Ahmad Hamad Pishtiwan and Khalil Mustafa Khadija.
Department of Biology, College of Education, Salahaddin University-Erbil, Kurdistan region, Iraq.
Correspondence to: Ahmad Hamad Pishtiwan. Department of Biology,
College of Education, Salahaddin University-Erbil, Kurdistan region,
Iraq. E-mail:
pishtiwan.hamad@su.edu.krd
Published: July 1, 2019
Received: March 8, 2019
Accepted: June 10, 2019
Mediterr J Hematol Infect Dis 2019, 11(1): e2019041 DOI
10.4084/MJHID.2019.041
This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0),
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
|
Abstract
Background:
Due to the recent appearance of organisms that are resistant to several
drugs (multidrug-resistant) like Enterobacteriaceae that produce
extended-spectrum β-lactamase (ESBL, concerns have remarkably increased
regarding the suitable treatment of infections. The present study was
an investigation into ESBL molecular characteristics among clinical
isolates of Klebsiella pneumoniae and Escherichia coli
resulting in urinary tract infections (UTIs) and their pattern of
antimicrobial resistance in order to come up with helpful information
on the epidemiology of these infections and risk factors accompanied
with them. Methods: In order to conduct the study, 20 K. pneumoniae and 48 E. coli were
isolated and retrieved from thalassemia center in Erbil, Iraq during
July 2016 and September 2016. The collected strains were analyzed and
the profile of their antimicrobial susceptibility was specified. In
order to spot β-lactamase genes (i.e. blaTEM, blaSHV, and blaCTX-M), polymerase chain reaction was conducted. Results: The findings obtained from multiplex PCR assay showed that out of the collected strains of ESBL-producing E. coli, had 81% blaTEM, 16.2% blaSHV, and 32.4% blaCTX-M genes. Similarly, 64.7% blaTEM, 35.2% blaSHV, and 41.1% blaCTX-M genes existed in the isolates of K. pneumoniae. It was found that antibiotic resistance pattern of E. coli and K. pneumoniae isolates to 20 antibiotics varied widely. It was also concluded that the majority of the K. pneumoniae and E. coli isolates were multi-drug resistant (MDR). Moreover, 75% and 87.5% of respectively K. pneumoniae and E. coli isolates showed the MDR phenotypes. Conclusion: TEM prevalence was high among other types of ESBLs. Over all, the most active antimicrobial agents in vitro remained to be the carbapenems.
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Introduction
It
has been reported that bacteria that belong to the Enterobacteriaceae
family are etiologic factors of numerous nosocomial infections all over
the world.[1] It is difficult to control diseases
induced by bacilli Enterobacteriaceae given the limitation of
therapeutic possibilities caused by constantly rising resistance of
such organisms to antibiotics. In fact, Ojdana et al. (2014) introduced
ESBLs as one of the most well-known resistance mechanisms in
Gram-negative bacilli.[2] ESBLs are a group of enzymes
that lead to resistance increase in Aztreonam, Ceftazidime, Cefotaxime,
related Oxyimino-β-lactams, cephalosporins, and penicillins, but
Clavulanic acid inhibits them. TEM, SHV, and CTX-M are the 3 main types
of ESBLs. CTX-M, which has become more prevalent than SHV and TEM,
includes a rapidly expanding family which has spread among a wide range
of clinically important bacteria and over wide geographic areas.[3]
Furthermore, strains that produce ESBL often demonstrate resistance to
antibiotics belonging to other classes (i.e. aminoglycosides,
quinolones, and sulfonamides), which makes strategies of treatment more
complex.[4]
In addition, Enterobacteriaceae family members such as Klebsiella pneumoniae and Escherichia coli often produce ESBLs; however, other genera of the Enterobacteriaceae
family have recently been reported to contain some other enzymes. A
higher level of resistance in such organisms was first observed in
patients with prolonged hospital stays in intensive care units in
Europe. However, isolates were identified in Africa, Asia, the Middle
East, and South and North Americas, and ESBL GNB soon became a global
problem and concern.[5]
Common ESBL genes coding for isolates of K. pneumoniae and E. coli
were determined as CTX-M (cefotaximase that preferentially hydrolyzes
cefotaxime), TEM (found and isolated in the early 80s from Teminora who
was a Greek patient), and SHV (for variable of sulphydryl which was
first observed in a single Klebsiella ozaenae strain
retrieved in Germany). These genes which are mediated by transposons,
plasmids, or chromosomes are all sporadically described all over the
world.[6]
Because there is an increase in the
rates of bacterial resistance every year, leading to rising global
concern, it is highly significant to understand susceptibility patterns
as hospital stays may prolong and mortality rates increase due to
inappropriate empirical antimicrobial therapy, which can be controlled
given appropriate therapy.[7] Acquiring additional
PBPs insensitive to ß-lactam or changing the normal PBPs are known as
the commonest cause of resistance in cocci such as MRSA and pneumococci
which are gram positive. However, a mixture of endogenous acquired
ß-lactamases with natural efflux and up-regulated impermeability is the
main reason for resistance in the gram-negative bugs.[8]
It should be noted that there are well-prepared documents on the fact
that routine disc-diffusion tests fail to detect ESBL production.
