BEST: International Journal of Humanities, Arts,

Medicine and Sciences (BEST: IJHAMS)

ISSN 2348-0521
Vol. 2, Issue 11, Nov 2014, 23-30
BEST Journals

PREVALENCE OFEXTENDED SPECTRUM BETA LACTAMASE IN CLINICAL

ISOLATES OF ESCHERICHIA COLI FROM SOME HOSPITALS IN
ALNASSIRIYAH CITY, THI QAR, IRAQ
INTIDHAARN .ABID & AHMED N. FAYAD
Department of Pathological Analysis, College of Science, Thi-Qar University, Thi Qar, Iraq

ABSTRACT
A total of 50 isolates of Escherichia coli collected from clinical specimens obtained from some hospitals in
Al Nassiriyah city, Thi Qar, Iraq. These isolates were tested for production of extended spectrum -lactamases
(ESBLs) by two ESBL phenotypic tests (ESBL screening test and confirmatory test (mDDST)). Out of 50 Escherichia coli
isolates34 (68%) and 30(60%) were positive ESBLs screening test for cefotaxime and ceftazidime respectively by using
disk diffusion method, 31(62%) and 36(72%) isolates were positive for cefotaxime and ceftazidime respectively by using
broth dilution method (MIC2g). 25(50%) of isolates were positive for ESBL by using modified confirmatory test
(mDDST)

with

extended

spectrum

cephalosporins

(ESCs)

and

disk

of

Amc

(AmoxicillinClavulanate).

All ESBL pruducers isolates (100%) were sensitive to imipenem. ESBL producers showed a high degree resistance to
cephalexin

(60%),

co-trimethaxazole

(80%),

cephalothin

(92%),

tetracycline

(92%),

cefixine

(100%)

and ampicillin (100%).

KEYWORDS: Cefotaxime and Ceftazidime, Monobactams

INTRODUCTION
Many bacteria become resistant to beta lactam antibiotics such as penicillins, monobactams, carbapenems
and cephalosporins by producing enzymes that destroy or inactivate the antibiotics, these enzymes are called lactamases
that break the beta lactam ring of the antibiotic, thus destroying the drug (1) lactamases have been found in a large
variety of enterobacteria (2). During the past 2 decades, broad spectrum cephalosporins, including oxymino- lactam
antibiotics, have been used worldwide, and antibiotic resistant strains that produce extended spectrum
lactamases (ESBLs) have emerged among the enterobacteriaceae, predominantly in E.coli (3). These bacteria are most
commonly pathogens that causing nosocomial infections (4), and community onset infections with ESBL producing
Escherichia coli are a major clinical concern in many countries (5). Infections by ESBL producers have been associated
with severe adverse clinical outcomes that have led to increased mortality, prolonged hospitalization, and rising medical
costs. Those a dverse outcomes have also been related, at least in part, to a delay in the administration of an effective
therapy (6,7). Because, the increase of infections that caused by E.coli, and they have resistance to lactam antibiotics in
our hospitals, the present study was carried out to estimate the presence of ESBLs in E..coli by phenotypic methods,
and evaluate treatment outcomes of some infections due to these bacteria to determine the appropriate use of antibiotics for
these infections.

MATERIALS AND METHODS

Specimens Collections
A total of 578 various clinical specimens (329 urine, 5 pus, 18 CSF, 38 burn swabs, 40 sputum, 32 pretonial fluid,
116 blood) were collected from out patients and hospitalized patients in each (Al- Hussein and Maternity & Children)

24

Intidhaarn .Abid & Ahmed N. Fayad

hospitals in Al- Nassiriyah city during the period from June 2011 to February 2012. MacCokey, s agar (Himedia, India)
was used to culture the specimens, and all isolates were identified using the API 20 E system (Bio Merieux, France).
Susceptibility Test
Antibiotic susceptibility testing was performed by the Kirby Bauer method on Mueller Hinton agar
(BD & BBL, USA) according to CLSI recommendations (8) The antibiotics ((symbol)(ing)) were a
moxicillin-clavulanate* ((AMC)(20/10)), cefotaxime** ((CTX)(30)), ceftazidime *((CAZ)(30)), imipenem * ((IPM)(10)),
gentamycin** ((CN)(10)), amikacin** ((AK)(30)), cephalothin * ((CF)(30)), trimethoprim** ((TR)(5)), nalidixic acid**
((NA)(30)), cefixime ** ((CFM)(30)), cephalexin** ((CL)(30)), cotrimethaxazole ** ((CO)(1.25-23.75)) ciprofloxacin**
((CIP)(5)), amipicillin* ((AM)(10)) and tetracycline** ((TE)(30)), cefepime ** ((CFP)(30)), ceftriaxone ** ((CRO)(30)),
aztreonam* ((ATM)(30)).
*:BD&BBL, USA
**:Bioanalyse, Turkey
ESBL Screening Testing
Disk diffusion tests for ceftazidime and cefotaxime were performed according to CLSI recommendations (9).
MICs were determined by the broth microdilution method, as described by NCCLS (10). An isolate was considered
positive for ESBL if the MIC for ceftazidime or cefotaxime was 2 g/ ml, if the inhibition zone for ceftazidime was
22mm, or the inhibition zone for cefotaxime was 27 mm(9).
ESBL Confirmatory Testing
It was performed using a modified double disk synergy test (mDDST)(11), by placing disks of ceftazidime,
cefotaxime, ceftriaxone, aztreonam and cefepime (30 g each) at distances of 20 mm (center to center) from a disk
containing amoxicillin -clavulanate (20 g /10 g).

