iMedPub Journals

Our Site: http://www.imedpub.com/

Copyright iMedPub 1
ARCHIVES OF CLINICAL MICROBIOLOGY
2013
Vol. 4 No. 5:2
doi: 10.3823/273
Characterization
of extended-spectrum
beta-lactamases
in some
enterobacteriaceae
clinical isolates from
Egyptian patients
1 Microbiology and Immunology
Dep., Faculty of Pharmacy,
Al-Azhar University.
2 Laboratory Unit, Molecular
Epidemiology Dep., Clinical
Trials and Military Service,
NAMRU.
3 Central public health
laboratories, MOH. Cairo-
Egypt.
Correspondencia:
amanyelsharif@gmail.com
Amany El-Sharif, Ph.D.,
Prof. Microbiology & Immunology.
Department of microbiology &
immunology, Faculty of pharmacy
(girls). Al-Azhar University
Yousef Abbas St. Nasr city, Cairo,
Egypt.
Telephone: 00202- 26716092.
Fax: 00202- 26716092.
Mail Address: Nasr City,
El-Sefarat area, block1 building
10, beside ministry of higher
education. Cairo-Egypt.
Salwa A
1
, Rania Abdel Khalek
2
, Amany Elsharif
1
, Marwa
Yousry
3
, Hanan El-Mohammady
2
and M. Seif Ashour
1
This article is available from:
www.acmicrob.com
Abstract
Objectives: to investigate and characterize ESBLs enzymes among Enterobacte-
riaceae clinical strains isolated from patients in Egypt.
Methods and ndings: ESBLs and AmpC producers were identied phenotypi-
cally. Beta-lactamases genotypic identication was performed for Escherichia, Kleb-
siella and Enterobacter spp. TEM, CTX-M-I CTX-M-IV, OXA-IIIand SHV genes were
detected. This study reported Gram negative bacilli as the predominant pathogens
as 343 (89.5%) isolates were recovered from 419 clinical samples isolated from
public hospitals, Enterobacteriaceae represent 84.2% of the identied isolates.
Escherichia spp. were the most frequent isolates, other isolates were identied as
Klebsiella, Enterobacter and Proteusspecies in frequency of 49.48%, 37.02 %, 5.88
% and 3.46% respectively. TEM was found as the most predominant gene produc-
ing ESBLs among Escherichia spp. (81.9%) and less frequent among Klebsiella and
Enterobacter spp., 40.5% & 41%respectively.
CTX-M-gp I genes were detected among 72.2%, of Escherichia and56.1% of Kleb-
siella spp., while CTX-M-IV genes were detected among 52.9% of Enterobacter
spp. Regarding OXA-III genes, these were detected in 44.4%, 28.1% & 11.7% of
examined Escherichia, Klebsiella and Enterobacter species respectively. While SHV
genes were the least frequent among Escherichia spp., SHV was the predominant
gene among Klebsiella spp. and moderate in Enterobacter spp. by 6.9%, 72% &
29.4% respectively. TEM-type allele is the most common ESBLs.

Conclusion: this is the rst report demonstrate different types of ESBLs genes
among ESBL and AmpC positive Enterobacteriaceae clinical isolates in Egypt. Our
nding reported that Enterobacteriaceae isolates are more predominant among
hospital clinical isolates. ESBLs and AmpC producing Enterobacteriaceae are be-
coming increasingly emerged and involved in infection.
Key words: Enterobacteriaceae, ESBL, AmpC, Escherichia, Klebsiella and Entero-
bacter.
iMedPub Journals
Our Site: http://www.imedpub.com/
ARCHIVES OF CLINICAL MICROBIOLOGY
2 Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
Introduction
Gram-negative bacteria are important etiological agents of
nosocomial and community acquired infections. Over the
past few decades the treatment of Gram-negative bacteria
infection has become a challenge due to their ability to ac-
quire antimicrobial resistance [2, 9].
Over the last few years, many new beta-lactam antibiotics
have been developed. However, with each new class of an-
tibiotics that has been used to treat patients, new beta-lac-
tamases have been emerged and cause resistance to that class
of antibiotics [6]. The selective pressure and overuse of new
antibiotics in the treatment of patients has led to new variants
of beta-lactamases. Unfortunately, resistance to expanded-
spectrum beta-lactam antibiotics due to beta-lactamases has
emerged rapidly. Because of their increased spectrum of ac-
tivity, especially against the oxyimino-cephalosporins, these
enzymes were called extended- spectrum beta-lactamases
(ESBLs). Today, over 150 different ESBLs have been described.
These beta-lactamases have been found worldwide in many
different genera of Enterobacteriaceae [5].
Most ESBLs are derivatives of TEM or SHV enzymes. There
are more than 90 TEM-type beta-lactamases and more than
25 SHV-type enzymes. TEM is the most commonly reported
beta-lactamase in Gram-negative bacteria [27].
