 tacccgcgac aggacggaaa gaccccatgg agctttactg tagtttgata ttgagtgttt 2101 gtaccacatg tacaggatag gtaggagccg atgagaccgg aacgctagtt tcggaggagg 2161 cgctggtggg atactaccct tgtgttatga accctctaac ccgcaccact aatcgtggtg 2221 ggagacagtg tcagatgggc agtttgactg gggcggtcgc ctcctaaaag gtaacggagg 2281 cgcccaaagg ttccctcaga atggttggaa atcattcgaa gagtgtaaag gcagaaggga 2341 gcttgactgc gagacctaca agtcgagcag ggacgaaagt cgggcttagt gatccggtgg 2401 ttccgcatgg aagggccatc gctcaacgga taaaagctac cctggggata acaggcttat 2461 ctcccccaag agtccacatc gacggggagg tttggcacct cgatgtcggc tcgtcgcatc 2521 ctggggctgt agtcggtccc aagggttggg ctgttcgccc attaaagcgg cacgcgagct 2581 gggttcagaa acgtcgtgag acagttcggt ccctatccgt cgcgggcgtt ggaaatttga 2641 gaggagctgt ccttagtacg agaggaccgg gatggactta ccgctggtgt accagttgtt 2701 ctgccaaggg cattgctggg tagctatgta gggaagggat aaacgctgaa agcatctaag 2761 tgtgaagccc acctcaagat gagatttccc atttctttaa gaaagtaaga cccctgagag 2821 atgatcaggt agataggttg gaaagtggaa ggctagtgnn gttggagcgg accaatacta 2881 atcg
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16S and 23S rRNA sequence of Enterococcus faecalis IJ-12 LOCUS DEFINITION EU547777 1246 bp DNA linear BCT 23-APR-2008 Enterococcus faecalis strain IJ-12 16S ribosomal RNA gene, partial sequence. ACCESSION EU547777 VERSION EU547777.1 GI:183673517 KEYWORDS . SOURCE Enterococcus faecalis ORGANISM Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1246) AUTHORS Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1246 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-12" /db_xref="taxon:1351" <1..>1246/product="16S ribosomal RNA" rRNA ORIGIN 1 ggcgtgccta atacatgcaa gtcgaacgct tctttcctcc cgagtgcttg cactcaattg 61 gaaagaggag tggcggacgg gtgagtaaca cgtgggtaac ctacccatca gagggggata 121 acacttggaa acaggtgcta ataccgcata acagtttatg ccgcatggca taagagtgaa 181 aggcgctttc gggtgtcgct gatggatgga cccgcggtgc attagctagt tggtgaggta 241 acggctcacc aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac 301 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 361 agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 421 tagagaagaa caaggacgtt agtaactgaa cgtcccctga cggtatctaa ccagaaagcc 481 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 541 attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 601 ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 661 gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 721 tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 781 gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 841 caaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 901 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 961 taccaggtct tgacatcctt tgaccactct agagatagag ctttcccttc ggggacaaag 1021 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1081 acgagcgcaa cccttattgt tagttgccat catttagttg ggcactctag cgagactgcc 1141 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1201 ctacacacgt gctacaatgg gaagtacaac gagtcgctag accgcg LOCUS DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM EU547785 2901 bp DNA linear BCT 23-APR-2008 Enterococcus faecalis strain IJ-12 23S ribosomal RNA gene, partial sequence. EU547785 EU547785.1 GI:183673632 . Enterococcus faecalis Enterococcus faecalis Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. 1 (bases 1 to 2901) Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San
REFERENCE AUTHORS TITLE JOURNAL
11
FEATURES source
rRNA ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881
Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA Location/Qualifiers 1..2901 /organism="Enterococcus faecalis" /mol_type="genomic DNA" /strain="IJ-12" /db_xref="taxon:1351" <1..>2901 /product="23S ribosomal RNA" ggcgcacggt ctttggggag ttttatagga gaaacatctt gaaacgggaa tagtctgtta agacgaaatg tcggaatccg agtaccgtga gtgtgcctac ccggcgagtt gtctgaatag ccaggttgaa ggggatgagg cgaaatagct ctaggggccc cgggagtcag gctaaggtcc atgttggctt cctgcgccga gtgtagtggt gcttagaagt atgactaagg ggccgatagg gaacaatgga aatgagtctt aaataatagt aactgcccgt gaactctcgt ttcggtcagc aaatcgtaag ggttagcttc gtcctaaggt tttgggcact acccgcgaca taccacatgt gctggtggga gagacagtgt gcccaaaggt cttgactgcg tccgcatgga tcccccaaga tggggctgta ggttcagaac ggagctgtcc gccaagggca tgaagcccac gatcaggtag cgncgaggac ggatgccttg ctgtaagtaa tattactttt agtacctgca gagcccaaac gtatagttga ctaacaacac cggggaccat gggaaaggtg aacaagtcaa acgattgcat ggcgaatgag ggtgcggtaa tgtgggtagc ttagggctag atctcgggtt actgcgagtg caaaatatat agaagcagcc aaatgtaccg aggagagcgt gagaatgccg tttcctgggg cgtaggcgat gggacgcagt gagtagagtt agcgaagttc accgtaaacc taaggaactc cgcagtgaat atgaagtata ggcgaagctc agcgaaattc gtctcaacga ggacggaaag acaggatagg tactaccctt cagatgggca tccctcagaa agacctacaa agggccatcg gtccacatcg gtcggtccca gtcgtgagac ttagtacgag ttgctgggta ctcaagatga ataggttgga a gcactaggag gctatgatcc cagtgaatac ggaagagaaa caacaagctt aggatttgga ctaggaggat cccgcaaggc aaaagcaccc agctcgttaa gcgaggttaa tatgtagtcg aacgcactgg ggagaaattc cctcggaatt accgaattca ataagatccg gttaagtgga accatttaaa gggctaaaca tctaagggcg gtatgagtag aaaggctcgt ggacaacagg aggctaagga aaatgcttta ctgatgtcac gacacaggta ggcaaaatga aggcccaagc ggggctgacg agaattgaag cttgtcgggt gagactcggt accccatgga taggagccga gtgttatgaa gtttgactgg tggttggaaa gtcgagcagg ctcaacggat acggggaggt agggttgggc agttcggtcc aggaccggga gctatgtagg gatttcccat agtggaaggc ccgatgaagg agagatttcc atagctgatt gaaaattcga gcttgttggg aaattccgct cctgagtacg taaatactcc cggaagggga tgagtgatgg gtcgaagaga tagacccgaa aggaccgaac caaacgaact gagaatgatg gataaactcc tagtcgaaag aaaggatgtg gagtgcgtaa tattaccgaa ttgaaggtcg cgaaagacag ccgcccaggg ttgatattcc atgcatgcga ctctttaagg actgccaaga gtcgaggaga ccccgtaact gactgtttat cctgcccggt ccccagtaaa aagttccgac gaaattttag gctttactgt tgagaccgga ccctctaacc ggcggtcgcc tcattcgaag gacgaaagtc aaaagctacc ttggcacctc tgttcgccca ctatccgtcg tggacttacc gaagggataa ttctttaaga tagtgatagt acgggactaa gaatggggga agaggtagac ttccctgagt gttgtaggac aaagagggtg gcggaacacg ctagtgaccg gtgaaataga cgtgcctttt cggagccgca accatgtgat ccacgtacgt tggagatagc gaggtagagc gaatgccatt ggaaacagcc gggttgcaca tagctcacta gctgtggact atcgtgagga gtgagaatcc ttagtcggga tgtaccagtt ttggaagtgc acaagttgtg aaagcttcta gtatcctaag tcgggagaag caaaaacaca gctggaaggt cggcggccgt ccgcacgaaa tacctgtgaa agtttgatat acgctagttt cgcaccacta tcctaaaagg agtgtaaagg gggcttagtg ctggggataa gatgtcggct ttaaagcggc cgggcgttgg gctggtgtac acgctgaaag aagtaagacc tggagcggac
aagtgnnnag caccgatatg acccaatatc gcagagaact agcggcgagc tccaatatgg aaagccccgt agaaattccg atagtgaacc tcctgaaacc gtagaatgaa gcgaaagcga ctacccatgt tgaaaagtgc tggttctctc actgtttgga catttatatc cagaccacca gacaactagg gtcgagtgac acaccattag cggctggagc tgtccaccgt cctaagccga gtttttgttt atgtccaagc acggggagcg gtgagaaaac gtgagcgagc gggtgctgac ggtctctgca taagaggatg aactataacg ggcgtaacga gatgcaggtt tgagtgtttg cggaggaggc atcgtggtgg taacggaggc cagaagggag atccggtggt caggcttatc cgtcgcatcc acgcgagctg aaatttgaga cagttgttct catctaagtg cctgagagat caatactaat
12
16S and 23S rRNA sequence of Enterococcus faecium IJ-21 LOCUS EU547778 1165 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain IJ-21 16S ribosomal RNA gene, partial sequence. ACCESSION EU547778 VERSION EU547778.1 GI:183673522 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1165) AUTHORS Javed,I., Ali,M.I., Ahmad,B., Ghumro,P.B., Chaudry,G.J., Hameed,A. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1165 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-21" /db_xref="taxon:1352" <1..