Original research
Detection of carbapenem
resistance and virulence genes among Acinetobacter baumannii isolated
from hospital environments in center of Iran
Mohsen Nazari1, Zohreh Youzbashi2, Mansoor
Khaledi3, Javad Fathi4, Hamed Afkhami3,*
1Department of Bacteriology, Pasteur Institute of Iran,
Tehran, Iran
2Department of Biology, Damghan
Branch, Islamic Azad University, Damghan, Iran
3Department of Medical Microbiology, Faculty of Medicine, Shahed University of Medical Science, Tehran, Iran
4Department of Bacteriology and Virology, School of
Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Corresponding
author: Hamed Afkhami, Ph.D
Department of Medical
Microbiology, Faculty of Medicine, Shahed University
of Medical Science, Tehran, Iran
Tel/Fax: +98 919
6652678; Email: hamedafkhami70@gmail.com; http://orcid.org/0000-0002-1110-6447
Received: October, 27,
2020; Accepted: December, 11, 2020
Abstract
Carbapenem-resistant Acinetobacter baumannii
are the top urgent antibiotic resistance threat in the world. The aims of this study were the determination of carbapenem-resistant
genes and virulence genes among isolates from hospital environments. In
this study, A. baumannii isolated from hospital environments and
evaluated its antibiotic resistance, virulence factors, and resistance genes.
Of 258 samples, 58 showed growth of the target organism. Antibiotic
susceptibility test results considered all the A. baumannii to be
multidrug-resistant isolates with the highest resistance being 36.2% to
ciprofloxacin; while the most effective antibiotics with 98.3% susceptibility
was piperacillin-tazobactam. Of these 58 hospital environment isolates, 18
isolates were positive for Metallo beta-lactamase.
Overall, 65% of the isolates from hospital environments had many virulence
factors. PCR assays demonstrated the highest and lowest positive results in csgA and cvaC
gene among hospital environment isolates. Results indicate that the determination
of carbapenem-resistant genes and virulence genes among isolates from hospital
environments is very important.
Keywords: Carbapenem resistance, Virulence gene,
Non-clinical isolates, Acinetobacter baumannii
1. Introduction
Multidrug-resistant Acinetobacter baumannii is an opportunistic
pathogen that causes nosocomial infections [1-3]. Infections caused by this bacterium have a high
prevalence in hospitalized and immunocompromised patients who are admitted to
intensive care units [4, 5]. These infections are ventilator-associated
pneumonia, soft-tissue, urinary tract, and meningitis infections [6-8]. A. baumannii has an ability to survive for
long periods the
surfaces, sometimes even for
several years [9]. Due to resistance to a broad range of
antimicrobial agents can long-term persistence in the clinical settings,
surviving on nutrient sources and transmission by healthcare staff [10, 11].
Carbapenems and colistin are the last choices of antibiotic therapies
against multi-drug resistant (MDR) A. baumannii strains [12]. The first carbapenem-resistant A. baumannii
(CRAB) originated in the USA and was reported in 1991 [13]. Recent studies have reported that CRAB is a major
causative organism in hospital-acquired infections [14]. Several mechanisms are responsible for resistance
to carbapenems in CRAB [15]. The production of carbapenemase enzymes is one of
the important mechanisms of carbapenem resistance. These enzymes are class A, B,
and D according to molecular Ambler
classification. The members of class A carbapenemases include SME, IMI, NMC,
GES, SFC, and KPC families. Also, class B enzymes are called metallo-beta-lactamases (MBLs), including IMP, VIM, SIM,
and NDM. Class D β-lactamases referred to OXA-type enzymes or oxacillinases which are the most prevalent carbapenemases
in A. baumannii [16, 17]. Prevalence of virulence Factors (VF) is
contributed to pathogenesis in A. baumannii [18]. Some of the most significant VF of the A.
baumannii strains are curli fibers (csg), colicin V production
(cvaC), siderophores
like aerobactin (iutA), and cytotoxic
necrotizing factor (cnf) [18, 19]. The characterization of latent virulence genes,
antimicrobial resistance, and molecular detection of carbapenemases of this
bacterium in the hospital environments on abiotic and biotic surfaces are the
important epidemiological issue. The aims of this study were the determination
of antimicrobial resistance and molecular detection of antibiotic resistance
and virulence genes among A. baumannii isolated from hospital
environments in Qom hospitals.
