Extended Spectrum β-Lactamases in Escherichia coli & Klebsiella pneumoniae, their molecular characterization & associated risk factors.

Abstract

ABSTRACT:
Background & objectives: Extended spectrum β-lactamase (ESBL) production is increasing all over the world with limited treatment options. This study aimed to determine the occurrence of ESBLs in Escherichia coli and Klebsiella pneumoniae, their molecular types and associated risk factors
Methods: Total 86 clinical isolates of E. coli (n=46) and K. pneumoniae (n=40) collected between January and June 2010 , were screened for production of ESBL using combined disk methods and Microscan . ESBL strains were further typed for the bla TEM/SHV/CTX-M genes by PCR using specific primers. Resistance to other antimicrobial agents was also studied. Various risk factors associated with ESBL infections were analyzed by logistic regressions.
Results: ESBLs were found in 52.2 % E. coli and 60 % K. pneumoniae isolates. Majority of the typeable isolates harboured two or more ESBL genes (55.3%). Overall blaCTX-M was the commonest genotype (78.9%) followed by blaTEM (57.9%) and blaSHV (34.2%) either alone or in combination. Sensitivity of ESBL isolates to other antibiotics was impenum and meropenum ( 100%) , tygacillin 95% , Amikacin 79% , pipracillin /tazocin 58% and ciprofloxacin 31.6% while only 5% of strains were susceptible to gentamycin. Prior antibiotic exposure, use of intravenous device and urinary catheter, renal insufficiency and ICU admission were associated with ESBL infection on univariate analysis.
Interpretation & conclusions: Our study showed high ESBL occurrence with CTX-M as the emerging type. High co-resistance to other non B-lactam antibiotics is a major challenge for management of ESBL infections.

Full text

Egyptian Journal of Medical Microbiology Vol 20, (3):35-45 (2011)
Extended Spectrum β-Lactamases in Escherichia coli & Klebsiella pneumoniae,
their molecular characterization & associated risk factors.
1 Sahar M. Ali and 2 Zakia Abu Zahab
1Microbiology and Immunology department, Faculty of Medicine, Menufiya University, Egypt.
2Clinical pathology department Faculty of Mediciene Al Azhar university (girls) Egypt.
2
ABSTRACT:
Background & objectives: Extended spectrum β-lactamase (ESBL) production is increasing all over the
world with limited treatment options. This study aimed to determine the occurrence of ESBLs in
Escherichia coli and Klebsiella pneumoniae, their molecular types and associated risk factors
Methods: Total 86 clinical isolates of E. coli (n=46) and K. pneumoniae (n=40) collected between
January and June 2010 , were screened for production of ESBL using combined disk methods and
Microscan . ESBL strains were further typed for the bla TEM/SHV/CTX-M genes by PCR using specific
primers. Resistance to other antimicrobial agents was also studied. Various risk factors associated with
ESBL infections were analyzed by logistic regressions.
Results: ESBLs were found in 52.2 % E. coli and 60 % K. pneumoniae isolates. Majority of the typeable
isolates harboured two or more ESBL genes (55.3%). Overall blaCTX-M was the commonest genotype
(78.9%) followed by blaTEM (57.9%) and blaSHV (34.2%) either alone or in combination. Sensitivity of
ESBL isolates to other antibiotics was impenum and meropenum ( 100%) , tygacillin 95% , Amikacin
79% , pipracillin /tazocin 58% and ciprofloxacin 31.6% while only 5% of strains were susceptible to
gentamycin. Prior antibiotic exposure, use of intravenous device and urinary catheter, renal insufficiency
and ICU admission were associated with ESBL infection on univariate analysis.
Interpretation & conclusions: Our study showed high ESBL occurrence with CTX-M as the emerging
type. High co-resistance to other non B-lactam antibiotics is a major challenge for management of ESBL
infections.