>1165 rRNA /product="16S ribosomal RNA" ORIGIN 1 ggcgtgccta atacatgcaa gtcgaacgct tctttttcca ccggagcttg ctccaccgga 61 aaaagaggag tggcgaacgg gtgagtaaca cgtgggtaac ctgcccatca gaaggggata 121 acacttggaa acaggtgcta ataccgtata acaatcaaaa ccgcatggtt ttgatttgaa 181 aggcgctttc gggtgtcgct gatggatgga cccgcggtgc attagctagt tggtgaggta 241 acggctcacc aaggccacga tgcatagccg acctgagagg gtgatcggcc acattgggac 301 tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 361 agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 421 tagagaagaa caaggatgag agtaactgtt catcccttga cggtatctaa ccagaaagcc 481 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 541 attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 601 ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 661 gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 721 tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 781 gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 841 ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 901 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 961 taccaggtct tgacatcctt tgaccactct agagatagag cttccccttc gggggcaaag 1021 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1081 acgagcgcaa cccttattgt tagttgccat cattcagttg ggcactctag caagactgcc 1141 ggtgacaaac cggaggaagg tgggg LOCUS DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM EU547786 2880 bp DNA linear BCT 23-APR-2008 Enterococcus faecium strain IJ-21 23S ribosomal RNA gene, partial sequence. EU547786 EU547786.1 GI:183673643 . Enterococcus faecium Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. 1 (bases 1 to 2880) Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San
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FEATURES source
rRNA ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821
Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA Location/Qualifiers 1..2880 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-21" /db_xref="taxon:1352" <1..>2880 /product="23S ribosomal RNA" ggatgccttg ctgtacgtaa tgttacgatt agtacctgca aagcccaaac agatagtcga tggaagacac ggaggaccat gggaaaggtg aacaagtcaa acgattgcat ggcgtttgag ggtgcggtaa tgtgggtagc ttagggctag atctcgggtt actgtgagtg caaaatatat agaagcagcc aaatgtaccg aggagagcgt gagaatgccg tttcctgggg gtaggcgatg ggacgcagga agaggagtca ccgaagttcc ccgcaaaccg aaggaactcg ccgcagtgaa gatgaagtat aagcgaaggt tagcgaaatt tgtctcaacg aggacggaaa tacaggatag atactacccc tcagatgggc ttccctcaga gagaccaaca aagggccatc agtccacatc agtcggtccc acgtcgtgag ccttagtacg cattgctggg acctcaagat agataggtca gcactaggag gctatgatcc gtgtgaatac ggaagagaaa cagcaagctt atgacttgga ctaggaggat ctcccaaggc aaaagcaccc agcccgttaa gcgaggttaa tatgtagtcg aacgcactgg ggagaaattc cctcggaatt accgaattca ataagatcca gttaagtgga accatttaaa gggctaaaca tctaagggcg gtatgagtag aaggctcgtc gataacaggt ggctaaggaa aatgcttctt tgatgtcaca acacaggtag gcaaaatgac taggcccaag aggggctgac acgaattgaa ccttgtcggg agagactcgg gaccccatgg gtaggagccg tgcgttatgg agtttgactg atggttggaa agtcgagcag gctcaacgga gacggggagg aagggttggg acagttcggt agaggaccgg tagctatgta gagatttccc ggagtggaag ccgatgaagg agagatttcc atagcacatt gaaaattcga gcttgttggg aaagtcagtc cctgagtacg taaatactcc cggaagggga tgggtgatgg gttgaagaga tagacccgaa aggaccgaac caaacgaact gagaatgatg gataaactcc tagtcgaaag aaaggatgtg gagtgcgtaa tattaccgaa ttgaaggcag cgaaagacag cgcccagggt tgatattcct tgcagacgat ttcttaagga ctgccgagaa tcgaggagag cccgtaactt cgactgttta gcctgcccgg gccccagtaa taagttccga tgaaatttta agctttactg tagaaatcgg ccactctaac gggcggtcgc atcattcgaa ggacgaaagt taaaagctac tttggcacct ctgttcgccc ccctatccgt gatggactta gggaagggat atttctttaa tacagtgatg acgggactaa gaatggggga cgaggtagac ttccctgagt gttgtaggac aaagagggta gcggaacacg ctagtgaccg gtgaaataga cgtgcctttt cggagccgca accatgtgat ccacgtacgt tggagatagc gaggtagagc gaatgccatt ggaaacagcc gggttgcaca tagctcacta gctgtggagt