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Detection of metallo- -lactamase in Pseudomonas aeruginosa from Tripoli, Libya

Mohamed S Ellabib1, Mohamed A Aboshkiwa1, , Nagwa A Almargani, 2 ; Abdulaziz A. Zorgani1, , Allaaeddin El Salabi3,4 and Mohammed El-Gumati1
1Department of Medical Microbiology and Immunology, Faculty of Medicine, Al-Fateh University, Tripoli, Libya, 2 Academy of Graduate Science, Tripoli, Libya, 3 Department of Infection, Immunity & Biochemistry, School of Medicine, University Hospital of Wales, Heath Park, Heath, Cardiff, CF14 4XN, UK, 4  Department of Environmental Health, Faculty of Public Health, Medical Campus, University of Benghazi, Benghazi, Libya. Correspondence:

sallbros@yahoo.ca

Abstract
Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates have been reported to be an important cause of nosocomial infections. As very little information of the antibiotic resistance in Libya is known, this work was undertaken. The present study was conducted to study the incidence of MBLs in P. aeruginosa isolated from patients admitted to Tripoli Medical Center (TMC) and to Burn and plastic Surgery Hospital (BPSH). Three hundred and twelve isolates of P. aeruginosa were cultured from different samples from patients. Isolates were identified using Conventuals method and API20 NE. The isolates were subjected to susceptibility testing using CLSI guidelines. They were further screened for the production of MBLs by disc potentiating testing using EDTA-impregnated imipenem discs. Of the total 312 isolates of P. aeruginosa, antimicrobial susceptibly tests to fifteen anti-pseudomonal antibiotic showed that the percent of resistance was as follows; Amikacin (23.1%), Aztreonam (22.8%), Carbenicillin (51.9%), Cefepime (28.2%), Cefotaxime (51%), Ceftazidime (29.5%), Ciprofloxacin (26.9%), Gentamicin (35.3%), Imipenem (13.8%), Meropenem (15.7%), Piperacillin (39.1%), Piperacillin/tazobactam (27.6%), Polymyxin B (13.8%), Ticarcillin (53.8%), Tobromycin (31.1). Thirty four of imipenem resistant isolates were found to be MBL positive (10.9%). The study showed that MBL screening tests are simple and easy to perform as routine diagnostic tests for the detection of MBL production. EDTA disk screening test is simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM non susceptible P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test and that PCR confirmation be performed at a regional laboratory.

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Keywords: Carbapenem resistance, metallo- -lactamase, Pseudomonas aeruginosa, Tripoli, Libya.

Introduction
The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial infections caused by beta-lactamase resistant bacteria [1]. This is due to the broad spectrum activities and stability to hydrolysis by most beta-lactamase enzymes, which have made carbapenems the drug of choice for the treatment of infections Copyright iMedPub

caused by penicillin or cephalosporin-resistant Gram-negative bacilli, particularly extended spectrum beta-lactamase (ESBL) [2]. However in recent years emergence of carbapenem-hydrolysing metallo-beta-lactamase (MBL) in P aeruginosa has been described worldwide [3]. With respect to infection with MBL-producing strains represents a therapeutic problem due to their resistance to all beta-lactams except monobactams [4]. Several types of MBL enzymes have been identified and