Introduction:-
The decreasing bacterial susceptibility is an important issue related to antibiotics, where bacteria are
capable of developing novel survival strategies that allow some strains to thrive in the presence of
antibiotics to which they were previously susceptible. Some bacteria produce potent enzymes known as
beta- lactamases that inactivate many beta- lactam antibiotics. Among the family Enterobacteriacae, the
production of plasmid-mediated extended-spectrum β-lactamase (ESBLs) has emerged as an important
mechanism of resistance to β-lactam drugs that account for approximately 50% of antibiotic
consumption. Extended spectrum β - lactamases (ESBLs) are enzymes that mediate resistance to
extended- spectrum cephalosporins, such as cefotaxime (CTX ), ceftriaxone (CRO ), and ceftazidime
(CAZ ) and the monobactam aztreonam (1). ESBLs are usually plasmid-mediated β-lactamases, most
commonly found in Klebsiella pneumoniae, Escherichia coli and other Gram-negative bacilli (2) . Since
1983 the number of ESBL variants has been constantly growing; at present more than 300 different
ESBL variants are known (3). These have been classified into nine distinct structural / evolutionary
families based on their amino acid sequences. TEM and SHV enzymes form major families. However,
CTX-M type has emerged in many countries of the world (4). Currently the CTX-M family includes
more than 40 β-lactamases, which are grouped on the basis of sequence similarity into five distinct
clusters (subtypes) epitomized by CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-253.

Infections caused by ESBL-producing bacteria often involve immune-compromised patients, making it
difficult to eradicate these organisms in high-risk wards, such as intensive care unites (5)
Reduced susceptibility or increase in resistance to extended-spectrum cephalosporins and/or
monobactams represents the first indicator of ESBL production, but confirmation is dependent on
synergy between clavulanate and the selected β-lactams, using double-disk diffusion method, or E-test
(6)The expression of an extended-spectrum enzyme does not always involve a phenotype that can be
interpreted as resistant by the routine MICs and disk diffusion methods that follow Clinical and
Laboratory Standards Institute (CLSI) breakpoint.(7) Accordingly, ESBL-positive strains should be
reported as resistant even if drug MICs are below breakpoints established for cephalosporins and
aztreonam. This is defined for both Klebsiella spp, and E.coli, but not established for the other
Enterobacteriaceae (8)
Aim of the work :
The present study was carried out to explore the occurrence of ESBLs in E. coli and K. pneumoniae
strains isolated from various clinical specimens. To detect the commonly prevalent molecular genotypes
of ESBL (bla TEM/SHV/CTX-M) by PCR.To detect the sensitivity of ESBL strains to other
antimicrobial groups and to analyze various risk factors associated with the occurrence of infection by
ESBL producing strains.
Materials and Methods:-
This study was conducted over 6 months in the period from January to June (2010) , from patients in Al
Ansar Hospital Al Madina Almounawarh . Swabs from 1876 cases in different departments were sent to
the microbiological laboratory from them 546 cases had positive culture including 206 case having
infections due to gram negative bacteria. 86 from 206 gram negative isolets were E.coli and Klebsiella
pneumonia from them 46 were identified as ESBL producing strains which were included in this study.
The 46 patients with E coli and Klebsiella pneumonia ESBL producing strains were ; (23 ) males and
(25) females. Data from the patients were collected including : Demographic data, Intensive care unit
admission , history of antibiotic exposure in last 1 month , Use of urinary catheter, Use of intravenous
devices, Use of ventilator, Renal insufficiency (creatinine >2 mg/dl), Diabetes mellitus, Malignancy and
Corticosteroid use.
