Mostafa Alavi-Moghaddam, Mehran Dolati, Abdolreza Javadi, Bahareh Ghobadi Saki,Morteza Karami-Zarandi, Saeed Khoshnood

1Emergency Medicine Departent, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2Clinical Research Development Unit, Imam Hossein Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

4Student Research Committee, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

ABSTRACT Objective: To determine patterns of antimicrobial resistance,analyze the prevalence of oxacillinase and molecular typing of strains of Acinetobacter baumannii (A. baumannii).Methods: A total of 121 strains of A. baumannii were obtained from patients admitted to Imam Hossein and Imam Khomeini Hospitals, Tehran, Iran, from January 2016 to November 2018.Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute recommendations. The presence of oxacillinase genes was assessed by polymerase chain reaction(PCR). To determine clonal relatedness, all isolates were subjected to repetitive sequence-based PCR (REP-PCR).Results: The isolates were obtained from 56 (46.3%) males and 65 (53.7%) females with the mean age of 39.5 years. Colistin with 100.0% sensitivity rate had the highest effect, while ceftriaxone with 16.5% sensitivity rate had the least effect on A. baumannii isolates. In addition, 96 (79.3%) and 99 (81.8%) isolates were resistant to imipenem and meropenem, respectively. A total of 109 isolates (90.0%) exhibited multiple drug resistance with 10 different resistotypes. In total, 75 (75.7%) of carbapenem resistant isolates were positive for blaOXA-23-like, and 14 (14.1%) for blaOXA-24-like gene. The five main clones A, B, C, D, and E were detected in 25 (25.2%), 36 (36.4%), 10 (10.1%), 8 (8.0%), and 6 (6.1%) of isolates, respectively.Conclusions: Carbapenem-resistant A. baumannii strains are high in the current study. To control the spread of carbapenem-resistant A. baumannii strains, regular monitoring programs are needed.

KEYWORDS: Acinetobacter baumannii; Multidrug resistance;Oxacillinase; Repetitive sequence-based PCR

1. Introduction

Acinetobacter baumannii (A. baumannii), an opportunistic Gramnegative, aerobic, non-fermenting cocobacilli, is mostly found in many healthcare environments, different water sources and soil.This bacterium is ample in mucous membranes and normal flora of skin in humans and able to cause different infections such as septicemia, meningitis, urinary tract infection, upper respiratory tract infections, pneumonia and especially in intensive care units(ICUs) worldwide[1].

A. baumannii is resistant to a number of commonly used antibacterial drugs intrinsically, and has a significant capacity to develop antibiotic resistance via different mechanisms[2]. Plasmids,integrons and transposones are the most important interchangeable genetic agents that play an important role in the transfer of antibiotic resistance genes. Resistance to various antibiotics such as carbapenems, fluoroquinolones, ami noglycosides and broad spectrum β-lactams are observed in this bacterium, which complicates the treatment of infections. Nowadays, A. baumannii became more widespread as an important pathogen, specifically as a multidrug-resistant agent (MDR)[3,4].

Therapeutic issues caused by MDR A. baumannii and long-lasting nature in hospital environments and also the ability to transfer between living and non-living things has caused enhancment in the appearance of the bacteria and its increasing infection. Thus,mortality in infections caused by A. baumannii is estimated to be about 75%[5].

Resistance to carbapenem is now observed globally in A.baumannii and these isolates are typically resistant to various classes of antibacterial agents. Several outbreaks caused by carbapenem resistant among A. baumannii isolates are reported from different countries of the world which could be an alarm for global health[6]. A. baumannii has different mechanisms for resistance to carbapenems, which include changes in penicillin binding protein,efflux pumps and mostly by production of oxacillinases (OXAs) and less common by metallo β-lactamase (MBLs) genes, AmpC stable derepression and reduced permeability[7,8].

The first report of OXA-type beta-lactamases in A. baumannii was related to Acinetobacter resistant to imipenem (ARI-1) that later designated as OXA-23[4]. Today, OXA-type carbapenemases have eight subgroups, four of which in A. baumannii have been reported:OXA-23-like consisting of OXA-23, OXA-27 and OXA-49; OXA-24-like (OXA-24, OXA-25, OXA-26, OXA-40 and OXA-72);OXA-51-like and OXA-58[9]. The transmission of isolates resistant to carbapenem A. baumannii is shown in the hospital. For control of resistant isolates and epidemiological aims, fast and accurate differentiation of epidemic strains from the many incidental strains is important[10,11].