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described in P. aeruginosa such as IMP, VIM. [2, 5]. P. aeruginosa is uniquely problematic due to it is ability to inherent and acquire resistance to many drug classes via mutation to all relevant treatments [4]. Furthermore, few isolates of P. aeruginosa are resistant to all reliable antibiotics and this problem seems likely to grow with the emergence of class 1 integrons encoding both carbapenems and amikacin resistance genes [6].The rates of the occurrence of metallo- -lactamase mediated resistance in P. aeruginosa have escalated since 2000 and severely affected treatment options in Asia, Europe and Latin America to non- -lactam antibiotics [8]. Clinical isolates harboring metallo- -lactamases have also recently been reported in western Canada and in Texas, which highlight the need for development of accurate diagnostic tests by clinical laboratories to detect the presence of new, and more potent antimicrobial agents [9]. In this respect nosocomial infections caused by P. aeruginosa remains one of the most difficult to treat and to control [9]. In vitro antimicrobial susceptibility data are required for successful therapy, because acquired resistance to such antimicrobials as -lactams, fluoroquinolones and aminoglycosides is so prevalent in P. aeruginosa [10-11]. Strategies for controlling P. aeruginosa infections include; early detection of P. aeruginosa as the causative pathogen, determination of its antimicrobial susceptibilities, initiation of effective and adequate therapy as well as strict infection control practice such as hand hygiene [11]. Up to our knowledge these information and such studies in Libya are not well known. For that reason this work aims to obtain some insight view on the detection and prevalence of such important type of antibiotic resistance mechanisms in P. aeruginosa in two hospitals; one major teaching Tripoli Medical Center (TMC) and the other is an important setting hospital; Burn and Plastic Surgery Hospital (BPSH).

and Laboratory Standard Institute recommendations (CLSI) [13]. The following anti-pseudomonal antibiotics used were aminoglycosides [amikacin (30g), gentamicin (10g), and tobramycin (10g)], cephalosporins [cefepime (30g), ceftazidime (30g) and cefotaxone (30g)], fluoroquinolones [ciprofloxacin (5g)], carbapenems [imipenem (10g) and meropenem (10g)] ,carbenicillin (100g) ticacillin (75g), pipercillin (100g), piperacillin/tazobactam (100/10g), aztreonam (30g) and polymyxin B (100g). Isolates were considered to be carbapenem sensitive when the zone size around imipenem was 13 mm, intermediate 14-15 mm and resistant to 16 mm. MBL-producing P. aeruginosa isolates were suspected when the isolate was resistant to imipenem. Screening and confirmation for the detection of MBL was carried out by disc potentiating test with EDTA-impregnated imipenem discs [14, 15, 16].

Metallo- -lactamase screening test

All P. aeruginosa isolates which showed resistance to imipenem were submitted for further examination for the detection of MBLs production using EDTA (750 g) combined disk. The IMP-EDTA combined disk test was performed as described by [16] Test organisms were inoculated onto plates of Mueller-Hinton agar and optical density was adjusted to 0.5 McFarland standards. A 0.5M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA 2H 2 O in 1000 ml of distilled water and adjusting it to pH 8.0 by using NaOH and the mixture was sterilized by autoclaving. Two 10g imipenem discs were placed on the plate and 5l (750g) of EDTA solution was added to one of the discs. The inhibition zones of the imipenem and imipenem-EDTA discs were compared after 16-18 h of incubation at 35C. An increase in the zone size of at least 7 mm around the imipenem-EDTA was recorded as an MBL-positive isolate.

Materials and Methods

Over the period of this study from August 2008 to august 2009, 312 non duplicate isolates of P. aeruginosa were obtained and isolated from different clinical samples collected from patients admitted to TMC and BPSH hospitals. Samples were transported and processed in the microbiology laboratory without delay. Isolates were identified by standard laboratory techniques using conventional method and Identification was confirmed with the API 20NE system (BioMerieux, Marcy lEtoile, France) as well as oxidase test [12]. Antimicrobial sensitivity testing was performed on Mueller Hinton Agar plates with commercially available discs obtained from (Oxoid) using Kirby Bauer Disc Diffusion method. The results were recorded and interpreted following Clinical

Detection of Metallo- - lactamase by EDTA

Differences between imipenem discs and imipenem/EDTA discs were regarded positive if the P value was observed < 0.05. Statistically significant difference was found in the resistance pattern of MBL positive and negative isolates for Imipenem depended on (P <0.05),. Increased zone inhibition to 7mm was considered as MBL positive. These results were clearly discriminated using combined disc cut off 7mm increase in inhibition zone by using imipenem-EDTA statistical methods to prove the change in inhibitory zone before and after using EDTA. P value of less than < 0.05 indicates statistical significance results for this change. Statistical analysis was performed by linear regression with the help of SPSS 17 .0 statistic software (SPSS Inc).