2. Materials and Methods
2.1 Bacterial strain collection and identification
The study was conducted at Pasteur Institute in the period from
October 2019 to December 2019. Totally, 58 isolates were collected from different hospital environments of Qom
hospitals (Kamkar and Beheshti
hospitals) (Table 1).
Samples were washed with PBS and transferred to brain heart infusion (BHI)
media for further incubation at 37 °C. Samples were inoculated into blood agar
and MacConkey agar for standard aerobic growth and
placed at 37 °C overnight. The isolates were identified by the
standard biochemical tests. The final confirmation of A.
baumannii isolates was performed by PCR blaOXA-51-like
gene [20].
2.2 Antimicrobial susceptibility testing
According to the CLSI 2019 guidelines, the disk
diffusion test was performed on Mueller-Hinton agar using a panel of nine antibiotic disks including ciprofloxacin
(CIP), levofloxacin (LVX), gentamicin (GM), imipenem (IMI), piperacillin-tazobactam (PTZ),
ampicillin-sulbactam (SAM), ceftriaxone (CRO), and
trimethoprim-sulfamethoxazole (SXT) (Mast
Diagnostic, UK).
2.3 Phenotypic detection of MBL production
Combined disk diffusion test (CDDT) was performed using an imipenem disk (Mast Diagnostic, UK) and in combination with EDTA
(Sigma, UK) to identify MBLs. The
inhibition zones of the imipenem, and imipenem + EDTA disks were compared after 20 h of incubation at 37 °C. In the combined disc test, if the increase in
inhibition zone with the imipenem + EDTA disk was >7 mm than the imipenem disk alone, it was considered as MBL
positive [21].
2.4 Detection of carbapenem resistance and virulence genes
Genomic DNA extractions were performed based on the protocol as
described elsewhere [22]. PCR reaction mixtures were prepared in total
volumes of 25 μl. The presence of the
carbapenemase-encoding genes including blaOXA23-like, blaOXA24-like, blaOXA58-like, blaIMP, blaNDM, and blaVIM genes and virulence genes
cnf1, csgA, cvaC,
iutA were investigated by PCR assays in all
isolates [23-28]. All PCR primers are shown in Table 2.
3. Results
Antibiotic susceptibility testing showed out of 58
hospital environment isolated strains, the resistance against to CIP, LVX, GM, IMI,
SXT, SAM, CRO, and PTZ were 36.2% (21/58), 31% (18/58), 18.9% (11/58), 15.5% (9/58), 8.6% (5/58), 8.6% (5/58), 5.1% (3/58) and 1.7%
(1/45), respectively.
Of these 58 hospital environment isolates, 21/58 (36.2%) showed resistance to imipenem and were
therefore further tested for MBL production. Eighteen of these isolates showed
positive results for MBL production by CDDT method as shown in Table 3.
PCR analysis in all carbapenem-resistant isolates revealed that
prevalence of blaVIM,
blaOXA-23-like, blaOXA-24-like, and blaIMP were
10/21 (47.6%), 3/21 (14.3%), 3/21 (14.3%), and 1/21 (4.7 %) of the strains,
respectively. None of the hospital environment isolates carried blaOXA-58-like
or blaNDM (Table 3).
Also, PCR assays demonstrated positive results in 43.1% (25/58) of strains for csgA, 32.7% (19/58) of strains
for cnf1, 12%
(7/58) of strains for iutA, and 3.4% (2/58) of
strains for cvaC genes among hospital environment isolates (Table 3).