Moreover, the significance and detection method of ESBLs are not fully
recognized by many clinical laboratories; therefore, there may be a
lack of enough resources in laboratories to reduce the spread of these
mechanisms of resistance.[9]
A wide variety of
ESBLs including SHV, TEM, OXA, CTX, AmpC, and so forth exist; however,
most of them are derivatives of SHV, TEM, and CTX-M enzymes which are
most often found in K. pneumoniae and E. coli. In this regard, the current study was aimed at determining the prevalence of the ESBL phenotype and examines the existence of blaSHV, blaCTX-M, and blaTEM genes in isolates.
Materials and Methods
Isolates of bacteria. In total, 68 consecutive non-duplicate of K. pneumoniae and E. coli
isolates (n = 20 and 48, respectively) were retrieved from specimens of
urine at a Thalassemia center in Erbil, Iraq. The samples were obtained
from both outpatients and inpatients between July 2016 and September
2016. Standard microbiological techniques were used for isolation.[10]
Conventional microbiological procedures were employed to identify the
isolates. Besides, the VITEK 2 compact system was utilized to
re-identify them (BioMerieux, France).
Antimicrobial susceptibility testing.
According to the guidelines of the Clinical and Laboratory Standards
Institute (CLSI), the isolates were screened by the disc diffusion
method (Kirby-Bauer disc diffusion method) on Mueller-Hinton agar (MHA)
plates in order to test their antimicrobial susceptibility.[11]
The utilized antimicrobials included Amoxicillin+Clavulanic acid
(20+10μg), Amikacin (10 μg), Azithromycin (15μg), Cefixime (5μg),
Cefotaxime (30μg), Chloramphenicol (30μg), Ceftazidime (30μg),
Ciprofloxacin (10μg), Doxycycline (30μg), Imipenem (10μg), Gentamicin
(10μg), Kanamycin (30μg), Nalidixic acid (30μg), Meropenem (10μg),
Nitrofurantoin (100μg), Norfloxacin(10μg), Ofloxacin (5μg),
Streptomycin (25μg), Piperacillin (100μg), and Tobramycin (10μg).
Testing for production of ESBL (MDDST).
Using a disc of Amoxicillin-Clavulanate (20/10 μg) with four
cephalosporins of Ceftriaxone, 3GC-Cefotaxime, 4GC-Cefepime, and
Cefpodoxime, the Modified Double Disc Synergy Test (MDDST) was employed
to test all strains in terms of their production of Extended Spectrum
Beta-Lactamase (ESBL). A lawn culture belonging to the organisms was
created on a Mueller-Hinton agar plate following the recommendations by
CLSI.[11] A disc that contained
Amoxicillin-Clavulanate (20/10 μg) was put in the middle of the plate.
The 3GC and 4GC discs were placed respectively 15mm and 20mm
center-to-center apart from the center of the amoxicillin-clavulanate
disc.[12] Any increase or distortion in the zone
toward the Amoxicillin-Clavulanate disc was regarded positive for the
production of ESBL. According to CLSI guidelines, the combined disc
test was used to confirm ESBL production.
Detection of ESBL genotypes by multiplex PCR amplification.
