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Pakzad I, Kazemian H. Identification of Carbapenemase genes and evaluation of their prevalence among E.coli and Klebsiella pneumoniae Isolated from patients who admitted to Ilam hospitals by phenotypic and genotypic method. Journal title 2019; 2 (1)
URL: http://newresearch.medilam.ac.ir/article-1-434-en.html
URL: http://newresearch.medilam.ac.ir/article-1-434-en.html
Clinical Microbiology Research Center, Ilam University of Medical sciences, Ilam, Iran
Abstract: (2215 Views)
Abstract
Objectives: Drug resistance among gram-negative bacteria
is a worldwide challenge. Due to the importance of drugresistant
Klebsiella pneumoniae and Escherichia coli strains in
hospital-acquired infections, we aimed to determine the
phenotypic and genotypic characteristics of ESBL-, AmpC-,
and carbapenemase-producing isolates obtained from hospitalized
patients in Tehran and Ilam (Iran). Materials and
Methods: In total, 90 K. pneumoniae isolates and 65 E. coli
isolates were collected from various infections. Phenotypic
identification of bacterial isolates was performed using standard
methods. Phenotypic screening of ESBL, AmpC, and
carbapenemase enzymes was carried out. Detection of ESBL,
AmpC, and carbapenemase genes was also performed by
the PCR method. Results: Phenotypic detection tests showed
that 36 (40%) K. pneumoniae and 23 (35.4%) E. coli isolates
were ESBL producers. Moreover, 18 (20%) and 6 (9.2%) K.
pneumoniae and E. coli isolates were AmpC producers, respectively.
Modified Hodge test results indicated that 39
H.K. and H.H. contributed equally to this work as first authors.
This is an Open Access article licensed under the Creative Commons
Attribution-NonCommercial-4.0 International License (CC BY-NC)
(http://www.karger.com/Services/OpenAccessLicense), applicable to
the online version of the article only. Usage and distribution for commercial
purposes requires written permission.
2 Med Princ Pract Kazemian et al.
DOI: 10.1159/000500311
(43.3%) K. pneumoniae and 18 (27.7%) E. coli isolates produced
carbapenemase. Molecular tests showed that 40% of
K. pneumoniae and 36.9% of E. coli isolates were ESBL positive.
AmpC was detected in 24.4 and 13.8% of K. pneumoniae
and E. coli isolates. Carbapenemase was detected in 34
(37.8%) K. pneumoniae and 13 (20%) E. coli isolates.
Conclusion:
In this study, 3 K. pneumoniae isolates simultaneously
carried ESBL, AmpC, and carbapenemase genes. Upto-
date strategies such as combination therapy or utilization
of new antimicrobial agents might help to combat such
drug-resistant organisms. © 2019 The Author(s)
Published by S. Karger AG, Basel
Introduction
Drug resistance in gram-negative bacteria is a worldwide
challenge [1]. Major resistance to gram-negative
pathogens is related to Enterobacteriaceae, Acinetobacter
baumannii, and Pseudomonas aeruginosa [2]. In the Enterobacteriaceae
family, Klebsiella pneumoniae and Escherichia
coli have notable drug resistance. Also, they are the
common cause of hospital- and community-acquired infections
[3, 4].
The wide distribution of extended-spectrum β-lactamases
(ESBLs) amongst Enterobacteriaceae has been
attributed to the over- or misuse of these antibiotics [5].
ESBLs are able to hydrolyze various types of β-lactam antibiotics,
including cephalosporins and monobactams.
Therefore, treating infections caused by ESBL-producing
bacteria has become a complicated issue [5, 6]. Dissemination
of hospital-acquired infections can be prevented
via early detection of infections caused by such microorganisms
[7].
Carbapenems have been considered as the treatment
of choice for ESBL-producing gram-negative bacterial infections.
Carbapenemase-producing strains cause serious
infections in hospitalized patients and are associated with
mortality [8]; hence, the use of molecular techniques
could be helpful in accurately diagnosing infections
caused by ESBL- and carbapenemase-producing organisms.
Drug-resistant strains of K. pneumoniae and E. coli in
hospital-acquired infections are important [9]; thus, the
aim of the present study was to determine the phenotypic
and genotypic characterization of ESBL-, AmpC-, and
carbapenemase-producing isolates of K. pneumoniae and
E. coli obtained from hospital-associated infections in
Tehran and Ilam (Iran).