4. Discussion
A. baumannii is an opportunistic
pathogen and also has the ability to cause nosocomial diseases due to
antibiotic resistance and can survive on surfaces, the body of the treatment
staff, and patients [29, 30]. This bacterium has the
ability to survive on different surfaces and objects, so it has a high
potential for spread and colonization in hospitalized patients [31]. Through
the mechanisms of acquisition of determinants of resistance and upregulation of intrinsic resistance mechanisms, this bacterium
is able to resistance against a wide range of available antibiotics [30]. A. baumannii with
multidrug resistance causes severe infections and high mortality, especially in
patients with impaired immune systems or immunocompromised [2, 32]. Although the virulence
factors and pathogenicity mechanism of A. baumannii are not fully
understood and require further study and research, but this bacterium has the ability to cause a wide
range of infections and deaths in hospitals. The virulence factors of this
bacterium play an important role in resisting the host's defense mechanism [33, 34]. Also these factors are
important role in binding and invasion bacteria to host cells [28]. According to the contents,
it is necessary to study the prevalence of antibiotic resistance and prepare a
useful antibiotic treatment model to control and treat diseases caused by A.
baumannii. The most important factor in resistance to carbapenem
antibiotics is the presence of the 𝑏𝑙𝑎OXA genes, which causes the
production of carbapenem hydrolyzing enzymes. In the present study, out of 58
isolates, the rate of antibiotic resistance in A. baumannii based on
antibiotic susceptibility tests was the highest and lowest resistance for CIP
and PTZ antibiotics, respectively. According to Nourbakhsh et al. (2018) studies, the highest resistance is related to CIP (97.2%) [35], which is similar to our
study in terms of the highest antibiotic resistance. In the study of Shakibaie et al., resistance to CIP and PTZ was reported to be 66% and 93.3%,
respectively [36]. Although the bacterial
resistance to CIP was as high as in our study, the resistance to PTZ differed
greatly from our study. According to a study by Shirmohammadlou
et al., A. baumannii is completely resistant to CIP [37], and this result is
consistent with our study. These results are consistent with the study of Kabbaj et al., which showed that MBL production is among
the 74% of bacteria resistant to imipenem [38].
Among carbapenem-resistant isolates, the frequency
of blaVIM and blaIMP genes
had the highest and lowest, respectively. Also none of the hospital environment
isolates carried blaOXA-58-like or blaNDM.
In the study of Amudhan et al. [39], the most resistance is
related to blaVIM in 45%, and in
the study of Shirmohammadlou et al. [37], resistance to blaIMP was less common, which met with
our study.
Regard to prevalence of virulence genes, the
results of our study were similar to those of Al-Kadmy et al. (Highest frequency was csgA 66.7% and lowest frequency was cvaC 9.5%) [40], Momtaz et
al. (Highest frequency was csgA 55% and lowest frequency was cvaC 10%) [41], although our results
contradicted the results of Darvishi study (highest frequency was cnf1, 35.53% and lowest frequency was csgA 12.39%) [28].
The present study had some limitations. Mainly,
this was a two-center study; therefore, the generalization of the results to
other regions requires further investigations.
In conclusion, increasing antibiotic resistance due
to the acquisition of resistance genes and mutations due to selective pressures
is a global problem. The increase in antibiotic resistance in A. baumannii
is spreading, especially against effective antibiotics. Therefore, Studying and
determining the pattern of antibiotic resistance for the preparation of a
treatment protocol and the use of appropriate antibiotics is effective and
reduces the increase in antibiotic resistance. In the current study, we isolated A. baumannii
from hospital environments and determinated its
antibiotic resistance, virulence factor, and resistance gene. Antibiotic
susceptibility testing showed the most resistance and sensitivity was against CIP and PTZ respectively. PCR analysis in all
carbapenem-resistant isolates revealed a high prevalence of blaVIM and blaIMP. Also, results
demonstrated the highest and lowest positive results for csgA
and cvaC gene among hospital environment isolates.
Acknowledgments
We would like to thank the Pasteur Institute of Iran and Kamkar and Beheshti hospitals of
Qom for supporting this study.
Author Contributions
MN, HA: conceived and design the study. JF, MN, MK: supervised data
collectors. MK, JF, ZY and HA: drafting the article or revisiting it critically
for important intellectual content. All authors read and approved the final
manuscript.
Conflict of Interest
We declare that we have no conflict of interest.
Ethical declarations
This study was in accordance with the declaration of Helsinki and
ethical permission was sought from the institutional Ethics Committee of
Pasteur Institute of Iran, Tehran, Iran (Ethical Code: 1398057). However, because we only used leftovers
from clinical specimens, the local ethics committee waived the need for
informed consent.
Financial Support
This work was supported
by Pasteur Institute of Iran, Tehran, Iran (grants No. 41443).
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