Using the method utilized by Monstein et al. (2007) with slight
modifications, multiplex PCR was employed to examine the positive
isolates in the initial screening test for ESBL production for the
existence of blaSHV, blaCTX-M, and blaTEM genes.[13]
Freshly cultured isolates bacteria were used to prepare template
deoxyribonucleic acid (DNA) was prepared using PrestoTM Mini gDNA
bacterial kit. All reactions of PCR were conducted by utilizing 2 μl
DNA template (density of 10 ng/µl), the Master Mix consisting of 3 mM
MgCl2, 0.2% Tween® 20, 20 mM Tris-HCl pH 8.5, (NH4)2S04,
0.4 mM of each dNTP, 0.4 μM of each primer, and 0.2 units/µl Ampliqon
Taq DNA polymerase. The conditions of polymerase chain reaction
amplification were set up as follow: primary denaturation step for 10
minutes at 95°C; 30 denaturation cycles for 30 seconds at 94°C,
annealing 30 seconds at 60°C for, extension for 2 minutes at 72°C, and
a final extension step for 10 minutes at 72°C. Using agarose gel
electrophoresis, size separation PCR amplicons were utilized to detect
respective genes (Table1).
 |
Table 1. List of primers used for Multiplex PCR amplification. |
Results
Antimicrobial susceptibility profile. In total, 68 consecutive non-duplicate of K. pneumoniae and E. coli
isolates (n = 20 and 48, respectively) were retrieved, and their
antimicrobial resistance profile against 20 different antimicrobial
agents was tested. The current results revealed that K. pneumoniae and E. coli isolates vary widely to different antimicrobials. The resistance rates of isolates of K. pneumoniae and E. coli against the selected 20 antimicrobial agents obtained from urine samples. It was found that a majority of the K. pneumoniae and E. coli isolates were resistant to several drugs (multi-drug resistant: MDR) where a total of 87.5% and 75% of respectively E. coli and K. pneumoniae isolates indicated MDR phenotypes.
Furthermore, the results of the antimicrobial susceptibility test against E. coli revealed that E. coli showed 81.25% resistance to Amoxicillin+Clavulanic acid (Table 2),
whereas susceptibility to doxycycline decreased to 39.5%. Similar
patterns were observed for Piperacillin. Substantial decrease of
16.6–37.5% was observed in the susceptibility for all Cephalosporins.
Imipenem, Meropenem, Amikacin, Gentamicin, Ciprofloxacin, Tobramycin
and Ofloxacin with susceptibility rates of respectively 100%, 100%,
95.8%, 95.8%, 95.8%, 95.2%, and 93.75% were the most active agents
against E. coli. Resistance to Azithromycin Norfloxacin and Streptomycin was comparatively less (10.5%, for them). On the other hand, E. coli
showed a different sensitive rate to Chloramphenicol, Kanamycin,
Nalidixic acid and Nitrofurantoin with 85.4%, 62.5%, 50% and 75%,
respectively. Meanwhile, similar results were observed for K. pneumoniae which revealed that K. pneumoniae showed 65% resistant to Amoxicillin+Clavulanic acid, whereas susceptibility to Piperacillin dropped to 50% (Table 2).
 |
Table 2. Antibiotic resistance pattern of K. pneumoniae and E. coli isolates. |
Similar patterns
were noticed for Nalidixic acid. In addition, a substantial drop of
30–40% was observed in the susceptibility for all Cephalosporins.
Nevertheless, K. pneumoniae
showed a different sensitive rate to Chloramphenicol, Doxycycline,
Streptomycin, Azithromycin, Kanamycin and Nitrofurantoin with 95%, 95%,
95%, 90%, 90% and 70% respectively.
ESBL screening of E. coli and K. pneumoniae. Out of the 48 E. coli
isolates, a total of 37 isolates (77%) showed positive results in
initial screening test of ESBL production by MDDST and phenotypic
confirmatory test of ESBL production. Meanwhile, out of the 20 K. pneumoniae
isolates, a total of 17 isolates (85%) showed positive results in
initial screening test of ESBL production and phenotypic confirmatory
test of ESBL production.
