Multidrug resistant AmpC β-lactamase producing Escherichia coli isolated from a paediatric hospital

Abstract

Objective
: The objective of the study was to observe the antimicrobial resistance of AmpC β-lactamase producing E. coli.
Methods: Six hundred and seventy E. coli were isolated from 20,257 various pathological samples collected from The Children’s Hospital and Institute of Child Health, Lahore, Pakistan. The isolates showed resistance to ceftazidime which were further examined for AmpC β-lactamase activity by Disc Potentiation method.
Results: There were 670 isolates of E. coli out of which 85 (12.6%) were AmpC β-lactamase producers. Risk factors like intravenous line (76.5%), endotracheal tube (22.4%), surgery (12.9%) and urinary catheters (7.1%) were found to be associated with infection caused by AmpC β-lactamase producing E. coli. Antimicrobial resistance pattern revealed that AmpC producing E. coli were highly resistant to co-amoxiclav, ceftazidime, cefotaxime, cefuroxime, cefixime, ceftriaxone and cefoxitin (100% each). Least resistance was observed against sulbactam-cefoperazone (14.1%), cefepime (7.1%), piperacillin-tazobactam (5.9%) and none of the isolates were resistant to imipenem and meropenem.
Conclusion: The minimum use of invasive devices and strict antibiotic policies can reduce the spread of AmpC β-lactamase producing E. coli.

Full text

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Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk 181
INTRODUCTION
β-lactam antibiotics account for approximately
50% of global antibiotic consumption which
has considerably increased the resistance in
Gram negative bacteria.
1
AmpC β-lactamase
production is one of the commonest causes of
resistance to β-lactam antibiotics among Gram-
negative bacteria. AmpC β-lactamases are
resistant to aminopenicillins, carboxypenicillins,
ureidopenicillins, cephalosporins, broad as well as
extended spectrum cephalosporins (cephamycin)
and monobactams (aztreonam).
2,3
AmpC
β-lactamases are resistant to β-lactamase inhibitors
like clavulanic acid.
4
E. coli is a major organism among normal ora
and it causes a wide variety of intestinal and extra-
intestinal diseases, such as diarrhea, urinary tract
infections, septicemia and neonatal meningitis.
5
It
is resistant to a wide variety of clinically important
antibiotics due to production of AmpC β-lactamase
enzyme.
6
Most of the risk factors of AmpC
producing E. coli infections include prolonged
hospital and intensive care unit stay, use of urinary,
arterial or venous catheters, ventilator assistance,
1. Noor-ul-Ain Jameel, (M.Phil),
2. Hasan Ejaz, (M.Phil),
3. Aizza Zafar, (M.Phil),
4. Hafsa Amin, (M.Phil),
1, 4: Institute of Molecular Biology and Biotechnology,
The University of Lahore, Lahore, Pakistan.
2, 3: Department of Microbiology,
The Children’s Hospital and Institute of Child Health,
Lahore, Pakistan.
Correspondence:
Hasan Ejaz,
Department of Microbiology,
The Children’s Hospital & Institute of Child Health,
Lahore, Pakistan.
E-mail: hasanmicro@gmail.com
* Received for Publication: July 3, 2013
* Revision Received: November 1, 2013
* Revision Accepted: November 6, 2013
Original Article
Multidrug resistant AmpC β-lactamase producing
Escherichia coli isolated from a paediatric hospital
Noor-ul-Ain Jameel
1
, Hasan Ejaz
2
, Aizza Zafar
3
, Hafsa Amin
4
ABSTRACT
Objective: The objective of the study was to observe the antimicrobial resistance of AmpC β-lactamase
producing E. coli.
Methods: Six hundred and seventy E. coli were isolated from 20,257 various pathological samples collected
from The Children’s Hospital and Institute of Child Health, Lahore, Pakistan. The isolates showed resistance
to ceftazidime which were further examined for AmpC β-lactamase activity by Disc Potentiation method.
Results: There were 670 isolates of E. coli out of which 85 (12.6%) were AmpC β-lactamase producers.
Risk factors like intravenous line (76.5%), endotracheal tube (22.4%), surgery (12.9%) and urinary
catheters (7.1%) were found to be associated with infection caused by AmpC β-lactamase producing E.
coli. Antimicrobial resistance pattern revealed that AmpC producing E. coli were highly resistant to co-
amoxiclav, ceftazidime, cefotaxime, cefuroxime, cexime, ceftriaxone and cefoxitin (100% each). Least
resistance was observed against sulbactam-cefoperazone (14.1%), cefepime (7.1%), piperacillin-tazobactam
(5.9%) and none of the isolates were resistant to imipenem and meropenem.
Conclusion: The minimum use of invasive devices and strict antibiotic policies can reduce the spread of AmpC
β-lactamase producing E. coli.
KEY WORDS: E. coli, AmpC β-lactamase, Antimicrobial resistance, Multidrug resistant E. coli.
doi: http://dx.doi.org/10.12669/pjms.301.4045
How to cite this:
Jameel NA, Ejaz H, Zafar A, Amin H. Multidrug resistant AmpC β-lactamase producing Escherichia coli isolated from a paediatric
hospital. Pak J Med Sci 2014;30(1):181-184. doi: http://dx.doi.org/10.12669/pjms.301.4045
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