atcgtgagga gtgagaatcc tagtcgggac gtacccgttg cggaaatgtc caagctgtga aagcttctag aatcctaagg cgggagaagg tcaaaaacac tgctggaagg acggcggccg cccgcacgaa gtacctgtga tagtttgata aacgctagtt ccgcaccact ctcctaaaag gagtgtaaag cgggcttagt cctggggata cgatgtcggc attaaagcgg cgcgggcgtt ccgctggtgt aaacgctgaa gaaagtaaga tatggagcgg caccgatatg acccaacatc gcagagaact agcggcgagc tccaatatgg aaaaccccgt agaaattccg atagtgaacc acctgaaacc gtagaatgaa gcgaaagcga ctacccatgt tgaaaagtgc tggttctctc actgtttgga cattcatatc cagaccacca gacaactagg gtcgagtgac acacctttag ctgctggagc tgtccaccga ctaagccgag tttttgtttg tgtccaagca tggggaggga tgagaaaaca tgagcgagag ggtgctgatc aggtctctgc ttaagaggag taactataac aggcgtaacg agatgcaggt ttgagtgtct tcgatggagg aatcgtggtg gtaacggagg gcagaaggga gatccggtgg acaggcttat tcgtcgcatc cacgcgagct ggaaatttga accagttgtt agcatctaag tccctgagag actgatacna
ggcgcacggt ctttggggag tttaatagga gaaacatcta gaaacgggaa tagttctttc agacgaaatg tcggaatccg agtaccgtga gtgtgcctac ccggcgagtt gtctgaatag ccaggttgaa ggggatgagg cgaaatagct ctaggggccc cgggagtcag gctaaggtcc atgttggctt cctgcgccga gtgtattggt gcttagaagt atgactaagg gccgacaggc agcaatggag gtaagtctga aataatagta gcggcccgta aactctcgtt atacgatcag aaaatcgtaa tgcttagcgc ggtcctaagg atttgggcac tacccgcgac gtaccgcatg cgctggtggg ggagacagtg cgcccaaagg gcttgactgc ttccgcatgg ctcccccaag ctggggctgt gggttcagaa gaggagctgt ctgccaaggg tgtgaagccc atgatcaggt
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16S and 23S rRNA sequence of Enterococcus faecium IJ-31 LOCUS EU547779 1473 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain IJ-31 16S ribosomal RNA gene, partial sequence. ACCESSION EU547779 VERSION EU547779.1 GI:183673537 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1473) AUTHORS Javed,I., Ali,M.I., Ahmad,B., Raiz,M., Chaudry,G.J., Hameed,A. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1473 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-31" /db_xref="taxon:1352" <1..>1473 rRNA /product="16S ribosomal RNA" ORIGIN 1 ggcgtgccta atacatgcaa gtcgaacgct tctttttcca ccggagcttg ctccaccgga 61 aaaagaggag tggcgaacgg gtgagtaaca cgtgggtaac ctgcccatca gaaggggata 121 acacttggaa acaggtgcta ataccgtata acaatcaaaa ccgcatggtt ttgatttgaa 181 aggcgctttc gggtgtcgct gatggatgga cccgcggtgc attagctagt tggtgaggta 241 acggctcacc aaggccacga tgcatagccg acctgagagg gtgatcggcc acattgggac 301 tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 361 agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 421 tagagaagaa caaggatgag agtaactgtt catcccttga cggtatctaa ccagaaagcc 481 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 541 attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 601 ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 661 gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 721 tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 781 gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 841 ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 901 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 961 taccaggtct tgacatcctt tgaccactct agagatagag cttccccttc gggggcaaag 1021 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1081 acgagcgcaa cccttattgt tagttgccat cattcagttg ggcactctag caagactgcc 1141 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1201 ctacacacgt gctacaatgg gaagtacaac gagttgcgaa gtcgcgaggc taagctaatc 1261 tcttaaagct tctctcagtt cggattgcag gctgcaactc gcctgcatga agccggaatc 1321 gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc 1381 ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt tggagccagc 1441 cgcctaaggt gggatagatg attggggtga agt LOCUS EU547787 2876 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain IJ-31 23S ribosomal RNA gene, partial sequence. ACCESSION EU547787 VERSION EU547787.1 GI:183673657 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 2876)