Bacterial Isolates:-
These bacteria were isolated from various clinical specimens such as blood culture , urine , sputum and
other miscellaneous sources as wound, pus, ear swabs, eye swabs, peritoneal fluid These isolates were
recovered from the specimens as follow:-
Blood (5-10 ml) was collected from each patient and inoculated into BC bottles (Plus+ Aerobic
[product no.5117294; BD] and Plus+ Anaerobic [product no. 4204742 ; BD]) and were monitored with
the BACTEC 9120 instrument (Becton Dickinson, Cockeysville, MD, USA) by the use of the standard
growth detection algorithms provided with the system. Positive blood cultures set were examined by
Gram stain. Only blood culture sets with Gram- negative enteric bacillus – like morphology were
included and a subculture was made for that sample on both blood agar plates and MacConkey agar
plates. Other specimens are cultured on the specific media( Mac Coney agar, blood agar , chocolate agar and
CLED agar ) (Oxoid,USA) under the optimal conditions, and were identified to genus and species using
standard methods (conventional manual methods and Microscan ) (9 ). Multiple samples or isolates from
the same patient were excluded from the study. These isolates were stored on brain heart infusion at -80
till doing the PCR.
Screening and confirmation of ESBLs producing isolates:-
Test organisms from stock culture were activated by inoculation in to Mueller Hinton broth (BD, USA)
and incubated at 37°C for 24 hours. The concentration of the bacterial suspension was adjusted to be
equivalent to 0.5 McFarland standards. The test organism was seeded on the surface of freshly prepared
Mueller Hinton agar (BD, USA), in three directions using a sterile swab, according to the
recommendations of Kirby-Bauer Disk Diffusion method (10 ) and CLSI guide lines (7) . The plates were

allowed to stand at room temperature for 15 minutes prior to the application of antibiotic containing
disks.
ESBL-producing isolates were detected using the Double Disk Diffusion method (Double Disk
Approximation Test) (11). Disks containing aztreonam, ceftazidime, ceftriaxone, and cefotaxime (30 µg
each) were placed around a central disk of Amoxicillin-Clavulanic acid 30 ug (20:10 resp.) (Oxoid, UK),
30 mm center to center on Mueller Hinton agar plates. Plates were incubated aerobically at 37°C for 18–
24 hours, and the diameter of the inhibition zone (if any) around the antimicrobial disks was measured in
mm using a ruler. Enhancement of the inhibition zone toward the amoxicillin-plus-CA disk was was
recorded, and the organism was thus considered as an ESBL producer. Results were interpreted according
to CLSI criteria for the isolates of Klebsiella and E. coli spp. An inhibition zone of 22mm for ceftazidime
and 27mm for cefotaxime indicate that the strain probably produces ESBL. ESBLs producing isolates are
generally cefoxitin sensitive and so were screened by the disk diffusion test using 30 ug cefoxitin disks.
Two control organisms, E.coli ATCC 25922 and K.pneumoniae ATCC700603, were inoculated in each
set of tests for quality control.
Figure (2)
Single disk diffusion and double disk diffusion test
MicroScan ESBL test:-
The MicroScan instrument (WalkAway/ 40 instruments with version1.51 of the Data Management
System) was used for all reading and interpretation of panel results. MicroScan Dried Overnight Neg
Combo Type 35 panels and MicroScan ESBL plus were used for Gram- negative isolates. The
MicroScan panels were performed in accordance with the guidelines of the manufacturer. The results
were obtained after overnight incubation. Panels of ESBL plus were read manually following 16 to 20 h
of incubation.
Antimicrobial susceptibility testing:-
Susceptibility tests To other antibiotics were performed by using the results obtained from both the
Kirby-Bauer disc diffusion method and following CLSI guidelines and that obtained from the
MicroScan instrument. the antimicrobials tested were: Ampicillin; Ampicillin –clavulinc acid ;
Piperacillin- tazobactam; Cefazolin ; Cefuraxim ; Cefepime; Amikacin; Impenium; Meropenium ;
Ciprofloxacin , Tygacilline and Gentamicin.