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Results
During the period of this study, a total of 312 non duplicate isolates of P. aeruginosa isolates were isolated; 170 isolates (54.5%) were from patients admitted to TMCH and 142 (45.5%) were from patients admitted to BPSH. Specimens site source from TMC were mostly obtained from urine; 50 (92.6%), while most specimens from BPSH were obtained from wound swab; 86 (74.8%). Susceptibility pattern showed that the most effective antibiotics with less frequency of resistance against P. aeruginosa isolates were found to be polymyxin B followed by imipenem and meropenem. The less active antibiotics were ticarcillin, carbenicillin and cefotaxime. Table 1 represents the frequency of resistant P. aeruginosa to 15 anti-pseudomonal antibiotics. Considering carbapenem resistance during this study, it was found that out of 312 isolates of P. aeruginosa, 42 (13.5%) were found resistant to imipenem and out of this 34 (10.9%) were found to be MBL producers as confirmed by the disc potentiating method. Frequency of MBLS in both hospitals are presented in table 2. Resistance pattern of MBL positive and negative isolates against antibiotics are presented in table 3. The results clearly showed that the resistance pattern was higher for both MBLs positive and negative isolates toward other drugs ( Table 3). Table 1. A  ntibiotic sensitivity pattern of 312 P. aeruginosa strains to most common used anti-pseudomonas antibiotics.
% of resistant 53.8 51.9 51 39.1 35.3 31.1 29.5 28.2 27.6 26.9 23.1 22.8 16.4 13.8 13.5 N=312. Antibiotics Potency (g) Ticarcillin (75) Carbenicillin (100) Cefotaxime (30) Pipercilline (100) Gentamicin (200) Tobromycin 30 Ceftazidime (30) Cefepime (30) Piperacillin/tazobactam (100) Ciprofloxacin (15) Amikacin (30) Aztreonam (30) Meropenem (10) Polymyxin B (300) Imipenem (10)

Table 2. Prevalence of metallo -lactamase in TMC & BPSH.

Antimicrobial IMP/EDTA resistant 10.8% 7.6% 14.7% HOSPTIAL TMC+BPSH TMC BPSH

Changing patterns and differences of susceptibility between both IMP resistant and sensitive isolates was observed. Most of the isolates which showed resistance to imipenem and either was MBL positive or negative were found highly resistant to other antibiotics compared to isolates which were sensitive to imipenem. It was found that the resistance rate among IMP resistant isolates toward ceftazidime was (69%), piperacillin/tazobactam (66.6%), and cefepime (69%) respectively as illustrated in Table (4) compared to sensitive isolates. Differences in resistance patterns between both hospitals are shown in table 4.

Discussion
In the last few years MBLs have been identified from clinical isolates worldwide with increasing frequency over the past few years [7, 8, 17]. Strains producing these enzymes have been responsible for prolonged nosocomial outbreaks that were accompanied by serious infections [16]. The occurrence of an MBL-positive isolate in a hospital setting possesses a therapeutic problem, as well as a serious concern for infection control management [17]. For that reason accurate identification, detection and reporting of MBL-producing P. aeruginosa will aid infection control practitioners in preventing the spread of these multi-drug-resistant isolates [18]. The results of this study showed that P. aeruginosa isolates were highly resistant to most usable anti pseudomonal antibiotics in both hospitals particularly for carbenicillin, cefotaxime, piperacillin and ticarcillin. These findings are similar to other studies reported worldwide [19]. Further higher resistance rates were observed in BPSH for many anti pseudomonals such as carbenicillin, cefepime, ceftazidime, gentamicin, piperacillin, ticarcillin and tobramycin. The high incidence of resistance at BPSH may be attributed to fact that many of anti pseudomonal drugs were introduced long time ago in this hospital than the TMC. Furthermore, there are an intensive use of these antibiotics among patients attending this hospital and high prevalence of these bacteria in burn patients. The results of this study is in consistence with other studies reported the increasing incidence of P. aeruginosa resistance