15
Javed,I., Ali,M.I., Ahmad,B., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..2876 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="IJ-31" /db_xref="taxon:1352" <1..>2876 rRNA /product="23S ribosomal RNA" ORIGIN 1 ggcgcacggt ggatgccttg gcactaggag ccgatgaagg acgggactaa caccgatatg 61 ctttggggag ctgtacgtaa gctatgatcc agagatttcc gaatggggga acccaacatc 121 tttaatagga tgttacgatt gtgtgaatac atagcacatt cgaggtagac gcagagaact 181 gaaacatcta agtacctgca ggaagagaaa gaaaattcga ttccctgagt agcggcgagc 241 gaaacgggaa aagcccaaac cagcaagctt gcttgttggg gttgtaggac tccaatatgg 301 tagttctttc agatagtcga atgacttgga aaagtcagtc aaagagggta aaaaccccgt 361 agacgaaatg tggaagacac ctaggaggat cctgagtacg gcggaacacg agaaattccg 421 tcggaatccg ggaggaccat ctcccaaggc taaatactcc ctagtgaccg atagtgaacc 481 agtaccgtga gggaaaggtg aaaagcaccc cggaagggga gtgaaataga acctgaaacc 541 gtgtgcctac aacaagtcaa agcccgttaa tgggtgatgg cgtgcctttt gtagaatgaa 601 ccggcgagtt acgattgcat gcgaggttaa gttgaagaga cggagccgca gcgaaagcga 661 gtctgaatag ggcgtttgag tatgtagtcg tagacccgaa accatgtgat ctacccatgt 721 ccaggttgaa ggtgcggtaa aacgcactgg aggaccgaac ccacgtacgt tgaaaagtgc 781 ggggatgagg tgtgggtagc ggagaaattc caaacgaact tggagatagc tggttctctc 841 cgaaatagct ttagggctag cctcggaatt gagaatgatg gaggtagagc actgtttgga 901 ctaggggccc atctcgggtt accgaattca gataaactcc gaatgccatt cattcatatc 961 cgggagtcag actgtgagtg ataagatcca tagtcgaaag ggaaacagcc cagaccacca 1021 gctaaggtcc caaaatatat gttaagtgga aaaggatgtg gggttgcaca gacaactagg 1081 atgttggctt agaagcagcc accatttaaa gagtgcgtaa tagctcacta gtcgagtgac 1141 cctgcgccga aaatgtaccg gggctaaaca tattaccgaa gctgtggagt acacctttag 1201 gtgtattggt aggagagcgt tctaagggcg tcgaaggcag atcgtgagga ctgctggagc 1261 gcttagaagt gagaatgccg gtatgagtag cgaaagacag gtgagaatcc tgtccaccga 1321 atgactaagg tttcctgggg aaggctcgtc cgcccagggt tagtcgggac ctaagccgag 1381 gccgacaggc gtaggcgatg gataacaggt tgatattcct gtacccgttg tttttgtttg 1441 agcaatggag ggacgcagga ggctaaggaa tgcagacgat cggaaatgtc tgtccaagca 1501 gtaagtctga agaggagtca aatgcttctt ttcttaagga caagctgtga tggggaggga 1561 aataatagta ccgaagttcc tgatgtcaca ctgccgagaa aagcttctag tgagaaaaca 1621 gcggcccgta ccgcaaaccg acacaggtag tcgaggagag aatcctaagg tgagcgagag 1681 aactctcgtt aaggaactcg gcaaaatgac cccgtaactt cgggagaagg ggtgctgatc 1741 atacgatcag ccgcagtgaa taggcccaag cgactgttta tcaaaaacac aggtctctgc 1801 aaaatcgtaa gatgaagtat aggggctgac gcctgcccgg tgctggaagg ttaagaggag 1861 tgcttagcgc aagcgaaggt acgaattgaa gccccagtaa acggcggccg taactataac 1921 ggtcctaagg tagcgaaatt ccttgtcggg taagttccga cccgcacgaa aggcgtaacg 1981 atttgggcac tgtctcaacg agagactcgg tgaaatttta gtacctgtga agatgcaggt 2041 tacccgcgac aggacggaaa gaccccatgg agctttactg tagtttgata ttgagtgtct 2101 gtaccgcatg tacaggatag gtaggagccg tagaaatcgg aacgctagtt tcgatggagg 2161 cgctggtggg atactacccc tgcgttatgg ccactctaac ccgcaccact aatcgtggtg 2221 ggagacagtg tcagatgggc agtttgactg ggggcggtcg cctcctaaaa ggtaacggag 2281 gcgcccaaag gttccctcag aatggttgga aatcattcga agagtgtaaa ggcagaaggg 2341 agcttgactg cgagaccaac aagtcgagca gggacgaaag tcgggcttag tgatccggtg 2401 gttccgcatg gaagggccat cgctcaacgg ataaaagcta ccctggggat aacaggctta 2461 tctcccccaa gagtccacat cgacggggag gtttggcacc tcgatgtcgg ctcgtcgcat 2521 cctggggctg tagtcggtcc caagggttgg gctgttcgcc cattaaagcg gcacgcgagc 2581 tgggttcaga acgtcgtgag acagttcggt ccctatccgt cgcgggcgtt ggaaatttga 2641 gaggagctgt ccttagtacg agaggaccgg gatggactta ccgctggtgt accagttgtt 2701 ctgccaaggg cattgctggg tagctatgta gggaagggat aaacgctgaa agcatctaag 2761 tgtgaagccc acctcaagat gagatttccc atttctttaa gaaagtaaga tccctgagag 2821 atgatcaggt agataggtca ggagtggaag tacagtgatg tatggagcgg actgat
16