Molecular analysis (PCR amplification of bla genes) :-
Reference confirmation for ESBL production was by molecular characterization, i.e. by PCR analysis for
ß-lactamase genes of the families CTX-M, SHVand TEM genes. DNA from E.coli and Klebsiella
pneumoniae ESBL producing strains was extracted from overnight bacterial growth at 37°C by
extraction Kit, pharmacia GFX spin column DNA protocol. After preparation of the kit contents the
procedure were done as follow:
20µl of protease was pipet into 1.5 ml tube, 200µl of sample were added to the tube, and 400µl of lysis
buffer . The tube was incubated at room temperature for 30 minutes. Then the mixture was applied to the
spin column (in a 2 ml collection tube) and centrifuged at 10000 rpm for 1 minute and flowthrough was
discarded. 500µl of lysis buffer were added to the spin column and again centrifuged at 10000 rpm for 1
minute and flowthrough was discarded. 500µl wash buffer was added to the column and centriguged at
8000 rpm for 1 minuute and flowthrough was discarded. The spin column was placed in a new 1.5 ml
microcentrifuge tube, and 125µl elution buffer was added and incubated at room temperature for 1 minute
and then centrifuged at 10000 rpm for 1 minute.
Extracted DNA of all phenotypic confirmed E.coli and Klebsiella pneumoniae ESBL producing strains
was subjected to amplification by PCR using blaTEM/SHV/CTX-M specific primers. The primers to
amplify the targeted genes were chosen from earlier published studies (12). The primers were procured
from OPERON Biotechnologies, Germany. PCR Kit (The commercial MMX invetrogen master mix Cat
No 492056A).
Table (1) : PCR primers
Target
Primer
name
Primer sequence (5`-3`)
Product
size(bp)
BlaTEM
TEM-F
TEM-R
TCCGCTCATGAGACAATAACC
TTGGTCTGACAGTTACCAATGC
931
BlaSHV
SHV-F
SHV-R
TGGTTATGCGTTATATTCGCC
GGTTAGCGTTGCCAGTGCT
868
BlaCTX-M
CTX-F
CTX-R
TCTTCCAGAATAAGGAATCCC
CCGTTTCCGCTATTACAAAC
909
Master mix preparation: final reaction volume 25µl was prepared with 12.5 µl readymade master mix, 20
pmol of both primers and 5 µl of extracted DNA and 5.5 µl H2O (Mili-Q grade) . Amplification reactions
were carried out in a thermocycler (PTC-100, MJ Research, USA) under the following conditions: initial
denaturation at 96 ºC for 3 min, followed by 35 cycle of denaturation at 96ºC for 30 sec, annealing at 58
ºC for 45 sec and elongation at 72ºC for 30 sec. The final elongation step was extended to 3 min at 72ºC.
Five microliters aliquots of PCR products were analyzed by gel electrophoresis with 1% agarose gels
(BioRad, Hercules, Calif.) in tris acetate EDTA (TAE) buffer pH 7.8 . Gels were stained with ethidium
bromide (10 mg/L) and visualized by UV transilluminator.
Statistical methods:
The results were statistically analyzed by correlation analysis.The risk factors were analyzed using SPSS
statistical software, version 12.0 (SPSS Inc., Chicago, IL, USA) by univariate logistic regression analysis
Results:-
Gram negative bacterial species isolated from clinical samples:
During our study period, a total of 206 patients were identified as having infections due to gram negative
bacteria . Escherichia coli was the most common species isolated from these specimens, comprising 46/
206 (22.3%). This organism was the major isolate recovered from urine samples, representing 30/46 (

65.2%) . Klebsiella pneumoniae was the second isolated pathogen from the tested samples, constituting
40/ 206 (19.4 %) of the total isolates, where 18/40 (45 %) of the Klebsiella pneumoniae isolates were
recovered from urine , 14/40 (35 %) from sputum, 6/40 (15 %) from blood and the remaining 2/40 (5 %)
from the other miscellaneous sources.Pseudomonas spp were also isolated from 39/206 (18.9 %) of the
total isolates, proteus spp represent29/ 206 ( 14.1 %) and Acietobacter spp represented 28/206 (13.6 %)
of the total isolates.