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Table 3. (%) Resistant pattern of MBL positive and negative isolate strains from both TMCH and BPSH.
BPSH N=23 N=2 100% 100% 66.6% 66.6% 66.6% 66.6% 100% 100% 66.6% 100% 100% 100% 66.6% 0% 100% P=21 100% 85.7% 66.6% 76% 80.9% 53.3% 80.9% 90.4% 61.9% 85.7% 61.6% 85.7% 61.9% 9.5% 95.2% N=5 100% 100% 100% 60% 100% 40% 100% 60% 60% 60% 60% 60% 60% 20% 40% TMCH N=18 P=13 100% 69% 61.5% 61.5% 53.8% 46.1% 76.9% 76.9% 46.1% 46.1% 46.1% 46.1% 53.8% 7.6% 76.9% TMCH &BPSH N=41 N=7 100% 100% 87.5% 62.5% 87.5% 50% 100% 75% 62.5% 75% 75% 75% 62% 12% 62.5% p=34 100% 79.4% 64.7% 70% 70% 50% 79% 85.2% 55.8% 70.5% 55.8% 58.8% 55.8% 8.8% 88.2%

Hospital Antibiotic Imipenem (10) Meropenem (10) Ceftazidime (30) Pip/tazobactam (110) Cefepime (30) Aztreonam (30) Cefotaxime (30) Ticarcillin (75) Amikacin (30) Pipercilline (100) Tobramycin (30) Ciprofloxacin (15) Gentamicin (200) Polymyxin B (300) Carbenicillin (100)

Table 4. (%) Susceptibility pattern of IMP resistance and sensitive strains to anti-pseudomonas drugs.
BPSH n=142 IMPS 118 0% 9.5% 32.2% 29% 26.2% 30.5% 55.0% 69% 23.7% 44.0% 35.5% 30.5% 43.2% 23.7% 53.3% IMPR 24 100% 62.5% 70.8% 75% 75% 58% 66.6% 66.6% 62.5% 62.5% 66.6% 70.8% 45.11% 8.2% 70.8% IMPS 152 0% 1% 14.4% 12.5% 12.5% 7.8% 34.2% 34.2% 12.5% 18.5% 14.4% 13.1% 27.6% 9.2% 35.5% TMCH n=170 IMPR 18 100% 77.7% 61.1% 61.1% 61.1% 44.4% 83.3% 72.2% 50% 50% 55% 50% 55% 22.2% 61.1% IMPS 271 0 4.4% 22.2% 20% 18.5 17.7% 43.3% 49.6% 17.4% 29.6% 23.7% 20.7% 34.4% 15.5% 43% TMCH&BPSH n=312 IMPR 41 100% 69% 69% 66.6% 69% 61.9% 73.8% 69% 57.1% 59.5% 14.4% 13.1% 50% 14.4% 66.6% Hospital Antibiotics potency (g) Imipenem (10) Meropenem (10) Ceftazidime (30) Pip/tazobactam(110 Cefepime (30) Aztreonam (30) Cefotaxime (30) Ticarcillin (75) Amikacin(30) Pipercilline (100) Tobramycin (30) Ciprofloxacin (15) Gentamicin (200) Polymyxin B (300) Carbenicillin (100)