16S and 23S rRNA sequence of Enterococcus faecium ATCC 27273 LOCUS EU547780 1471 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain ATCC 27273 16S ribosomal RNA gene, partial sequence. ACCESSION EU547780 VERSION EU547780.1 GI:183673554 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus. REFERENCE 1 (bases 1 to 1471) AUTHORS Javed,I., Chaudry,G.J., Hameed,A. and Ahmed,S. TITLE Direct Submission JOURNAL Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA FEATURES Location/Qualifiers source 1..1471 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="ATCC 27273" /db_xref="ATCC:27273" /db_xref="taxon:1352" <1..>1471 rRNA /product="16S ribosomal RNA" ORIGIN 1 ggcgtgccta atacatgcaa gtcgaacgct tctttttcca ccggagcttg ctccaccgga 61 aaaagaggag tggcgaacgg gtgagtaaca cgtgggtaac ctgcccatca gaaggggata 121 acacttggaa acaggtgcta ataccgtata acaatcaaaa ccgcatggtt ttgatttgaa 181 aggcgctttc gggtgtcgct gatggatgga cccgcggtgc attagctagt tggtgaggta 241 acggctcacc aaggccacga tgcatagccg acctgagagg gtgatcggcc acattgggac 301 tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa 361 agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt 421 tagagaagaa caaggatgag agtaactgtt catcccttga cggtatctaa ccagaaagcc 481 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 541 attgggcgta aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa 601 ccggggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt ggaattccat 661 gtgtagcggt gaaatgcgta gatatatgga ggaacaccag tggcgaaggc ggctctctgg 721 tctgtaactg acgctgaggc tcgaaagcgt ggggagcaaa caggattaga taccctggta 781 gtccacgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 841 ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 901 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 961 taccaggtct tgacatcctt tgaccactct agagatagag cttccccttc gggggcaaag 1021 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1081 acgagcgcaa cccttattgt tagttgccat cattcagttg ggcactctag caagactgcc 1141 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1201 ctacacacgt gctacaatgg gaagtacaac gagttgcgaa gtcgcgaggc taagctaatc 1261 tcttaaagct tctctcagtt cggattgcag gctgcaactc gcctgcatga agccggaatc 1321 gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc 1381 ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt tggagccagc 1441 cgcctaaggt gggatagatg attggggtga a LOCUS EU547788 2876 bp DNA linear BCT 23-APR-2008 DEFINITION Enterococcus faecium strain ATCC 27273 23S ribosomal RNA gene, partial sequence. ACCESSION EU547788 VERSION EU547788.1 GI:183673673 KEYWORDS . SOURCE Enterococcus faecium ORGANISM Enterococcus faecium Bacteria; Firmicutes; Lactobacillales; Enterococcaceae; Enterococcus.
17
FEATURES source
rRNA ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821
1 (bases 1 to 2876) Javed,I., Ahmad,B., Ali,M.I., Hameed,A., Chaudry,G.J. and Ahmed,S. Direct Submission Submitted (05-MAR-2008) South Texas Centre for Emerging Infectious Diseases, Department of Biology, University of Texas at San Antonio, BSE 3.404, One UTSA Circle, San Antonio, TX 78249, USA Location/Qualifiers 1..2876 /organism="Enterococcus faecium" /mol_type="genomic DNA" /strain="ATCC 27273" /db_xref="ATCC:27273" /db_xref="taxon:1352" <1..>2876/product="23S ribosomal RNA" ggcgcacggt ctttggggag tttaatagga gaaacatcta gaaacgggaa tagttctttc agacgaaatg tcggaatccg agtaccgtga gtgtgcctac ccggcgagtt gtctgaatag ccaggttgaa ggggatgagg cgaaatagct ctaggggccc cgggagtcag gctaaggtcc atgttggctt cctgcgccga gtgtattggt gcttagaagt atgactaagg gccgacaggc agcaatggag gtaagtctga aataatagta gcggcccgta aactctcgtt atacgatcag aaaatcgtaa tgcttagcgc ggtcctaagg atttgggcac tacccgcgac gtaccgcatg cgctggtggg ggagacagtg gcgcccaaag agcttgactg gttccgcatg tctcccccaa cctggggctg tgggttcaga gaggagctgt ctgccaaggg tgtgaagccc atgatcaggt ggatgccttg