RESULTS
E.coli isolated from different specimens, were pus 2(40%), burn swab 5 (13.15%), CSF 2(11.1%), urine
37(10.33%), pretonial fluid 2(6.25%), sputum 2(5%). The results were appeared, E.coli not registered any presence for it in
blood specimens (Table 1).
Table 1: Numbers & Percentages of E.coli Isolates from Different Clinical Specimens
Type of
Specimens
Urine
Pus
CSF
Burns swabs
Sputum
Peritoneal fluid
Blood
Total

No. of
Specimens
329
5
18
38
40
32
116
578

No. & (%) of

E.coli Isolates
37(11.24)
2(40)
2 (11.1)
5(13.15)
2(5)
2(6.25)
0(0)
50(8.65)

Table 2 shows the numbers and percentages of isolates with the extended spectrum lactamase. 34(68%)
isolates of E.coli had a positive ESBLs screening test for cefotaxime by using disk diffusion method, while 30(60%)
isolates of E.coli were positive ESBLs screening test for ceftazidime, and 31(62%) and 36(72%) isolates were positive for
ceftazidime and cefotaxime respectively by broth dilution method (MIC 2g/ml). Confirmatory test of ESBLs (mDDST)

Prevalence Ofextended Spectrum Beta Lactamase in clinical Isolates of

Escherichia coli from some Hospitals in AlNassiriyah City, Thi Qar, Iraq

25

with extended spectrum cephalosporins, aztreonam and disk of amoxicillin clavulanate was positive for 25(50%) isolates
of E.coli (Table2)
Table 2: Numbers & Percentages of Isolates with the ESBL
No.& Percentages of Positive Isolates (N=50)
ESBLs Screening Test
Cefotaximea
34(68%)
Cefotaximeb
36(72%)
Ceftazidimec
30(60%)
d
Ceftazidime
31(62%)
ESBLs confirmatory test(mDDST)
25(50%)
a=Disk diffusion (inhibition zone 27mm)
b,d=Broth dilution (MIC 2g/ ml), c=Disk diffusion (inhibition zone 22mm)
Table 3 was appeared the antibiotic susceptibility of ESBLs producing isolates (n=25). The results showed that all
isolates (100%) of E.coli which producing ESBLs were founded to be sensitive to imipenem. The resistance of
gentamycin, amikacin, trimethoprim, nalidixic acid and ciprofloxacin was 20%, 12%, 30%, 8% and 28% respectively. The
high rate of resistance was observed in cephalexin, co- trimethaxazole, cephalothin, tetracycline, cefixime and ampicillin,
that registered 60%, 80%, 92%, 92%, 100% and 100% respectively.
Table 3: Antibiotic Susceptibility of ESBLS Producing Isolates (N=25)
No. &(%)of
Resistant Isolates

Antibiotics
Imipenem
Gentamycin
Amikacin
Cephalothin
Trimethoprim
Nalidix acid
Cefixime
Cephalexin
Co-trimethaxazole
Ciprofloxacin
Amipicillin
Tetracycline

5(20%)
3 (12%)
23(92%)
9(36%)
2(8%)
25(100%)
15(60%)
20(80%)
7(28%)
25(100%)
23(92%)

No. &(%)of
Sensitive Isolates
25(100%)
20(80%)
22(88%)
2(8%)
16(64%)
23(92%)
10(40%)
5(20%)
18(72%)
2(8%)

All (100%) extended spectrum lactamase producing isolates (n=25) were obtained from the specimens of urine
(Table 4).
Table 4: Distribution of ESBLS Producing Isolates on Different Specimens
Type of
Specimens
Urine
Pus
CSF
Burns swabs
Sputum
Peritoneal fluid
Blood
Total

No. of
E.Coliisolates
37
2
2
5
2
2
0
50

No.& (%) of ESBLS

Producing Isolates
25(100)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
25(50%)