:K. pneumoniaeproducing -and ESBL E. coliproducing -Prevalence of ESBL -:2 Figure
Percent of ESBL occurance in E .coli were as follow 75.00, 46.67, 0.00 and 100.00 compared to 66.67,
66.67, 57.14 and 0.00 in klebsiella pneumonia obtained from blood ,urine , sputum and other sources
respectively as shown in figure 2 . From 206 clinical isolates of gram negative bacilli, 86 isoltes of E.Coli and Klebsiella were included in
this study to test for ESBL production. ESBL production was very common among Klebsiella
pneumoniae, where out of the 40 Klebsiella pneumoniae isolates, 24 (60%) were ESBL producers.
However, within E coli isolates, 24 out of 46 isolates were ESBL-producers, representing (52.2)%.
TABLE(2):.
Demographics and specimen types from patients who had infections due to ESBL-producing E. coli or
K. pneumonia:
Parameter
Value
% of ESBL oroducing organisms
Chi-square
E. coli
(n =24)
K.
pneumoniae
(n = 24)
Total
ESBL
(n=48)
X2
P-value
N
%
N
%
N
%
Age group (yr)
<20
4
16.67
3
12.50
7
14.58
0.932
0.988
21-40
7
29.17
8
33.33
15
31.25
41-60
8
33.33
10
41.67
18
37.50
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
Blood
Urine
Sputum
other
Total
E.coli
Klebsiella pneumoniae

>60
5
20.83
3
12.50
8
16.67
Sex
Male
10
41.67
13
54.17
23
47.92
2.109
0.348
Female
14
58.33
11
45.83
15
31.25
Hospital unit
Medicine
3
12.50
4
16.67
7
14.58
2.175
0.975
Surgery
4
16.67
2
8.33
6
12.50
ICU
15
62.50
16
66.67
31
64.58
Outpatient
0
0.00
1
4.17
1
2.08
Other
2
8.33
1
4.17
3
6.25
Specimen type
Blood
6
25.00
4
16.67
10
20.83
12.554
0.05*
Sputum
0
0.00
8
33.33
8
16.67
Urine
14
58.33
12
50.00
26
54.17
Other
4
16.67
0
0.00
4
8.33
Out of 48 ESBL producing strains of E coli and Klebsiella pneumoniae 18 cases are present in age
group 41-60 years (37.5%) , male to female ratio is 23/25 , ICU admission constitutes 31 /48 (64.6%)
and the urine constitute 26/48 (54.2%).
Susceptibility tests To other antibiotics were performed and the results show that All strains were
susceptible to impenum and meropenum ( 100%) , 95% of strains were suscptable to tygacillin , 79%
were susceptible to Amikacin , 58% were susceptible to pipracillin /tazocin and 31.6% were susceptible
to ciprofloxacin while only 5% of strains were susceptible to gentamycin.
-:3Table
Risk factors associated with ESBL infection on univariate analysis:-
ESBL
Total isolets
P-
value
Odd
ratio
CI 95%
N
%
N
%
Value
L
U
Antibiotic
exposure in last 1
month
NO
8
16.67
39
45.35
0.001
4.1489
1.738
9.90
Yes
40
83.33
47
54.65
Use of urinary
catheter
NO
13
27.08
50
58.14
0.0011
3.739
1.736
8.054
Yes
35
72.92
36
41.86
Use of intravenous
devices
NO
6
12.50
28
32.56
0.013
0.306
0.117
0.804
Yes
42
87.50
60
69.77
Use of ventilator
NO
20
41.67
46
53.49
0.211
0.621
0.304
1.268
Yes
28
58.33
40
46.51
Renal insuffi
ciency
(creatinine >2
mg/dl)
NO
22
45.83
58
67.44
0.0237
2.4481
1.1855
.0552
Yes
26
54.17
28
32.56
Admission in
intensive care unit
NO
17
35.42
46
53.49
0.049
0.477
0.230
0.987
Yes
31
64.58
40
46.51
Diabetes mellitus
NO
37
77.08
70
81.40
0.654
0.769
0.324
1.826
Yes
11
22.92
16
18.60
Corticosteroid use
NO
39
81.25
71
82.56
1.000
0.915
0.367
2.284
Yes
9
18.75
15
17.44
Malignancy
NO
38
79.17
70
81.40
0.821
0.869
0.359
2.101

Yes
10
20.83
16
18.60
Various risk factors were analyzed to determine their association with infection by ESBL producing
organisms. Inpatient use of intravenous device, antibiotic therapy in last 1 month, ICU admission and
renal insufficiency (creatinine >2 mg/dl) were significantly associated with infections by ESBL producing
isolates on univariate analysis.
