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 Afkhami^3,*

 

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

 

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 bla_OXA-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 bla_OXA23-like, bla_OXA24-like, bla_OXA58-like, bla_IMP, bla_NDM, and bla_VIM 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 bla_VIM, bla_OXA-23-like, bla_OXA-24-like, and bla_IMP 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 bla_OXA-58-like or bla_NDM (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 𝑏𝑙𝑎_OXA 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 bla_VIM and bla_IMP genes had the highest and lowest, respectively. Also none of the hospital environment isolates carried bla_OXA-58-like or bla_NDM. In the study of Amudhan et al. [39], the most resistance is related to bla_VIM in 45%, and in the study of Shirmohammadlou et al. [37], resistance to bla_IMP 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 bla_VIM and bla_IMP. 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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