It has been suggested that the naturally occurring TEM-type
ESBLs are the result of uctuating selective pressure from
several beta-lactams within a given institution rather than
selection with a single agent. Although TEM-typebeta-lac-
tamases are most often found in E. coli and K. pneumonia
[6, 9], they are also found in other species of Gram-negative
bacteria with increasing frequency such as Enterobacteraero-
genes, Morganella morganii and Proteus species [22]. Unlike
the TEM typebeta-lactamases, there are relatively few deriva-
tives of SHV-1. The majority of SHV-type ESBLs are found in
strains of K. pneumonia [27]. However, these enzymes have
also been found in Citrobacter diversus, E. coli, and P. aeru-
ginosa [6]. Plasmid-mediated ESBLs called CTX-M hydrolyze
cefotaxime and they have mainly been found in strains of
Salmonellatyphimurium. Recently OXA-type ESBLs are de-
tected in Enterobacteriaceae [7] and they are characterized
by their poor inhibition by clavulanic acid [18]. Some studies
have shown the prevalence of multidrug resistant and ESBLs
Enterobacteriaceae in hospitals in Middle Eastcountries in-
cluding Egypt [2, 5].
Until recently, no data were available regarding molecular
characterization and frequency of ESBLs in clinical samples
isolated from Egyptian patients. In this study, we investigated
the prevalence of ESBL senzymes, their antimicrobial-resis-
tance proles and detection of predominant resistance genes
among Enterobacteriaceae clinical strains.
Material and Methods
Study design
In the present study 419 clinical samples (urine, blood, spu-
tum, body uids, and wound swabs) were collected from
inpatients, before administration of antibiotics, attending
Abou-Elreesh Hospital (AEH) and Central Public Health Labo-
ratories (CPHL) in Cairo, Egypt. Ethical approval to perform
the study was obtained from the management board.
Clinical samples were submitted for bacterial cultures and
identication for both Gram negative and Gram Positive
bacteria using standard microbiological techniques. Bacterial
isolates were tested for susceptibility to antibiotics by the disc
diffusion method recommended by the Clinical Laboratory
Standards Institute [31].
ESBL phenotypic detectionDisc diffusion method by the fol-
lowing antimicrobial discs was used: ceftazidime, aztreonam,
cefotaxime, or ceftriaxonediscs (30g per each). Conrmation
was done using CLSI combined method test [31]. ESBL was
detected by an increase in the diameter of inhibition zone
to 5-mm for either ceftazidime or cefotaxime in combina-
tion with clavulanic acid versus its zone when tested alone.
Double-disc synergy test (DDST) was also used. Discs were
used include aztreonam, ceftazidime, cefpodoxime and ce-
fotaxime (30 g/each) [12].
AmpC phenotypic detectionScreening with cephamycin
(cefoxitin) disc resistance for the presence of AmpC type
enzyme was used. Cefoxitin/cefotaxime or ceftazidime disc
antagonism tests was also used to detect inducible AmpC
which can be recognized because of blunting of the CAZ
or CTX zone adjacent to the FOX disc [11]. The antibiotic
discs used throughout this study were the product of Oxoid
Laboratories, UK.
Molecular charecterization of ESBLs Enterobacteriaceaeby
polymerase chain reaction (PCR)The DNA template was pre-
pared by boiling method. ESBL-positive isolates were tested
for the genes encoding TEM, SHV, CTX-M and OXA III beta-
lactamases by PCR using the primers listed in Table 1. PCRs
were performed using 6.25 L of Dream Taq green PCR Mas-
ter Mix (Fermentas) in a full automated Applied Biosystem/
Gene Amp PCR system 9700.
ARCHIVES OF CLINICAL MICROBIOLOGY
Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
3
iMedPub Journals
Our Site: http://www.imedpub.com/
The amplied PCR product were analyzed by gel electropho-
resis (BioRad Power PAC 300) with 2% agarose stained with
Gel red (Phenix research) and visualized by UV transillumina-
tion (BioRad). Gene Ruler- 100 bp plus DNA ladder from (Fer-
mentas) was used as a marker according to the product size.
Results
Identication of isolates
In this study, a total of 419 clinical samples included urine,
blood, sputum, body uids and wound swabs were collected
according to physicians request before administration of any
antibiotics to perform bacterial culture and sensitivity test in
Central public health laboratories, Cairo-Egypt. Out of the
375 recovered isolates, a number of 343 isolates (91.4 %)
were found to be Gram- negative bacilli. From which 289
(84.2%) were Enterobacteriaceae species. Escherichia spp.
was the most frequently isolated organisms among Entero-
bacteriaceae isolates (49.48%). Other frequently isolates
were Klebsiella spp. (37.02 %), Enterobacter spp. (5.88 %),
Proteus spp. (3.46%) and Salmonella spp. (1.38%). Each of
Citrobacter spp. and Yersinia spp. were represented by 1.03%
and Morganella spp. was 0.69%, table 2.