ctgtacgtaa tgttacgatt agtacctgca aagcccaaac agatagtcga tggaagacac ggaggaccat gggaaaggtg aacaagtcaa acgattgcat ggcgtttgag ggtgcggtaa tgtgggtagc ttagggctag atctcgggtt actgtgagtg caaaatatat agaagcagcc aaatgtaccg aggagagcgt gagaatgccg tttcctgggg gtaggcgatg ggacgcagga agaggagtca ccgaagttcc ccgcaaaccg aaggaactcg ccgcagtgaa gatgaagtat aagcgaaggt tagcgaaatt tgtctcaacg aggacggaaa tacaggatag atactacccc tcagatgggc gttccctcag cgagaccaac gaagggccat gagtccacat tagtcggtcc acgtcgtgag ccttagtacg cattgctggg acctcaagat agataggtca gcactaggag gctatgatcc gtgtgaatac ggaagagaaa cagcaagctt atgacttgga ctaggaggat ctcccaaggc aaaagcaccc agcccgttaa gcgaggttaa tatgtagtcg aacgcactgg ggagaaattc cctcggaatt accgaattca ataagatcca gttaagtgga accatttaaa gggctaaaca tctaagggcg gtatgagtag aaggctcgtc gataacaggt ggctaaggaa aatgcttctt tgatgtcaca acacaggtag gcaaaatgac taggcccaag aggggctgac acgaattgaa ccttgtcggg agagactcgg gaccccatgg gtaggagccg tgcgttatgg agtttgactg aatggttgga aagtcgagca cgctcaacgg cgacggggag caagggttgg acagttcggt agaggaccgg tagctatgta gagatttccc ggagtggaag ccgatgaagg agagatttcc atagcacatt gaaaattcga gcttgttggg aaagtcagtc cctgagtacg taaatactcc cggaagggga tgggtgatgg gttgaagaga tagacccgaa aggaccgaac caaacgaact gagaatgatg gataaactcc tagtcgaaag aaaggatgtg gagtgcgtaa tattaccgaa ttgaaggcag cgaaagacag cgcccagggt tgatattcct tgcagacgat ttcttaagga ctgccgagaa tcgaggagag cccgtaactt cgactgttta gcctgcccgg gccccagtaa taagttccga tgaaatttta agctttactg tagaaatcgg ccactctaac ggggcggtcg aatcattcga gggacgaaag ataaaagcta gtttggcacc gctgttcgcc ccctatccgt gatggactta gggaagggat atttctttaa tacagtgatg acgggactaa gaatggggga cgaggtagac ttccctgagt gttgtaggac aaagagggta gcggaacacg ctagtgaccg gtgaaataga cgtgcctttt cggagccgca accatgtgat ccacgtacgt tggagatagc gaggtagagc gaatgccatt ggaaacagcc gggttgcaca tagctcacta gctgtggagt atcgtgagga gtgagaatcc tagtcgggac gtacccgttg cggaaatgtc caagctgtga aagcttctag aatcctaagg cgggagaagg tcaaaaacac tgctggaagg acggcggccg cccgcacgaa gtacctgtga tagtttgata aacgctagtt ccgcaccact cctcctaaaa agagtgtaaa tcgggcttag ccctggggat tcgatgtcgg cattaaagcg cgcgggcgtt ccgctggtgt aaacgctgaa gaaagtaaga tatggagcgg caccgatatg acccaacatc gcagagaact agcggcgagc tccaatatgg aaaaccccgt agaaattccg atagtgaacc acctgaaacc gtagaatgaa gcgaaagcga ctacccatgt tgaaaagtgc tggttctctc actgtttgga cattcatatc cagaccacca gacaactagg gtcgagtgac acacctttag ctgctggagc tgtccaccga ctaagccgag tttttgtttg tgtccaagca tggggaggga tgagaaaaca tgagcgagag ggtgctgatc aggtctctgc ttaagaggag taactataac aggcgtaacg agatgcaggt ttgagtgtct tcgatggagg aatcgtggtg ggtaacggag ggcagaaggg tgatccggtg aacaggctta ctcgtcgcat gcacgcgagc ggaaatttga accagttgtt agcatctaag tccctgagag actgat
18
Fig. 4.1a. Phylogenetic relationship of isolated strains of Enterococcus faecalis IJ-03, IJ-07, IJ-11 and IJ-12
19
Fig. 4.1b. Phylogenetic relationship of isolated strains of Enterococcus faecium IJ06, IJ-21 and IJ-31
20
Fig. 4.2a
Fig. 4.2b
Fig. 4.2a: Antimicrobial activity of Enterococcus faecium IJ31 against Bacillus cereus at incubation period of 24 hrs (A), 48 hrs (B), 72 hrs (C), 96 hrs (D) and 120 hrs (E) Fig. 4.2b: Antimicrobial activity of Enterococcus faecium IJ31 against Listeria monocytogenes at incubation period of 24 hrs (A), 48 hrs (B), 72 hrs (C), 96 hours (D) and 120 hrs (E)
Fig. 4.3a
Fig. 3b
Fig. 4.3a: Antimicrobial activity of Enterococcus faecium IJ-31 against Bacillus cereus at different pH after 48 hrs at pH 5 (A), pH 6 (B), pH 7 (C), pH 8 (D) and pH 9 (E) Fig. 4.3b: Antimicrobial activity of Enterococcus faecium IJ-31 against Listeria monocytogenes after 48 hrs at pH 5 (A), pH 6 (B), pH 7 (C), pH 8 (D) and pH 9 (E)
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Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated From Dairy Products