182 Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk
Noor-ul-Ain Jameel et al.
haemodialysis, emergency abdominal surgeries,
use of naso gastric tube and prior use of β-lactamase
antibiotic.
7,8
Clinical isolates of AmpC β-lactamase producing
E. coli and their antimicrobial resistance have been
described from different parts of the world.
9-11
However, there are only few studies from Pakistan,
which have systematically reported the role of
various interventions and antimicrobial resistance
of AmpC β-lactamase producing E. coli. This
study was undertaken to assess the risk factors
and antimicrobial resistance pattern of such E. coli
isolated from paediatric patients.
METHODS
This study was conducted at Microbiology
Department of The Children’s Hospital and
Institute of Child Health Lahore, Pakistan, during
March 2011 to February 2012. A total number of 670
E. coli strains were isolated from various clinical
specimens such as blood, pus, urine, sputum,
tracheal secretions and various tips. The isolates
were identied as E. coli by colonial morphology,
Gram’s stain, catalase test, oxidase test and API 20E
system (bioMerieux, France).
12
Isolates were screened for AmpC β-lactamase
production by disc diffusion method as described
by Clinical Laboratory Standards Institute (CLSI).
13
The E. coli which showed reduced susceptibility
to ceftazidime and cefotaxime were selected for
further conrmation by Disc Potentiation method
using 3-amino phenyl boronic acid (APB).
14
A suspension of each isolated AmpC β-lactamase
producing E. coli was made according to the 0.5
McFarland turbidity standard and antimicrobial
susceptibility testing was performed using two
plates on Mueller Hinton agar (90mm) for each
strain. The antibiotic discs of amikacin (30 µg),
aztreonam (30 µg), cefepime (30µg), cexime (5 µg),
cefotaxime (30 µg), cefoxitin (30 µg), cefpodoxime
(30 µg), ceftazidime (30 µg), cefuroxime (30 µg),
ciprooxacin (5 µg), co-amoxiclav (20/10 µg),
co-trimoxazole (1.25/23.75 µg), gentamycin
(10 µg), meropenem (10 µg), imipenem (10 µg),
piperacillin-tazobactam (100/10 µg) and sulbactam-
cefoperazone (75/30 µg) were placed on Mueller-
Hinton agar plates and incubated overnight at
37
o
C. After overnight incubation the diameter of
each zone of inhibition was measured in mm. The
antimicrobial susceptibility testing results were
noted according to the CLSI guidelines.
13
The clinical record of each patient was reviewed.
The patients were assessed for the various
interventions like intravenous line, endotracheal
tube, surgery, peritoneal dialysis catheters, nasal
gastric tube, urinary catheters and central venous
pressure line.
RESULTS
During the study period, 20,257 clinical samples
were processed for isolation of AmpC β-lactamase
producing E. coli. Out of 670 E. coli isolated from
these samples, there were 85 (12.6%) AmpC
β-lactamase producers.
The 85 patients infected with AmpC producing E.
coli had undergone through various interventions
during hospitalization as shown in Table-I. These
interventions included intravenous lines 65 (76.5%),
endotracheal tubes 19 (22.4%), surgeries 11 (12.9%),
peritoneal dialysis catheters 8 (9.4%), naso gastric
tubes 6 (7.1%) and central venous pressure lines 2
(2.4%).
Table-II: Antimicrobial resistance of
AmpC β-lactamase producing E. coli.
Antibiotics Resistant n (%)
Co-amoxiclav (20/10µg) 85 (100)
Ceftazidime (30µg) 85 (100)
Ceftriaxone (30µg) 85 (100)
Cefotaxime (30µg) 85 (100)
Cexime (5µg) 85 (100)
Cefuroxime (30µg) 85 (100)
Cefoxitin 85 (100)
Co-trimoxazole (1.25/23.75µg) 78 (91.8)
Cefpodoxime (30µg) 74 (87.1)
Aztreonam (30µg) 59 (69.4)
Gentamicin (10µg) 53 (62.4)
Amikacin (30µg) 52 (61.2)
Ciprooxacin (5µg) 29 (34.1)
Sulbactam-cefoperazone (75/30µg) 12 (14.1)
Cefepime (30µg) 6 (7.1)
Piperacillin-tazobactam (100/10µg) 5 (5.9)
Imipenem (10µg) 0 (0)
Meropenem (10µg) 0 (0)
Table-I: Various interventions among
AmpC positive E. coli patients (n=85).
Interventions AmpC positive E. coli
n %
Intravenous line 65 76.5
Endotracheal tube 19 22.4
Surgery 11 12.9
Peritoneal dialysis catheter 8 9.4
Naso gastric tubes 6 7.1
Urinary catheters 6 7.1
Central venous pressure line 2 2.4

Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk 183
All the 85 (100%) AmpC producing E. coli were
resistant to co-amoxiclav, ceftazidime, cefotaxime,
cefuroxime, cexime, ceftriaxone and cefoxitin.
AmpC producing E. coli showed less resistance
to sulbactam-cefoperazone 12 (14.1%), cefepime
6 (7.1%) and piperacillin-tazobactam 5 (5.9%).
None of the isolates were found to be resistant to
imipenem and meropenem (Table-II).
DISCUSSION
The emergence of resistance to the third
generation cephalosporins in Gram negative
bacteria is a major concern which is mostly caused
by AmpC β-lactamase. It is difcult to treat
multidrug resistant AmpC β-lactamase producing
E. coli. High frequency of AmpC β-lactamase
producing E. coli and their resistance to antibiotic
has been reported in many areas of the world and
which is continuously increasing.
2
In our study,
12.6% of AmpC producing E. coli were isolated from
paediatric patients. These observations are similar
to the studies carried out by some other workers.
15,16
Generally, hospital environment accounts high
number of resistance bacteria which frequently
transfers from one patient to another.
There are many factors such as various interven-
tions during hospitalization which are associated
with the transmission of AmpC β-lactamase pro-
ducing bacteria. In our study various such inter-
ventions were intravenous lines (76.5%), surgeries
(12.9%), peritoneal dialysis catheters (9.4%), naso
gastric tubes (7.1%), urinary catheters (7.1%) and
central venous pressure lines (2.4%). The risk fac-
tors associated with AmpC producing organism
have also been investigated in different studies. A
case control study on AmpC β-lactamase producing
E. coli was carried out among the patients who had
undergone various invasive procedures who had
bacteremia. These included urinary catheter (37%),
peritoneal dialysis catheter (6.3%) and intravenous
lines (3.7%).
17
Another study reported indwelling
urinary catheter (25.9%) and central venous cath-
eter (29.6%) as risk factors for infections caused by
AmpC β-lactamase producing strains.
18
These nd-
ings suggested that these risk factors posed a threat
for the patients to become colonized or infected
with AmpC β-lactamase producing strains. The
patients who receive these interventions like intra-
venous line, urinary catheters and other catheters
become susceptible to infections caused by AmpC
β-lactamase producing strains.
In the current study, AmpC β-lactamase producing
E. coli were multidrug resistant. All were resistant to
co-amoxiclav, ceftazidime, cefotaxime, ceftriaxone,
cexime, cefuroxime and cefoxitin. These ndings
are in accordance with the work done by some
researchers. One such study from Korea reported
all of the AmpC producing E. coli were resistant to
co-amoxiclav, ceftazidime, cefotaxime, ceftriaxone
and cefoxitin (100% each).
19
Similar observations
were found in another study from Spain.
20
These
ndings clearly show that AmpC β-lactamase
producing E. coli strains are highly resistant to
clinically important antibiotics. Continuous or
frequent use of these antibiotics probably leads to
higher resistance rates of AmpC-producing isolates,
especially in paediatric populations.
15
The isolated AmpC β-lactamases producing
E. coli found to be signicantly resistant to co-
trimoxazole (91.8%), cefpodoxime (87.1%),
aztreonam (69.4%), gentamicin (62.4%), amikacin
(61.2%) and ciprooxacin (34.1%). These ndings
are slightly different from other studies. One study
conducted in China observed antibiotic resistance of
AmpC β-lactamase producing E. coli from different
paediatric hospitals. The isolated strains were found
to be resistant to ciprooxacin (70%), gentamicin
(70%) and amikacin (30%).
15
Another study carried
out in France reported that AmpC β-lactamase
producing E. coli isolated from bacteremic patients
were considerably resistant to ciprooxacin (50%)
but less resistant to gentamicin (5.6%) and none of
the strain was resistant to amikacin.
21
In another
study from Korea, none of the AmpC β-lactamase
producing E. coli showed resistant to co-trimoxazole,
aztreonam, cefpodoxime, gentamicin, amikacin
and ciprooxacin.
19
Higher rates of resistance to
these antibiotics in our study could also be due to
other possible mechanisms like efux pump or loss
of porin.
AmpC β-lactamase producing E. coli were found
to be less resistant to sulbactam-cefoperazone
(14.1%), cefepime (7.1%) and piperacillin-
tazobactam (5.9%) in our study. Contrary to our
results, studies from Korea and Canada reported
none of AmpC β-lactamase producing E. coli
resistant to sulbactam-cefoperazone, cefepime and
piperacillin-tazobactam.
19,22
Mulvey et al reported,
4.7% of 65 AmpC β-lactamase producing E. coli
showed resistance to piperacillin-tazobactam.
16
None of the AmpC β-lactamase producing E. coli
was found resistant to imipenem and meropenem
in our study. Similar ndings have been reported in
other studies conducted in Japan, United States and
Spain.
20,23
A study from Pakistan reported resistance
of AmpC producing bacteria to gentamicin (75%),
Antimicrobial resistance of AmpC β-lactamase producing E. coli