Antimicrobial susceptibility testing
The antibiotic susceptibility pattern among the Enterobac-
teriaceae isolates is illustrated in table 3. Detection of anti-
microbial resistance to examined isolates revealed that high-
level of resistance to beta-lactam antimicrobial agents was
reported. The incidence of sensitivity of the third generation
(ceftriaxone, cefotaxime and ceftazidime) and fourth genera-
tion (cefepime) cephalosporins were 35.6%, 35.6%, 37.7%
and 43.9% respectively. For monobactams (aztreonam), the
incidence of sensitivity was 31.14 %. For carbapenems, and
Table 1. Primers used for detection of ESBLs encoding gene.
Target gene Primer sequence Product size (bp)
bla TEM [30]
TEM/F: 5- GAA GAC GAA AGG GCC TCG TG-3
TEM/R: 5- GGT CTG ACA GTT ACC AAT GC -3
1074
bla SHV [30]
SHV/F: 5- CGC CGG GTT ATT CTT ATT TGT CGC -3
SHV/R: 5- TCT TTC CGA TGC CGC CGC CAG TCA -3
1016
Bla CTX-M- gp-I [23]
CTXM1-F3: 5- GAC GAT GTC ACT GGC TGA GC -3
CTXM1-R2: 5,- AGC CGC CGA CGC TAA TAC A -3,
499
BlaCTX-M- gp-IV [23]
CTX-M914F: 5- GCT GGA GAA AAG CAG CGG AG -3
CTX-M914R: 5- GTA AGC TGA CGC AAC GTC TG
474
bla OXAIII-gp [15]
OXA-1-S: 5- AGC CGT TAA AAT TAA GCC C -3
OXA-1-AS: 5- CTT GAT TGA AGG GTT GGG CG -3
908
Table 2. Enterobacteriaceae species isolated from different
clinicalspecimens in Egypt.
Isolated organisms No. (%) of isolates
Klebsiella spp. 107
Klebsiella
pneumoniae
89 26%
Klebsiella
oxytoca
3 0.9%
Klebsiella
terrigena
14 4.08%
Klebsiella
ozaenae
1 0.29%
Escherichia spp. 143
Escherichia
coli
113 32.9%
Escherichia
coli (inactive)
29 8.45%
Escherichia
hermanii
1 0.29%
Enterobacter spp. 17

Enterobacter
cloacae
9 2.6%
Enterobacter
aerogenes
1 0.29%
Enterobacter
agglomerans
3 0.87%
Enterobacter
asburie
4 1.16%
Proteus spp. 10

Proteus
mirabilis
8 2.33%
Proteus
vulgaris
2 0.58%
Salmonella spp. 4 1.16%
Citrobacter freundii 3 0.87%
Yersinia enterocolitica 3 0.87%
Morganella morganii 2 0.58%
iMedPub Journals
Our Site: http://www.imedpub.com/
ARCHIVES OF CLINICAL MICROBIOLOGY
4 Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
quinolones used, it was observed that 92.7% of all Entero-
bacteriaceae isolate were sensitive to imipenem and 51.9%
were sensitive to ciprooxacin. For aminoglycosides about
25.9% and 35.3 % were susceptible to gentamicin and ami-
kacin respectively. Moreover folate inhibitors showed less
activity against Enterobacteriaceae isolates.
Detection of ESBL producing enterbacteriaceae
isolates
It was found that 72 (50.3%) isolates of Escherichiaspp., 89
(83.1%) isolates out of Klebsiellaspp., and 100% of Entero-
bacterspp. were supposed to carry ESBLs.CLSI phenotypic
conrmatory tests for ESBLs production were used in this
study for all Enterobacteriaceae. Double-disc synergy test
(Figure 3) was done to all Enterobacteriaceae isolates and
results of the two methods are tabulated collectively in table
4. It shows that more than one disc is recommended for
double disc method for detection of ESBLs in Enterobacte-
riaceae isolates.
Table 3. Antibiotic susceptibility pattern of Enterobacteriaceae species isolated from different clinical specimens in Egypt.
Antibiotics
No. (%) susceptible
Escherichia
spp.
Klebsiella
spp.
Enterobacter
spp.
Proteus
spp.
Salmonella
spp.
Citrobacter
spp.
Yersinia
spp.
Morganella
spp.