In PCR detection of ESBL genotypes, it was found that all of the ESBL screening positive K. pneumoniae and E. coli isolates had one or more ESBL genes that were tested in the present study. Overall, 85% (17/20) of K. pneumoniae and 77% (37/48) of E. coli isolates were positive for one or more ESBL genes. The multiplex PCR assay results indicated that 32.4% blaCTX-M genes, 16.2% blaSHV genes, and 81% blaTEM genes were detected in the E. coli isolates. Similarly, the isolates of K. pneumoniae contained 64.7% blaTEM, 35.2% blaSHV, and 41.1% blaCTX-M genes. The overall incidence of ESBL genotypes in K. pneumoniae and E. coli isolates is illustrated in Figure 1.
 |
Figure
1. The overall incidence of ESBL genotypes in screening positive K. pneumoniae and E. coli isolates. |
Discussion
As
a global challenge, antimicrobial resistance in pathogenic bacteria is
accompanied with high rates of mortality and morbidity. In addition,
because of multidrug resistant patterns, infections have been reported
to be difficult or even impossible to treat with conventional
antimicrobials. Because many healthcare centers fail to diagnose
causative microorganisms and their patterns of antimicrobial
susceptibility timely in patients with bacteremia and other serious
infections, antibiotics are broadly, liberally and mostly unnecessarily
used.[14]
In the current study, high prevalence of MDR isolates of K. pneumoniae and E. coli was noticed in the clinical samples. The overall prevalence of MDR phenotypes in K. pneumoniae and E. coli isolates was respectively 75% and 87.5%. Among the MDR isolates of E. coli and K. pneumoniae,
a majority of them were producers of ESBL. Similar to the results of
the present study, also in a research by Bora et al. (2014) reported
the same ratios.[15]
In the current study, the
antimicrobial susceptibility patterns were determined in all isolates,
and the results obtained from the test of antimicrobial susceptibility
against E. coli and K. pneumonia revealed that isolated bacteria were different in their susceptibility to the tested antimicrobials. Liao et al. (2017)[4] and Tabar et al. (2016) reported similar results.[16] Carbapenems are often the final influential therapy that exists for infections resulting from MDR Enterobacteriaceae.[17]
According to other studies, 100% sensitivity was seen with Imipenem and
Meropenem, which has been reported to be the most effective antibiotic
including the isolates that produce ESBLs. This is an important result
of the present study because many infections can be treated with
Carbapenemes. This result can be relevant to the fact that these
antibiotics are more expensive and thus used less in this region.
Paterson et al. (2001) stated that even if ESBL producers show an in vitro susceptibility, they are intrinsically resistant to all cephalosporins.[18]
In the present study, 9% and 13% of the producers of ESBL were found to
have false susceptibilities respectively to cefotaxime and Ceftazidime.
This can be attributed to the fact that different ESBL enzymes possess
various optimal substrate profiles.[19]
In fact,
ESBLs are reported to be a challenge among hospitalized patients all
over the world. It has also been reported that ESBLs have different
prevalence rates among clinical isolates in different parts of the
world, and there is a rapid continuous change in their prevalence rate
over time.[20] Given the increased prevalence of
ESBLs-producing Enterobacteriaceae, it is highly crucial to develop
laboratory testing methods in order to accurately diagnose the
existence of such enzymes in clinical isolates.[21] Among all ESBL detection methods, modified double disc synergy tests were the most sensitive ones.[22] A study carried out by[23]
presented similar findings and indicated positive MDDST in 40/40
isolates, while it was positive in 25/40 and 39/40 isolates
respectively in double disk synergy test (DDST) and phenotypic
confirmatory disc diffusion test (PCDDT).
By following the MDDST screening criteria for ESBL production, respectively 85% and 77% of K. pneumoniae and E. coli
isolates were screened for detecting production of ESBL. Existence of
one or more ESBL genes in all screened positive isolates revealed that K. pneumoniae and E. coli
isolates that produce ESBL are highly frequent in the geographical
region under investigation. In India, Kaur et al. (2013) observed that
63.4% E. coli and 60.3% K. pneumoniae isolates produced ESBL.[24]
Phenotypic tests for detection of ESBL can only confirm ESBL production
but fail to recognize the subtypes of ESBL. As reported by
Nüesch-Inderbinen et al. (1996), molecular methods have been proved to
be sensitive, but they costly and conducting them requires a long time,
expertise, and specialized equipment.[25] Ultimate
identification is only probable through methods of molecular detection.