Materials and Methods
Bacterial Strains
A total of 90 K. pneumoniae and 65 E. coli isolates were obtained
from various infections of hospitalized patients between
April 2016 and March 2017 in Tehran and Ilam. Phenotypic and
biochemical identification of bacterial isolates was performed according
to standard methods [10].
Phenotypic Screening of ESBL, AmpC, and Carbapenemase
Enzymes
All isolates were initially screened for ESBL production by a
combined disk method according to the guidelines of the Clinical
Laboratory Standards Institute (CLSI) [11]. Briefly, susceptibility
to cefotaxime (30 μg), cefotaxime/clavulanate (30/10 μg), ceftazidime
(30 μg), and ceftazidime/clavulanate (30/10 μg) (Mast Co.,
UK) was determined on Müller-Hinton agar (Merck Co, Germany).
ESBL-producing strains were recognized by an at least 5-mm
increase in zone diameter around cefotaxime/clavulanate and
ceftazidime/clavulanate disks in comparison with disks without
clavulanic acid. E. coli ATCC 35218 was used as the control strain
[11].
Cefoxitin disks (30 μg) were used to screen AmpC-producing
isolates according to CLSI recommendations [11]. A cefoxitincloxacillin
double-disk synergy test was carried out to confirm
AmpC production, as previously described [12].
The modified Hodge test (MHT) was performed to screen carbapenemase-
producing isolates [11]. K. pneumoniae ATCC BAA-
1705 and BAA-1706 were used as MHT-positive and -negative
controls [11].
Detection of ESBL-, AmpC-, and Carbapenemase-Related
Genes
Polymerase chain reaction was performed using specific primers
to detect genes encoding ESBLs (blaTEM, blaSHV, blaCTX-M, and
blaPER), AmpC (blaACC, blaDHA, blaEBC, blaFOX, blaMOX, and
blaCIT), and carbapenemase (blaIMP, blaVIM, blaNDM, blaKPC, and
blaOXA-48-like), as previously described [13–17]. The products were
separated by electrophoresis in 1% agarose gel with 1× TBE (Tris/
borate/EDTA) buffer, stained with safe stain load dye (CinnaGen
Co., Tehran, Iran) and visualized under ultraviolet illumination.
Statistical Analysis
The distribution of resistance genes among resistant and susceptible
isolates was calculated using χ2 and Fisher’s exact tests for
each gene. p values ≤0.05 were considered to be statistically significant.
Objectives: Drug resistance among gram-negative bacteria
is a worldwide challenge. Due to the importance of drugresistant
Klebsiella pneumoniae and Escherichia coli strains in
hospital-acquired infections, we aimed to determine the
phenotypic and genotypic characteristics of ESBL-, AmpC-,
and carbapenemase-producing isolates obtained from hospitalized
patients in Tehran and Ilam (Iran). Materials and
Methods: In total, 90 K. pneumoniae isolates and 65 E. coli
isolates were collected from various infections. Phenotypic
identification of bacterial isolates was performed using standard
methods. Phenotypic screening of ESBL, AmpC, and
carbapenemase enzymes was carried out. Detection of ESBL,
AmpC, and carbapenemase genes was also performed by
the PCR method. Results: Phenotypic detection tests showed
that 36 (40%) K. pneumoniae and 23 (35.4%) E. coli isolates
were ESBL producers. Moreover, 18 (20%) and 6 (9.2%) K.
pneumoniae and E. coli isolates were AmpC producers, respectively.
Modified Hodge test results indicated that 39
H.K. and H.H. contributed equally to this work as first authors.
This is an Open Access article licensed under the Creative Commons
Attribution-NonCommercial-4.0 International License (CC BY-NC)
(http://www.karger.com/Services/OpenAccessLicense), applicable to
the online version of the article only. Usage and distribution for commercial
purposes requires written permission.
2 Med Princ Pract Kazemian et al.
DOI: 10.1159/000500311
(43.3%) K. pneumoniae and 18 (27.7%) E. coli isolates produced
carbapenemase. Molecular tests showed that 40% of
K. pneumoniae and 36.9% of E. coli isolates were ESBL positive.
AmpC was detected in 24.4 and 13.8% of K. pneumoniae
and E. coli isolates. Carbapenemase was detected in 34
(37.8%) K. pneumoniae and 13 (20%) E. coli isolates.