% of
Susceptible
Strains
Ampicillin 0 0 0 0 0 0 0 0
Amoxicillin/
Clavulanic acid
0 0 0 6(60%) 0 0 0 0 6 (2.07%)
Ceftriaxone 70 (48.9%) 17(15.8%) 3 (17.6%) 9(90%) 1 (25%) 0 2(66.6%) 1(50%) 103(35.6%)
Cefotaxime 71 (48.9%) 17(15.8%) 3 (17.6%) 8(80%) 1 (25%) 0 2(66.6%) 1(50%) 103(35.6%)
Ceftazidime 74 (51.7%) 18(16.8%) 3 (17.6%) 10(100%) 1 (25%) 0 2(66.6%) 1(50%) 109(37.7%)
Cefepime 80 (55.9%) 23(21.4%) 8 (47.1%) 10(100%) 1 (25%) 1 (33.3%) 3(100%) 1(50%) 127(43.9%)
Aztreonam 72 (50.3%) 0 5 (29.4%) 10(12%) 1 (25%) 0 2(66.6%) 0 90(31.14%)
Imipenem
142
(99.9%)
91(85%) 13 (76.4%) 10(100%) 4 (100%) 3 (100% ) 3(100% ) 2 (100%) 268(92.7%)
Gentamicin 54 (37.7%) 4 (3.73%) 4 (23.5%) 8(80%) 2 (12.5%) 1 (33.3%) 1(33.3%) 1 (50%) 75(25.95%)
Amikacin 72 (50.3%) 11(10.2%) 5 (29.4%) 10(100%) 1 (25%) 1 (33.3%) 1(33.3%) 1 (50%) 102(35.3%)
Ciprooxacin 89 (62.2%) 31(28.9%) 10 (58.8%) 10(100%) 4 (100%) 1 (33.3%) 3(100%) 2 (100%) 150(51.9%)
Trimethoprim/
sulphamethoxazole.
37 (25.8%) 19(17.7%) 3 (17.6%) 8(80%) 2 (50%) 1 (33.3%) 1(33.3%) 1 (50%) 72(24.9%)
Figure 1. Detection of ESBL production by Double-disc synergy
test. Discs: ceftiaxone30 g; cefotaxime 30 g;
ceftazidime 30 g; aztreonam 30 g; cefepime 30
g; centre, amoxicillin/clavulanic acid, the organism
Enterobacter cloacae where there is an extension of
edge of inhibition zone of cefepime only towards
amoxicillin/clavulanic acid disc.
ARCHIVES OF CLINICAL MICROBIOLOGY
Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
5
iMedPub Journals
Our Site: http://www.imedpub.com/
Table 4. Detection of ESBLs by different phenotypic conrmatory tests among Enterobacteriaceae species isolated from
different clinical specimens in Egypt.
Phenotypic conrmatory tests Combined disc method [8] Double disc test [13]
Enterobacteriaceae isolates
CTX/CA
vs CTX
CAZ/CA vs
CAZ
CRO CTX CAZ ATM FEP
Escherichia spp.
62
(43.35%)
55
(38.46%)
57
(39.8%)
61
(42.6%)
58
(40.5%)
56
(39.1%)
61
(42.6%)
Klebsiella spp.
66
(61.68%)
72
(67.28%)
63
(58.8%)
63
(58.8%)
63
(58.8%
70
(65.45%
72
(67.3%
Enterobacter spp.
4
(23.5%)
4
(23.5%)
3
(17.6%)
4
(23.5%)
5
(29.4%)
4
(23.5%)
6
(35.2%)
Proteus spp. 0 0 0 0 0 0
1
(10%)
Salmonella spp.
3
(75%)
3
(75%)
2
(50%)
3
(75%)
3
(75%)
2
(50%)
3
(75%)
Citrobacter spp. 0 0 0 0 0 0 0
Yersinia spp. 0 0 0 0 0 0 0
Morganella spp.
1
(50%)
0
1
(50%)
1
(50%)
1
(50%)
1
(50%)
1
(50%)
Table 5. Distribution of ESBLs genes by PCR among positive ESBL for Escherichia spp., Klebsiellaspp. and Enterobacterspp.
isolated from different clinical specimens in Egypt.
No of positive screening for
ESBLs isolates
TEM No (%) SHV No (%) CTX-M-I No (%) CTX-M-IV No (%) OXA-III No (%)
Escherichia spp.(72)
59
(81.9%)
5
(6.9%)
52
(72.2%)
23
(31.9%)
32
(44.4%)
Klebsiella spp.(89)
36
(40.5%)
64
(72%)
42
(47.1%)
50
(56.1%)
25
(28.1%)
Enterobacter spp.(17)
7
(41.1%)
5
(29.4%)
2
(11.7%)
9
(52.9%)
2
(11.7%)
Total No. of isolates (178) 102 74 96 82 59
Table 6. Distribution of ESBLs genes by PCR among positive AmpC for examined Enterobacteriaceaeisolated from different
clinical specimens in Egypt.
Enterobacteriaceae isolates TEM SHV CTX-M-I CTX-M-IV OXA-III
Total no of positive screening for
AmpC isolates
N (%) N (%) N (%) N (%) N (%)
Escherichia spp.(13)
11
(84.6%)
0 9
(69.2%)
6
(46.15%)
7
(6.9%)
Klebsiella spp.(33)
7
(21.2%)
16
(48.5%)
12 (36.3%)
21
(63.6%)
8 (24.2%)
Enterobacter spp.(16)
6
(29.4%)
4
(25%)
1
(6.25%)
9
(56.25%)
1
(6.25%)
Total No. of isolates (62) 24 20 22 36 16
iMedPub Journals
Our Site: http://www.imedpub.com/
ARCHIVES OF CLINICAL MICROBIOLOGY
6 Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
Detection of AmpC inducible Enterobacteriaceae
isolates
Co-existence of both AmpC beta-lactamases and ESBL in
some Enterobacteriaceae strainshas been detected in this
study which led to false negative results.AmpC inducible
strains were detected in 13,33,16 and 3 isolates of Escheri-
chiaspp., Klebsiella spp., Enterobacter spp.& Citrobacter spp.
respectively..