-:4Table
Extended spectrum β-lactamase (ESBL) genotypes in E. coli and K. pneumoniae strains:
E. coli
K.pneumoniae
Chi-square
N
%
N
%
X2
P-value
A. Two or more ESBL
genes
12
57.14
9
50.00
0.383
0.536
blaTEM +blaSHV + blaCTX-M
3
14.29
3
16.67
0.020
0.888
blaTEM +blaCTX-M
6
28.57
4
22.22
0.296
0.587
blaTEM +blaSHV
1
4.76
0
0.00
0.924
0.336
blaSHV +blaCTX-M
2
9.52
2
11.11
0.012
0.911
B. Single ESBL gene
8
38.10
9
50.00
0.383
0.536
blaTEM only
1
4.76
4
22.22
2.459
0.117
blaSHV only
0
0.00
2
11.11
2.346
0.126
blaCTX-M only
7
33.33
3
16.67
1.642
0.200
Chi-square
X2
37.67
24.42
P-value
0.001*
0.002*
DNA isolated from 48 ESBL producing organisms (phenotypic confirmed) were subjected to PCR using
TEM, SHV and CTX-M specific primers. DNA from 38 (79.2%) isolates could be amplified either for
blaTEM, blaSHV , or blaCTX-M. Two or more genes for ESBL were present in 28/38 (55.3%) ESBL
typeable isolates, blaTEM + blaCTX-M being the most common combination (26.4%) followed by
blaTEM +blaSHV + blaCTX-M (15.8%), blaSHV + blaCTX-M (10.5%) and blaTEM + blaSHV (2.6%).
Among the isolates harbouring single ESBL gene, blaCTX-M was present in 26.3 % , blaTEM in 13.2 %

and blaSHV in 5.2% . Overall, blaCTX-M, blaTEM and blaSHV was detected in 78.9 % (30/38), 57.9 %
(22/38) and 34.2 % (13/38) isolates either alone or in combination .
Figure (3)
Detection of bla SHV , TEM and CTX-M by PCR
Discussion:
The spread of ESBL-producing bacteria has been strikingly rapid worldwide, indicating that continuous
monitoring systems and effective infection control measures are absolutely required (13). In our study
ESBLs were detected in 60 % in K. pneumoniae isolets and 52.2 % in E. coli isolates (overall 50 %).
Other studies had reported a high prevalence of ESBL production ranging from 41.0 to 63.6 per cent in
E. coli and 40 to 83.3 per cent in K. pneumonia (14, 15). In the West, ESBL production in
Enterobacteriaceae was less frequent varied from 5 to 52 per cent and in Asian countries from 10 to 46.5
% (16). 20% in Italian study (17), 39.5% in the Chinese study [18], and 13.3% in the Kuwaiti study (19).
Youssef et al., in 1999 (20) reported that 38% of Klebsiella pneumoniae isolates were ESBL producers
However, our results are nearly matching Jordanian study which reported that 70% of the K. pneumoniae
isolates recovered from the ICU of Jordan University Hospital to be ESBL producers (21). The high
percentage of ESBL production in our hospital may indicate the nature of patients being served as our
hospital is serving debilitated patients, and contain high percentage of patients with malignancy and
chronic diseases. Such findings impose the need for applying specific infection control measures to
eliminate this organism .