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in Burn patients to many antibiotics worldwide [20]. An early study conducted by Ellabib [21] on infection in burn patients showed that P. aeruginosa was the second leading pathogen in the BPSH and was highly resistant to most early anti pseudomonal drugs such as cefotaxime, carbenicillin, piperacillin and gentamicin. This study showed that all isolates were sensitive to ceftazidime, imipenem and aztreonam. These results are also similar somewhat to the findings of Ellabib and colleges at TMC [22]. Our study clearly demonstrated that there is an increase in the resistance pattern toward the new anti pseudomonal antibiotics used in this investigation such as aztreonam, Cefepime, ceftazidime, ciprofloxacin and piperacillin/tazobactam. These findings are similar to the findings of Shahid and co-authers who reported the incidence of AmpC enzymes responsible for the resistance of Indian isolates of P. aeruginosa [23]. Prevalence of MBL enzymes in our study showed that the incidence over all was (10.8%), this finding is in coherence with the findings of Pitout and co-authers [9]. Imipenem is recommended to test the occurrence of MBLs [24]. The results of MBL detection using EDTA combined disc were statistically significant for the three antibiotics used and recommended for such study, and this finding were in agreement with the results reported by [25]. For most antibiotics, pattern of imipenem resistant isolates positive to MBLs were higher in both hospitals compared to MBL negative isolates for most antibiotics. Resistance to ticarcillin, cefotaxime, cefepime, carbenicillin, ceftazidime and piperacillin/tazobactam were found between (70 and 90%) with some. These findings are due to the fact that resistant isolates due to MBL production usually possess multiple resistant to all -lactam antibiotics apart of the azetronam as a result of the broad spectrum hydrolysis of these enzymes to inactivate -lactam antibiotics [1]. Furthermore, these resistant isolates may also harbor other mechanisms of resistance to other classes of antibiotics such as; quinolones and aminoglycosides [1, 23]. Resistance to polymyxin B in this study was the lowest (13.8%) and was similar to other studies [18]. This is due to the mode of action of this antibiotic, which works at the level of cell membrane affecting its integrity as well as its limited external use [26]. Resistance of P. aeruginosa to the antibiotics (imipenem, meropenem and ceftazidime) used in this study compared with the sensitive isolates gives evidence to the occurrence of MBLs in addition to the other test results that confirmed such incidence. Our results showed that imipenem resistant isolates were highly resistant to all anti-pseudomonal drugs used in this study compared to sensitive ones. This is due to the remarkable ability of P. aeruginosa to acquire resistance mechanisms to many antimicrobial agents [28]. Such resistance mechanisms include chromosomal-mediated antibiotic

resistance genes, also more importantly to plasmid-mediated antibiotic resistance genes, class 1 integrons, transposons, insertion sequences, Efflux pumps and outer membrane proteins that may play an important role in such resistance to antibiotics [27, 28, 29]. Higher prevalence of such resistance mechanism was observed in BPSH compared to that found in TMC. It is not surprising that resistant isolates of P. aeruginosa were found as nosocomial pathogens in the hospital setting but the most interesting part of this study is the evidence of occurrence of high percentage of MBLs in isolates from patients admitted to intensive burn care unit as has been reported by Haung and co-workers in 2006 [30].

Conclusion
The rapid dissemination of MBL producers is worrisome and necessitates the implementation of surveillance and molecular genetic studies, and also to encourage the prudent use of antibiotics, in particular carbapenems which are very effective drugs to treat life threaten pseudomonas infections. The most active b -lactam antibiotic against MBL producing P.aeruginosa isolates was polymyxin. The study showed that MBL screening tests are simple and easy to perform as routine diagnostic tests for the detection of MBL production. DDST is easier and less expensive than other MBL screening tests such as E-test. The MBL-producing P. aeruginosa isolates were more resistant to various antimicrobial agents and were more prevalent in urine and wound cultures compared to other samples. We recommend that all imipenem and meropenem non susceptible or -resistant P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test as described in this study. Finally routine detection of MBLs will ensure optimal patient care and introduction of appropriate infection control procedures. Laboratories in Libya need to improve qualit control policies, introduce recent standard operating procedures (SOPs) and follow the international guidelines for detection of antimicrobial resistance in bacteria.

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