PCR for genotypic detection of ESBLs: (TEM/ SHV/
CTX-M-I CTX-M-IV/ OXA-III)
PCR test was done to positive ESBLs tests for Escherichia spp.,
Klebsiella spp. and Enterobacter spp. Results are tabulated
in table 5. TEM represents the most predominant gene pro-
ducing ESBLs among Escherichia spp. (81.9%) followed by
CTX-M-gp I (72.2%) while SHV represents only 6.9%. On the
other hand SHV was the predominant gene among Klebsiella
spp. (72%) followed by CTX-M-gp IV genes (56.1%) while
TEM was (40.5%) of ESBLs. CTX-M-IV genes was relatively
the most predominant among Enterobacter spp. (52.9%)
followed by TEM (41%) while SHV represents only (29.4%).
OXA-III genes were found in 28.1% of Klebsiella spp. isolates
and only in 11.7% of Enterobacter spp. while they were in
considerable percentage in Escherichia spp. (44.4%). Figures
2-6 show the pictures shot to the gel after electrophoresis,
showing lanes of positive bands of PCR products for each
enzymes group and ladders lanes in between each group
of samples.
Figure 2. Detection of bla TEM gene by conventional PCR where
positive isolates show bands at 1074 bp product size
Figure 3. Detection of bla SHV gene by conventional PCR where
positive isolates show bands at 1016 bp product size.
Figure 4. Detection of bla CTX-M-I gene by conventional
PCR where positive isolates show bands at 499 bp
product size.
Figure 5. Detection of bla CTX-M-IV gene by conventional PCR
where positive isolates show bands at 474 bp product
size.
ARCHIVES OF CLINICAL MICROBIOLOGY
Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
7
iMedPub Journals
Our Site: http://www.imedpub.com/
Regarding molecular characterization, we detected a variety
of beta-lactamases encoding genes among the examined
Enterobacteriaceae strains. The detected genes include TEM,
CTX-M-I, CTX-M-IV, SHV and OXA-III.
TEM-type was the most predominant ESBL in the present
study (52.5%) followed by equal frequency for both of CTX-
M-I&IV (49 % for each), while SHV (39%)and OXA-III (31.25%)
were the least but in considerable frequent detected among
examined Enterobacteriaceae. This indicates that TEM-type
allele is the most common ESBL in Egypt, this nding was
reported previously [9]. Different enzymes are reported as
a common ESBL in other settings, CTX-M was reported as
the predominance in Morocco [3, 4] and Portugal [17]. This
demonstrates the importance of studying the geographical
distribution and mobilization of resistance genes among clini-
cal bacterial isolates.
On genus level, there was a variation in distribution of dif-
ferent resistant genes. It was observed in our study that
TEM represents the most predominant gene producing ES-
BLs among Escherichia spp. (81.9%) followed by CTX-M-gp I
(72.2%) while SHV represents only 6.9%. On the other hand
SHV was the predominant gene among Klebsiella spp. (72%)
followed by CTX-M-gp IV genes (56.1%), while TEM repre-
sents about 40.5% of ESBLs encoding genes. For Enterobac-
ter spp., CTX-M-IV genes was relatively the most predomi-
nant (52.9%) followed by TEM (41%) while SHV represents
only (29.4%). These results are close to those reported by an
Indian study in 2010 [27].
It was reported that up to 90% of ampicillin resistance in
E. coli is due to the production of TEM-1[27] and it was the
rst TEM-type beta-lactamase that displayed the ESBL pheno-
type [21]. Although TEM-type beta-lactamases were reported
mostlyin Escherichia spp. [9] and Klebsiella spp.[21], they were
also detected in other species of Gram-negative bacteria with
increasing frequency as Enterobacteraerogenes [17].
Regarding CTX-M-type ESBL enzyme, it was detected in 49%
of examined isolates and it constitute a novel and rapidly
growing family of plasmid-mediated ESBLs that are currently
replacing mutant TEM or SHV ESBLs. They have become the
most prevalent type of ESBLs described during the last few
years [1, 4]. The types of the enzyme CTX-M-1 to 15are varied
with geographical distribution [4, 16, 20, 26].
In the present study, OXA-III enzymes encoding genes were
found in 31.25% of the examined isolates. These are plasmid-
encoded beta-lactamases which were reported as less sensi-
tive than TEM to clavulanic acid inhibition. OXA-type ESBLs
are increasingly reported recently [27, 29] and it was reported
among Egyptian clinical Enterobacteriaceae [24].
Figure 6. Detection of bla OXA-III gene by conventional PCR
where positive isolates show bands at 908 bp product
size.