Our results showed that a majority of patients were elderly (41-60years) 18/48(37.5%) . Urine
constituted the major source of ESBL producing strains 26/48 . It was not surprised that females had a
higher prevalence of infection due to ESBL producers than males, since females are more vulnerable to
urinary tract infection. ESBL-producing K. pneumoniae appeared to be an important cause of infection
among patients in intensive-care units. nearly same results are obtained from Kiratisin P et al , 2008 (12)
and Goyal A et al 2009 (22).
Using specific primers for TEM, SHV and CTX-M, only 38 (79.2%) of 48 ESBL isolates could be
typed for one or more genes. The negative amplification in the remaining isolates may be due to presence
of other ESBL genes, which we did not explore further. Two or more ESBL genes were present in 55.3 %
of the typeable isolates. Among 38 ESBL- typeable isolates, 55.3% carried several bla genes, which can
probably account for a high-level β-lactam-resistant phenotype.(12).
The blaSHV gene was only found alone in 5.2% (n=2/38) of ESBL-producing isolates. Similar results were
obtained by El Agammy et al , 2009 ( 23) where The blaSHV gene was only found alone in 6.8%
(n=15/220) of ESBL-producing isolates . The presence of SHV β-lactamase alone suggests that these
SHV genes are responsible for resistance to extended-spectrum cephalosporins in 5.2 % of ESBL-
producing isolates . However, the blaSHV gene was found to be associated with the blaTEM gene and the
blaCTX_M gene in 15.8% (n=6/38) . On the other hand, SHV β-lactamase was present with the CTX-M
enzyme in 10. 5% (n=4/38) of ESBL-producers similarly Goyal et al 2009 and El Agammy et al 2009
(22,23 ) report near results (11%) and ( 9.1% ) respectively. However, the SHV β-lactamase gene was
present with TEM in 2.6% only .
The blaTEM gene was only found alone in 13.2% (n=5/38) of ESBL-producing isolates. Similar results
were obtained by Goyal A et al ,2009 (22) where The blaTEM gene was only found alone in 8.5% (n=7/82)
of ESBL-producing isolates . However, the blaTEM gene was found to be associated with the blaCTX-M
gene in 26.4%(n=10/38) and in association with the blaSHV gene and the blaCTX_M gene in 15.8% (n=6/38)
compared to 28.1% and 15.9% reported by the Indian study (22) .From this result either SHV or TEM or
both are the ESBLs in 10 of 38 isolates producing ESBL(26.3%).
The blaCTX-M was the most common and was present either alone or in combination with other ESBL
type(s) in 78.9 %. Our findings support the hypothesis that CTX-M is emerging as the dominant ESBL
type in clinical isolates (24 ref 3pap1). Pournaras et al (24) reported 87 % prevalence of CTX-M enzyme
among ESBL producers in a tertiary care hospital of Greek. In a multi-centric study from Russia, CTX-M
gene was reported in 35.9 % of E. coli and 34.9 % of K. pneumonia ESBL strains (25). Brenwald et al
(26) reported an outbreak of CTX-M harbouring ESBL in UK. In a nationwide survey in Italy, CTX-M
producing strain were reported by, 10 of the 11 participating centers, with remarkably variable rates
among the centers (1.2 to 49.5% of the ESBL producers) (27).

TEM- and SHV-ESBL are derived from parental TEM-1 and SHV-1 by point mutations. TEM-1 and
SHV-1 are non-ESBL; however CTX-M enzymes are not derived from non ESBL and consequently all
CTX-M enzymes are ESBL (28). In this study, we did not determine which β-lactamase was responsible
for resistance to extended-spectrum cephalosporins because these genes were not sequenced. The DNA
sequence of these genes must be done to know the type of β-lactamase gene and the prevalence of ESBL
genes in ESBL-producing isolates .
Resistance to β-lactams, especially third-generation cephalosporins and non-β-lactams, among clinical
isolates of gram-negative bacteria is increasing worldwide (29,30). The therapeutic options for infections
due to ESBL producers have also become increasingly limited. (31,32) . In our study all ESBL
producing isolates were susceptible to impenum and meropenum ( 100%) , 95% of strains were
suscptable to tygacillin 79% were susceptible to Amikacin , 58% were susceptible to pipracillin /tazocin
and 31.6% were susceptible to ciprofloxacin while only 5% of strains were susceptible to gentamycin.