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Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated From Dairy Products

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Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated from Dairy Products

Department of Microbiology Quaid-i-Azam University, Islamabad 2009

Doctor of Philosophy (Ph.D) In Microbiology

Department of Microbiology Quaid-i-Azam University, Islamabad 2009

Doctor of Philosophy (Ph.D) in Microbiology

_____________________ (Prof. Dr. Fauzia Y. Hafeez)

_____________________ (Dr. Jahangir Arshad Khan)

_____________________ (Dr. Safia Ahmed)

_____________________ (Prof. Dr. Abdul Hameed)

Chapter 2 Review of Literature

Chapter 3 Materials and Methods

Chapter 5 Discussion References

Normality Noncompetitive indirect enzyme linked

immunosorbent assay OD Optical density

PAGE RNA rRNA SDS SB Tm TAE UV USDA VRE

Enterococcus spp. 3.4. 3.5. Composition of MRS broth 43

4.1. 4.2. 4.3.

Identification of isolated enterococci with API 20 Strep system 57 (bioMrieux, Germany)

Detection of virulence determinants and antibiotics resistance of 66 bacteriocin producing isolates

Antimicrobial activity of isolated Enterococcus strains

Effect of different concentrations of glucose and NaCl on 81 bacteriocin production

Potential strains for bacteriocins and PCR amplification of 86

Antibiotics suscptibility of isolated enterococci to commonly used 67 antibiotics.

Time course of bacteriocin production by selected bacterial isolates at pH 73 7.0 and 30 C

Time course of bacteriocin production by selected bacterial isolates at pH 73 7.0 and 35 C

Time course of bacteriocin production by selected bacterial isolates at pH 74 7.0 and 40 C

Effect of media pH for bacteriocin production form Ent. faecalis IJ-11 at 76 35 C

Effect of media pH for bacteriocin production from Ent. faecium IJ-21 at 76 35 C

Effect of media pH for bacteriocin production from Ent. faecium IJ-31 at 77 35 C

4.17. 4.18. 4.19.

Effect of different enzymes on the residual activity of partially purified 92 bacteriocin

Thermo-stability assay of partially purified bacteriocin

Antimicrobial assay SDS-PAGE Gel on Ent. faecalis IJ-11 as indicator 95 strain

Purification of bacteriocin by Sephadex G-50 column

1.1. Food Borne Diseases and Public Health

1.2. Microbial Solutions to Microbial Problems

1.3. Lactic Acid Bacteria (LAB)

1.4. Use of Lactic acid bacteria (LAB) in food Industry

1.5. Enterococcus as an Agent for Control of Listeria and Clostridia

1.6. Enterococci and Health Perspective

1.8. Identification and characterization of enterococci with modern molecular techniques

species specific clusters and allowed strain typing.

Aims and Objectives of the Research Project

Amino acid sequence of the purified enterocin

5009 5465 4630 5190 5178 3952 5328

Enterocin 1071B Enterocin 1071A Enterocin RJ-11 Enterocin M

E. faecalis BEF1071 E. faecalis BEF 1071 E. faecalis RJ-11 E. faecium AL40

3899 4286 5049 4628

GPGKWLPWLQPAYDFVTGLAKGIGKEGNKNKWKNV ESVFSKIGNAVGPAAYWILKGLGNMSDVNQAQDRINRKKH APAGLVAKFGRPIVKKYYKQIMQFIGEGSAINKIIPQWIARMWRT ATRSYGNGVYCNNSKCW-NVGEAKENIA GIVISGKASGL

2.2. Food safety issues

Materials and Methods

Materials and Methods

Materials and Methods

3.1.3. Molecular Characterization

Materials and Methods

Materials and Methods

16S rRNA Entrococcus spp.

Antisense, Reverse Sense, Forward Antisense, Reverse

LMG-23S-S1 23S rRNA Enterococcus faecium

5 GGTTAAGT AATAAGGGCGCACGG 3 2904

V583-23S-S250246 23S rRNA Enterococcus faecalis

Materials and Methods

Materials and Methods