Discussion
The current study showed that Gram-negative bacilli were the
predominant isolates (91.4%). Enterobacteriaceae spp. repre-
sented 84.2% while 15.8% were non-fermenters. Escherichia
spp. were the most frequent isolates, representing 49.48%.
Other less frequent isolates were identied as Klebsiella spp.
(37.02%) and Enterobacter spp. (5.88 %). Enterobacteriaceae
spp. were previously reported as the most commonly isolated
pathogens in clinical settings [2, 10, 29].
Upon observing susceptibility pattern in present study it was
found that, ESBL producing strains are not only showed high-
level of resistance to beta lactam antimicrobial agents but
also exhibited resistance to other groups of antimicrobials
like gentamicin (26%), amikacin (35%), ciprooxacin (52 %),
sulpha-methoxazole (24.9%). Other studies reported similar
nding where ESBL producing isolates showed a high degree
resistance to different groups of antimicrobial agents [20, 29].
This was attributed to the high mobility of genes encoding
ESBLs which are usually located on plasmids and hence can
harbor resistance genes to several other unrelated classes of
antimicrobials, such as the plasmid-mediated quinolone-re-
sistance genes and aminoglycoside-resistance genes [25, 32].
This means that resistance to two different kinds of antibiot-
ics could be a selective pressure for spreading such resistant
strains [17, 27].
Among isolated Escherichia spp. in the present study, 43.3%
were ESBL producers. The prevalence of ESBL among Kleb-
siella spp. and Enterobacter spp. were 65.42% and 17.6%
respectively. Prevalence of ESBLs among clinical strains of
these Enterobacteriaceae spp. were formerly reported [3, 14].
iMedPub Journals
Our Site: http://www.imedpub.com/
ARCHIVES OF CLINICAL MICROBIOLOGY
8 Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
Co-existence of different ESBL encoding genes within the
same isolate was commonly detected in the present study.
This association has been reported in other studies in differ-
ent countries [3, 19].
Co-existence of both AmpC beta-lactamases and ESBL in
some Enterobacteriaceae strains has also been detected in
the present study. Recently there have been numerous re-
ports of Enterobacterial strains harboring plasmid mediated
ESBLs, in addition to chromosomal Amp C-type ESBLs [11, 16,
28, 29]. The coexistence of both enzyme types in the same
strain in this study resulted in resistance to cephalosporins
and may result in false negative tests for the detection of
ESBLs and that was the reason for the elevated percentage
of false ESBLs upon screening and conrmation tests with
cephalosporins. The likely explanation is that AmpC-type
beta-lactamases resist inhibition by clavulanate and hence
obscure and mask the synergistic effect of clavulanate and
cephalosporins against ESBLs[11]. The effects of the non-
identication of ESBL-producing organisms may result in in-
appropriate dosing of extended-spectrum antibiotics, treat-
ment failure, an increase in hospital costs, length of stay, and
patient mortality [2].
The ndings of this report shed light on the current preva-
lence and molecular types of ESBL-producing Enterobacte-
riaceae in Egypt in clinical setting, which can contribute to
better understanding of the epidemiology of these resistance
enzymes at national level. Our report revealed increasing
prevalence of both AmpC beta-lactamases and ESBLs which
created an urgent need for accurate detection of currently
common ESBL producers which are rapidly spread among En-
terobacteriaceae isolates. Our results emphasize the necessity
for continuous updating of data of antimicrobial susceptibility
proles due to continuous evolution of antimicrobial resis-
tance mechanisms in bacteria. This ensures the safety and
efcacy of pathogen specic antimicrobial therapies.
Authors disclosures of potential
conicts of interest
Authors declare that they have no potential conicts of inter-
est. All authors read and approved the nal manuscript.
Acknowledgements
We are grateful to the management and medical stuff of
Department of Clinical Microbiology, Central Health Labo-
ratories, Ministry of Health and Population for assistance in
sample collection.
We also deeply indebted to staff of Bacteria Parasitic Dis-
ease Research Programin U.S. Naval Medical Research Unit-3
(NAMRU-3), Cairo, Egypt for the equipments and facilities
put at our disposal.
References
1. Al Sweih, N., Salama, M.F., Jamal, W., Al Hashem, G., Rotimi, VO. An
outbreak of CTX-M-15-producing Klebsiella pneumoniae isolates in
an intensive care unit of a teaching hospital in Kuwait. Indian J Med
Microbiol. 2011; 29 (2): 130-5.
2. Ashour, H.M., El-Sharif, A. Species distribution and antimicrobial
susceptibility of gram-negative aerobic bacteria in hospitalized cancer
patients. J Transl Med. 2009; 7: 14.
3. Barguigua, A., El Otmani, F., Talmi, M., Bourjilat, F., Haouzane, F.,
Zerouali, K. et al. Characterization of extended-spectrum beta-
lactamase-producing Escherichia coli and Klebsiella pneumoniae
isolates from the community in Morocco. J Med Microbiol. 2011; 60
(9): 1344-52.