184 Pak J Med Sci 2014 Vol. 30 No. 1 www.pjms.com.pk
ciprooxacin (75%), amikacin (65%) and sulbactam-
cefoperazone (32.5%) and none of strain was found
resistant to meropenem.
24
These nding suggest
that imipenem and meropenem might be useful
for the treatment of infections caused by AmpC
β-lactamase producing organisms.
Thus meropenem, imipenem, piperacillin-
tazobactam, cefepime and sulbactam-cefoperazone
could be drugs of choice for treating AmpC
β-lactamase producing E. coli infections. The
burden of AmpC producing E. coli strains can
be reduced by minimizing the use of invasive
devices and strict adherence of antibiotic policy.
Communication between the hospitals and the
other health institutions regarding the prevalence
of resistant bacteria, identiable risk factors and
controlled procedures can decrease the risk of
AmpC β-lactamase producing bacteria.
ACKNOWLEDGMENTS
We are thankful to the Management and Ethical
Committee of the Children’s Hospital & Institute of
Child Health, Lahore, Pakistan for providing us all
kind of facilities.
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Authors Contribution:
Noor-ul-Ain Jameel: Conceived the study,
performed experimental work and wrote the
manuscript.
Hasan Ejaz: Data analysis and critically reviewed
the manuscript for nal publication.
Aizza Zafar: Provided the facilities for experiments
and interpretation of results
Hafsa Amin: Helped in collection of isolates article
drafting.
Noor-ul-Ain Jameel et al.