26

Intidhaarn .Abid & Ahmed N. Fayad

DISCUSSIONS
E.coli is acommon hospital pathogen (12), although most strains of E.coli bacteria are harmless and live in the
intestine of healthy humans, several strains can produce powerful toxins and cause severe in humans. E.coli is capable of
causing wide variety of disease from urinary tract infection to meningitis (13). In this study, the results were appeared
that, the total infections rate of E.coli was 8.65% from total clinical specimens, and the results were indicated to presence
of E.coli in different percentages for varies specimens except blood specimens, which was appeared none presence of
bacteria. E.coli is a multipotent pathogen that has evolved the ability to cause disease in several body systems, at least in
the bowel, there are several different mechanisms of pathogenesis (14), these results are similar with results of study in
Pakistan (15) they isolated 121 E.coli samples from different clinical specimens were included urine, pus, cerebrospinal
fluid, sputum, tracheal secretions and catheter tips of hospitalized patients and out patients at the Pakistan institute of
medical science. ESBLs are increasingly being reported in hospital acquired infections and community associated
infections caused by Esherichia coli that produce CTX-M (16). The emergence of numerous CTX-M lactamase
producing strains, has changed thesusceptibility of these organisms, these enzymes confer resistance to penicillinsand
extended spectrum cephalosporins (i.e., MICs increase more for cefotaxime than for ceftazidime (17). The results of this
study showed a high frequency of ESBLs in the isolates of E.coli that examined by phenotype ESBLs screening test,
and the number of isolates had a positive screening test for cefotaxime by the disk diffusion were more than that for
ceftazidinethis may be result from isolates have CTX-enzymes, most of the CTX-M enzymes are much more active against
cefotaxime and ceftriaxone than against ceftazidime (18). ESBLs producers of our isolates had MIC >2g / ml to
ceftazidime and cefotaxime, this may be result from had high level of ESBLs in our isolates which caused of resistance to
both the drugs.
Confirmatory test was performed using a modified double disk synergy test (mDDST), this method revealed
that 25(50%) isolates were ESBL producers, these results are similar to results of (19), they found high occurrence of
ESBLs in E.coli (63.6%), isolates which shows an enhancement of the zone of inhibition on the augmentin
(amoxicillin clavulanic acid ) side of disc as compared to that which is seen on the side without augmentin were
confirmed as ESBL producers (20).
Jarlier, s DDST was modified by including a disk with cefepime and by simultaneous use of a disks with
cephalosorins and clavulanic acid (11). The use of cefepime increased the sensitivity of the DDST with ESCs for the
detection of ESBLs in enterobacters from 16 to 61%. when the disks were applied at the standard distance of 30 mm from
clavulanate and from 71 to 90% with the closer application of the disks. The greater efficacy of cefepime and cefpirome
instead of ESCs by ESBL detection methods on the basis of the inhibitory effect of clavulanic acid is expected, since the
former, but not the latter, retain activity against derepressed variant of enterobacters (21), our results revealed the numbers
of isolates that positive for ESBL by screening test of ESBL phenotype were more than that positive for ESBL by
confirmatory test (mDDST), these results may be due to production another enzymes of - lactamase that called
AmpC lactamase, either mediated by chromosomal depression or transferred by a plasmid for the Enterobacteriaceae
isolates carrying both the ESBL and AmpC enzymes, the phenotype appeared to be appositive screening test and negative
confirmatory test.
The AmpC enzyme can hydrolyze clavulanic acid, and thus make the confirmatory test negative (22).
All our isolates of E.coli that producing ESBL were sensitive to imipenem, these findings are in accordance with the
results of Italian study which appeared, the carbapenems were active against all ESBL producing enterobacteria(23).
Kolar et al. (2010) observed that 55.6% of ESBL producing E.coli isolates were resistant to tetracycline (24).

Prevalence Ofextended Spectrum Beta Lactamase in clinical Isolates of

Escherichia coli from some Hospitals in AlNassiriyah City, Thi Qar, Iraq

27

These results are in accordance with our study that appeared, resistance of ESBL producing E.coli isolates to tetracycline
was 92%. Our isolates also were appeared high resistance to all - lactam antibiotics that used in this study such as
cephalexin, cephalothin, cefixime and ampicillin. The NCCLs guide recommends that ESBL producers be reported as
resistant to all pencillins and cephalosporins as well as to aztreonam, even when they are shown to be susceptible to these
agents by conventional tests (25). We also noted in our results, the high resistance to co- trimethaxazle, conjugation
experiments demonstrated that resistance of ESBL producers to otherdrugs is associated with the presence of a conjugative
plasmid that confers resistance to these drugs such as co- trimethaxazole and aminoglycosides in addition to the ESBL(26).
The results in this study showed that the all ESBL producing isolates were from urine specimens, urinary tract infections
are extremely prevalent and not so simple to eradicate, although prophylactic therapies to decrease stone formation,
adherence of E.coli, and colonization with multidrug resistant strains might increase in importance in this new era of high
level drug resistance (27). In conclusion, this study demonstrates that a high incidence of ESBLs phenotype among E.coli
isolated in our hospitals, clinical attention must be focused on the ESBLs producers in our hospitals and highlighted the
need for using a correct phenotypic test to identify ESBL to selection proper antibiotics in the treatment of clinical
infection.

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