An Italian study,(33) showed that carbapenems were active against all ESBL-positive enterobacteria and
susceptibility to other drugs was as follows: amikacin 84.7 % piperacillin-tazobactam 84.4 %, gentamicin
48.0 %, and ciprofloxacin 32.8%. In Saudi Arabian study (34) the overall resistance rate of ESBL-
producing K pneumoniae isolates studied was alarmingly high to most antibiotics tested including
gentamicin, amikacin, cefepime, and trimethoprim/sulfamethoxazole. Imipenem followed by
ciprofloxacin were the antibiotics most active against ESBL-producers. K pneumoniae isolates harboring
ESBLs were more often resistant to flouroquinolones (35,36). In that study only 11.3% of ESBL-
producing K pneumoniae were resistant to ciprofloxacin.
High resistance to other non β-lactam antibiotics by ESBL producing strains poses a threat to treatment
failure by these drugs and also minimizes the therapeutic choice to carbapenem .
Various risk factors were also analyzed to determine their association with infection by ESBL producing
organisms. Inpatient use of intravenous device, use of urinary catheter, antibiotic therapy in last 1 month,
ICU admission and renal insufficiency (creatinine >2 mg/dl) were significantly associated with infections
by ESBL producing isolates on univariate analysis. ESBL infection was more frequent in patients who
required ventilator support; however, the difference was not significant. Significant association with prior
antibiotic therapy supports the hypothesis that selection pressure related to overuse of broad spectrum
antibiotics, especially third generation cephalosporins play important role for emergence of high level
resistance in the family Enterobacteriaceae. Graffunder et al (10) found that use of third generation
cephalosporins aminoglycosides and trimethoprim/ sulphamethoxazole were independently associated
with infections by ESBL producing strains and patients on prolonged use of ventilator were at the greatest
risk of having an ESBL organism. Several other studies had also shown strong association between

infection with ESBL-producing E. coli orK. pneumoniae and antibiotic use (14,13) . Reduction in use of
ceftazidime or all cephalosporins decreased the occurrence of infection by ESBL producing strains (10)
Conclusion
1-The blaCTX-M was the most common and was present either alone or in combination with other ESBL
type(s) in 78.9 %. Our findings support the hypothesis that CTX-M is emerging as the dominant ESBL
type in clinical isolates.
2- High resistance to other non β-lactam antibiotics by ESBL producing strains poses a threat to treatment
failure by these drugs and also minimizes the therapeutic choice to carbapenem .
3-The high percentage of ESBL production in our hospital may indicate the nature of patients being
served as our hospital is serving debilitated patients, and contain high percentage of patients with
malignancy and chronic diseases.
4- Inpatient use of intravenous device, use of urinary catheter, antibiotic therapy in last 1 month, ICU
admission and renal insufficiency (creatinine >2 mg/dl) were significantly associated with infections by
ESBL producing isolates on univariate analysis.
5- Significant association with prior antibiotic therapy supports the hypothesis that selection pressure
related to overuse of broad spectrum antibiotics, especially third generation cephalosporins play important
role for emergence of high level resistance in the family Enterobacteriaceae.
Recommendations :
1-In this study, we did not determine which β-lactamase was responsible for resistance to extended-
spectrum cephalosporins because these genes were not sequenced. The DNA sequence of these genes
must be done to know the type of β-lactamase gene and the prevalence of ESBL genes in ESBL-
producing isolates .
2- The high percentage of ESBL production in our hospital impose the need for applying specific
infection control measures to eliminate this organism.
3-Using specific primers for TEM, SHV and CTX-M, only 38 (79.2%) of 48 ESBL isolates could be
typed for one or more genes. The negative amplification in the remaining isolates may be due to presence
of other ESBL genes, which we did not explore further. More extended studies is recommended to detect
other genes responsible for ESBL production.
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