Conclusion:
In this study, 3 K. pneumoniae isolates simultaneously
carried ESBL, AmpC, and carbapenemase genes. Upto-
date strategies such as combination therapy or utilization
of new antimicrobial agents might help to combat such
drug-resistant organisms. © 2019 The Author(s)
Published by S. Karger AG, Basel
Introduction
Drug resistance in gram-negative bacteria is a worldwide
challenge [1]. Major resistance to gram-negative
pathogens is related to Enterobacteriaceae, Acinetobacter
baumannii, and Pseudomonas aeruginosa [2]. In the Enterobacteriaceae
family, Klebsiella pneumoniae and Escherichia
coli have notable drug resistance. Also, they are the
common cause of hospital- and community-acquired infections
[3, 4].
The wide distribution of extended-spectrum β-lactamases
(ESBLs) amongst Enterobacteriaceae has been
attributed to the over- or misuse of these antibiotics [5].
ESBLs are able to hydrolyze various types of β-lactam antibiotics,
including cephalosporins and monobactams.
Therefore, treating infections caused by ESBL-producing
bacteria has become a complicated issue [5, 6]. Dissemination
of hospital-acquired infections can be prevented
via early detection of infections caused by such microorganisms
[7].
Carbapenems have been considered as the treatment
of choice for ESBL-producing gram-negative bacterial infections.
Carbapenemase-producing strains cause serious
infections in hospitalized patients and are associated with
mortality [8]; hence, the use of molecular techniques
could be helpful in accurately diagnosing infections
caused by ESBL- and carbapenemase-producing organisms.
Drug-resistant strains of K. pneumoniae and E. coli in
hospital-acquired infections are important [9]; thus, the
aim of the present study was to determine the phenotypic
and genotypic characterization of ESBL-, AmpC-, and
carbapenemase-producing isolates of K. pneumoniae and
E. coli obtained from hospital-associated infections in
Tehran and Ilam (Iran).
Materials and Methods
Bacterial Strains
A total of 90 K. pneumoniae and 65 E. coli isolates were obtained
from various infections of hospitalized patients between
April 2016 and March 2017 in Tehran and Ilam. Phenotypic and
biochemical identification of bacterial isolates was performed according
to standard methods [10].
Phenotypic Screening of ESBL, AmpC, and Carbapenemase
Enzymes
All isolates were initially screened for ESBL production by a
combined disk method according to the guidelines of the Clinical
Laboratory Standards Institute (CLSI) [11]. Briefly, susceptibility
to cefotaxime (30 μg), cefotaxime/clavulanate (30/10 μg), ceftazidime
(30 μg), and ceftazidime/clavulanate (30/10 μg) (Mast Co.,
UK) was determined on Müller-Hinton agar (Merck Co, Germany).
ESBL-producing strains were recognized by an at least 5-mm
increase in zone diameter around cefotaxime/clavulanate and
ceftazidime/clavulanate disks in comparison with disks without
clavulanic acid. E. coli ATCC 35218 was used as the control strain
[11].
Cefoxitin disks (30 μg) were used to screen AmpC-producing
isolates according to CLSI recommendations [11]. A cefoxitincloxacillin
double-disk synergy test was carried out to confirm
AmpC production, as previously described [12].
The modified Hodge test (MHT) was performed to screen carbapenemase-
producing isolates [11]. K. pneumoniae ATCC BAA-
1705 and BAA-1706 were used as MHT-positive and -negative
controls [11].
Detection of ESBL-, AmpC-, and Carbapenemase-Related
Genes
Polymerase chain reaction was performed using specific primers
to detect genes encoding ESBLs (blaTEM, blaSHV, blaCTX-M, and
blaPER), AmpC (blaACC, blaDHA, blaEBC, blaFOX, blaMOX, and
blaCIT), and carbapenemase (blaIMP, blaVIM, blaNDM, blaKPC, and
blaOXA-48-like), as previously described [13–17]. The products were
separated by electrophoresis in 1% agarose gel with 1× TBE (Tris/
borate/EDTA) buffer, stained with safe stain load dye (CinnaGen
Co., Tehran, Iran) and visualized under ultraviolet illumination.
Statistical Analysis
The distribution of resistance genes among resistant and susceptible
isolates was calculated using χ2 and Fisher’s exact tests for
each gene. p values ≤0.05 were considered to be statistically significant.
: Cross sectional |
Subject:
General
Received: 2016/11/25 | Accepted: 2017/01/4 | Published: 2019/05/31
Received: 2016/11/25 | Accepted: 2017/01/4 | Published: 2019/05/31
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