4. Barguigua, A., El Otmani, F., Talmi, M., Bourjilat, F., Haouzane, F.,
Zerouali, K. et al. Prevalence and genotypic analysis of plasmid-
mediated beta-lactamases among urinary Klebsiella pneumoniae
isolates in Moroccan community. J Antibiot 2013; 66 (1): 11-6.
5. Bouchillon, S. K., Johnson, BM., Hoban, DJ., Johnson, JL., Dowzicky,
MJ., Wu, DH. et al. Determining incidence of extended spectrum
beta-lactamase producing Enterobacteriaceae, vancomycin-resistant
Enterococcus faecium and methicillin-resistant Staphylococcus aureus
in 38 centres from 17 countries: The PEARLS study 2001-2002. Int J
Antimicrob Agents. 2004; 24 (2): 119-24.
6. Bradford, P.A. Extended-spectrum beta-lactamases in the 21st
century: Characterization, epidemiology, and detection of this
important resistance threat. Clin Microbiol Rev. 2001; 14 (4): 933-51.
7. Castanheira, M., Deshpande, LM., Mathai, D., Bell, JM., Jones, RN.,
Mendes, RE. Early dissemination of NDM-1- and OXA-181-producing
Enterobacteriaceae in Indian hospitals: report from the SENTRY
Antimicrobial Surveillance Program, 2006-2007. Antimicrob Agents
Chemother 2011; 55 (3): 1274-8.
8. CLSI. Performance Standards for Antimicrobial Susceptibility Testing
Infobase. 2008.
9. El-Sharif, A., Raghdaa, A. Molecular detection of TEM-Type
lactamase producing Escherichia coli from diarrheic Egyptian children.
Archives of Clinical Microbiology 2012; 3 (5): 1-5.
10. Hoban, D.J., Bouchillon, SK., Hawser, SP., Badal, RE. Trends in the
frequency of multiple drug-resistant Enterobacteriaceae and their
susceptibility to ertapenem, imipenem, and other antimicrobial agents:
data from the Study for Monitoring Antimicrobial Resistance Trends
2002 to 2007. Diagn Microbiol Infect Dis. 2010; 66 (1): 78-86.
11. Jacoby, G.A. AmpC beta-lactamases. Clin Microbiol Rev. 2009; 22 (1):
161-82.
12. Jacoby, G.A., Walsh, KE, Walker, VJ. Identication of extended-
spectrum, AmpC, and carbapenem-hydrolyzing beta-lactamases
in Escherichia coli and Klebsiella pneumoniae by disk tests. J Clin
Microbiol. 2006; 44 (6): 1971-6.
13. Jarlier, V., Nicolas, MH., Fournier, G., Philippon, A. Extended broad-
spectrum beta-lactamases conferring transferable resistance to newer
beta-lactam agents in Enterobacteriaceae: Hospital prevalence and
susceptibility patterns. Rev Infect Dis. 1988; 10 (4): 867-78.
14. Jones, C.H., Tuckman, M., Keeney, D., Ruzin, A., Bradford, PA.
Characterization and sequence analysis of extended-spectrum-
{beta}-lactamase-encoding genes from Escherichia coli, Klebsiella
pneumoniae, and Proteus mirabilis isolates collected during tigecycline
phase 3 clinical trials. Antimicrob Agents Chemother 2009; 53 (2):
465-75.
ARCHIVES OF CLINICAL MICROBIOLOGY
Copyright iMedPub
2013
Vol. 4 No. 5:2
doi: 10.3823/273
9
iMedPub Journals
Our Site: http://www.imedpub.com/
15. Kim, J.Y., Jung, HI., An, YJ., Lee, JH., Kim, SJ., Jeong, SH. et al.
Structural basis for the extended substrate spectrum of CMY-10, a
plasmid-encoded class C beta-lactamase. Mol Microbiol. 2006; 60 (4):
907-16.
16. Livermore, D.M., Mushtaq, S., Nguyen, T., Warner, M. Strategies to
overcome extended-spectrum beta-lactamases (ESBLs) and AmpC
beta-lactamases in shigellae. Int J Antimicrob Agents 2011; 37 (5):
405-9.
17. Machado, E., Coque, TM., Canton, R., Novais, A., Sousa, JC., Baquero,
F. et al. High diversity of extended-spectrum beta-lactamases among
clinical isolates of Enterobacteriaceae from Portugal. J Antimicrob
Chemother 2007; 60 (6): 1370-4.
18. Maveyraud, L., Golemi, D., Kotra, LP., Tranier, S., Vakulenko, S.,
Mobashery, S. et al. Insights into class D beta-lactamases are revealed
by the crystal structure of the OXA10 enzyme from Pseudomonas
aeruginosa. Structure 2000; 8 (12): 1289-98.
19. Mishra, S., Sen, MR., Upadhyay, S., Bhattacharjee, A. Genetic linkage
of bla(NDM) among nosocomial isolates of Acinetobacter baumannii
from a tertiary referral hospital in northern India. Int J Antimicrob
Agents 2013.