The results of a study conducted by (Navon-Venezia et al., 2003)
revealed that it is necessary to periodically evaluate these phenotypic
tests because introduction of new enzyme can change their performance.[26]
In their study of phenotypic and genotypic methods of ESBL detection,
(Grover et al., 2006) stated that PCR is a reliable method for
detecting ESBL.[27] In the present study, multiplex PCR amplification assay was utilized to detect blaCTX-M, blaSHV, and blaTEM genes in the retrieved clinical isolates of K. pneumoniae and E. coli
because one of the advantages of this assay rapid screening of large
numbers of clinical isolates, moreover, if it is required, further
molecular epidemiological studies can take advantage of the DNA that is
isolated via this assay.[13]
Furthermore, it is
essential to identify beta-lactamase in order to conduct a reliable
epidemiological investigation into antimicrobial resistance. The
current study was conducted to survey antimicrobial drug resistance,
ESBL phenotypes, and blaSHV, blaTEM and blaCTX-M genes detection in K. pneumonia and E. coli isolates retrieved from urinary tract infections in Erbil, Iraq.
The
most globally common type of ESBL appeared to be CTX-M-type ESBLs with
their higher incidence in most locations compared to SHV and TEM ESBLs.[28] Among the three ESBL genotypes included in this study, the most prevalent one was found to be blaTEM (81%) and blaTEM (64.7%) respectively in ESBL-producing isolates of E. coli and K. pneumoniae. The less prevalent ESBL genotype was blaSHV, and the prevalence rate of blaSHV in ESBL-producing K. pneumoniae isolates (35.2%) was higher than E. coli isolates (16.2%). Also, the prevalence rate of blaCTX-M in ESBL-producing K. pneumoniae isolates (41.1%) was higher than E. coli
isolates (32.4%). It was found that all of the ESBL-producing isolates
of both organisms were positive for one or more ESBL genotypes. It was
observed that blaTEM alone was more prevalent in E. coli (62.16%, 23/37), and in K. pneumoniae (41.17%, 7/17), while blaCTX-M and blaTEM together predominated in E. coli (8.1%), while blaSHV, blaTEM, and blaCTX-M together predominated in isolates of K. pneumoniae (11.76%). A study conducted by Manoharan et al. (2011) reported similar findings.[29]
In the present study; however, TEM ESBL was the prevalent genotype and
CTX-M-type ESBL was not prevalent. The discrepancy is assumed to be
because of regional variations, since the strains collected and
evaluated in the current study were only from Erbil, Iraq.
Furthermore, in another study, Moghnieh et al. (2018) have reported that E. coli and Klebsiella spp resistance to third-generation cephalosporins is usual in whole countries, with outbreak reaching over 50% in Egypt and Syria[30]
and in our study, 30–40% was observed in the susceptibility for all
Cephalosporins, which this prevalence is close to other Arabia
countries, as well as in Moghnieh study, they reported that carbapenem
resistance is emerging, albeit with a prevalence of less than 10%.[30]
In parallel, we have found that the most active antimicrobial agents in
vitro remained to be the carbapenems. Khalaf and Al-Ouqaili et al.
(2018) in Baghdad, during a period one year demonstrated that SHV gene
was detected only in 12.5% E. coli, and 56.25% in K. pneumoniae.[31] Approximately, we found close to findings above that 16.2% SHV genes in E. coli and 35.2% SHV genes existed in the isolates of K. pneumoniae.
Of course, according the above findings several studies by Teawtrakul
et al. (2015) Girmenia et al. (2016), Ricciardi et al. (2016) and
Devrim et al. (2018) have shown that the rates and types of Klebsiella and Escherichia strains isolated are differed in other countries.[32-35]
These outcomes highlight require for structured national plans in the
zone to target infection prevention and antimicrobial supervision.
Conclusions
Knowledge
of the antimicrobial resistance patterns and resistance genes of
bacterial pathogens in a geographical area is important for control and
surveillance of antibiotic resistance. The results of the present study
revealed that MDR was highly prevalent. In addition, the Carbapenems,
Amikacin, and Ciprofloxacin were found to be the most to the least
active antimicrobial agents in vitro. Based on the results obtained in the present study, TEM was highly prevalent among other types of ESBLs.
Acknowledgment
This work was supported by the Biology Department, Education College, Salahaddin University and Thalassemia Hospital in Erbil.
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