20. Nedjai, S., Barguigua, A., Djahmi, N., Jamali, L., Zerouali, K., Dekhil,
M. et al. Prevalence and characterization of extended spectrum
beta-lactamases in Klebsiella-Enterobacter-Serratia group bacteria, in
Algeria. Med Mal Infect. 2012; 42 (1): 20-9.
21. Negri, M.C., Lipsitch, M., Blazquez, J., Levin, BR., Baquero, F.
Concentration-dependent selection of small phenotypic differences
in TEM beta-lactamase-mediated antibiotic resistance. Antimicrob
Agents Chemother 2011; 44 (9): 2485-91.
22. Perilli, M., Segatore, B., Mugnaioli, C., Celenza, G., Rossolini, GM.,
Stefani, S. et al. Persistence of TEM-52/TEM-92 and SHV-12 extended-
spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in
Italy. Microb Drug Resist. 2011; 17 (4): 521-4.
23. Pitout, J.D., Hossain, A., Hanson, ND. Phenotypic and molecular
detection of CTX-M-beta-lactamases produced by Escherichia coli and
Klebsiella spp. J Clin Microbiol 2004; 42 (12): 5715-21.
24. Poirel, L., Abdelaziz, MO., Bernabeu, S., Nordmann, P. Occurrence of
OXA-48 and VIM-1 carbapenemase-producing Enterobacteriaceae in
Egypt. Int J Antimicrob Agents 2013; 41 (1): 90-1.
25. Poirel, L., Van De Loo, M., Mammeri, H., Nordmann, P. Association of
plasmid-mediated quinolone resistance with extended-spectrum beta-
lactamase VEB-1. Antimicrob Agents Chemother 2005; 49 (7): 3091-4.
26. Qureshi, Z.A., Paterson, DL., Peleg, AY., Adams-Haduch, JM., Shutt,
KA., Pakstis, DL. et al. Clinical characteristics of bacteraemia caused by
extended-spectrum beta-lactamase-producing Enterobacteriaceae in
the era of CTX-M-type and KPC-type beta-lactamases. Clin Microbiol
Infect. 2012; 18 (9): 887-93.
27. Sharma, J., Sharma, M., Ray, P. Detection of TEM & SHV genes in
Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care
hospital from India. Indian J Med Res. 2010; 132: 332-6.
28. Singhal, S., Mathur, T., Khan, S., Upadhyay, DJ., Chugh, S., Gaind,
R. et al. Evaluation of methods for AmpC beta-lactamase in gram
negative clinical isolates from tertiary care hospitals. Indian J Med
Microbiol 2005; 23 (2): 120-4.
29. Taneja, N., Rao, P., Arora, J., Dogra, A. Occurrence of ESBL & Amp-C
beta-lactamases & susceptibility to newer antimicrobial agents in
complicated UTI. Indian J Med Res. 2008; 127 (1): 85-8.
30. Tasli, H. Bahar, IH. Molecular characterization of TEM- and SHV-
derived extended-spectrum beta-lactamases in hospital-based
Enterobacteriaceae in Turkey. Jpn J Infect Dis. 2005; 58 (3): 162-7.
31. Wang, P., Hu, F., Xiong, Z., Ye, X., Zhu, D., Wang, YF. et al.
Susceptibility of extended-spectrum-beta-lactamase-producing
Enterobacteriaceae according to the new CLSI breakpoints. J Clin
Microbiol 2011; 49 (9): 3127-31.
32. Zhanel, G.G., Decorby, M., Nichol, KA., Baudry, PJ., Karlowsky, JA.,
Lagace-Wiens, PR. et al. Characterization of methicillin-resistant
Staphylococcus aureus, vancomycin-resistant enterococci and
extended-spectrum beta-lactamase-producing Escherichia coli in
intensive care units in Canada: Results of the Canadian National
Intensive Care Unit (CAN-ICU) study (2005-2006). Can J Infect Dis
Med Microbiol 2008; 19 (3): 243-s Med Microbiol. 19(3): p. 243-9.
Archives of Clinical Microbiology (ACMicrob) is a new peer-reviewed,
international journal with world famous scientist on the editorial board.
ACMicrob is an open access journal with rapid publication of articles in
all felds and areas of microbiology and infectious diseases.
ACMicrob covers all aspects of basic and clinical microbiology relevant
to infectious diseases including current research on diagnosis, manage-
ment, treatment, preventive measures, vaccination, and methodology.
Clinical microbiology relevant inmmunology, pathophysiology,
genetics, epidemiological, and genomics studies are also welcome.
Submit your manuscript here:
http://www.acmicrob.com
Publish with iMedPub
http://www.imedpub.com
Follow us:
Where Doctors exchange clinical experiences,
review their cases and share clinical knowl-
edge. You can also access lots of medical
publications for free. Join Now!
http://medicalia.ning.com/
Medicalia.org