One of the molecular typing methods used to determine the clonal relatedness of A. baumannii is repetitive sequence-based polymerase chain reaction (REP-PCR)[12]. The aim of this study was to survey the antibiotic susceptibility, prevalence of oxacillinase genes and perform molecular typing by REP-PCR in A. baumannii clinical isolates.

2. Materials and methods

2.1. Ethics statement and informed consent

This research was confirmed by the Ethical Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran with ethics number: IR.SBMU.RETECH.REC.1397.464. Informed consent form was obtained from all of the patients.

2.2. Study design and specimens collection

This cross-sectional study was performed by using 121 A.baumannii strains isolated from patients admitted to Imam Hossein and Imam Khomeini hospitals located in Tehran, Iran, from January 2016 to November 2018. A questionnaire was designed and coded for each patient. The samples were taken from patients included blood, trachea, urine, cerebrospinal fluid, catheter and pleural fluid.

These samples were cultured on blood agar and MacConkey(Merck,Germany) to confirm the Gram-negative bacteria via Gram stain microscopy analysis. Bacterial isolates were initially identified as A. baumannii by biochemical tests [Oxidase, nitrate reduction, motility on sulfide indole motility, growth at 42℃and on MacConkey agar, oxidative-fermentative glucose, sugars fermentation on triple sugar iron agar, citrate utilization on Simon’s Citrate agar]. Also, the final confirmation of A. baumannii isolates was conducted by PCR of blaOXA-51-likegene[13,14].

2.3. Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI 2017) guidelines using the following antibiotics: amikacin (30 μg), imipenem (10μg), meropenem (10 μg), piperacillin-tazobactam (10/100 μg),cefepime (30 μg), ceftriaxone (30 μg), tetracycline (10 μg),tigecycline (15 μg), ciprofloxacing(5 μg), tobramycin (10 μg) and colistin (10 μg) (MAST, UK)[15]. A. baumannii ATCC19606 was used as the control strain, and Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as negative controls.

2.4. PCR amplification and detection of OXA genes

PCR was conducted to detect blaOXA-23-like, blaOXA-24-like, blaOXA-58-likeand blaOXA-143-like. All target genes and corresponding primers used for PCR amplification are shown in Table 1[16]. Bacterial DNA was obtained by boiling method[17]. The PCR mixture contained master mix (30 Mm of KCL: 10 Mm, 30 Mm, 10 Mm of Tris-HCL, Bioneer Company, Korea), the forward/reverse primers and DNA template.PCR conditions are included 30 cycles of amplification under the following conditions in Mastercycler Eppendorf (Eppendorf,Germany): denaturation at 95℃ for 5 min; annealing at 55℃ for 30 sec; and 45 sec for extension at 72℃, with a final extension at 72℃for 6 min.

Table 1. Primers used to detect genes encoding oxacillinases[16].

2.5. REP-PCR

Genetic relationship of all isolates was determined by REPPCR. In the REP-PCR method, the primer pair of REP-F(REP-Ⅰ, Ⅲ:GCGCCGICATCAGGC) and REP-R (REP-Ⅱ: ACGTCTTATCAGGCCTAC) was used and amplification PCRs were performed as previously described[18]. The amplified products were separated via electrophoresis on 1.5% agarose gels and compared by visual inspection. The amplified products were separated by electrophoresis on 1.2% agarose gel (SinaClon, Iran);after staining with ethidium bromide, they were visualized under UV gel documentation system; then they were photographed and compared together by visual inspection.

2.6. Statistical analysis

The results were entered and analyzed using the SPSSTM software,version 22.0 (IBM Corporation, Armonk, NY, USA) and Microsoft Excel 2016 (Microsoft Corporation, USA) statistical software to obtain frequencies and comparison among clones. Chi-square test was applied to analyze intergroup significance. In addition, P＜ 0.05 was considered statistically significant and the confidence interval was 95%.

3. Results

3.1. Occurrence of A. baumannii

A total of 121 (16.1%) non-duplicate A. baumannii isolates were collected and identified in 750 clinical specimens from different wards of Imam Hossein and Imam Khomeini Hospital in Tehran,Iran. The isolates were obtained from 56 (46.3%) males and 65(53.7%) females with the mean age of 39.5 years (ranged 9-79 years). The presence of blaOXA-51-likegene was confirmed in all isolates by PCR.The majority of the isolates were collected from intensive care unit (49.6%) and surgery (14.0%), respectively. The frequencies of collected isolates in terms of the hospital ward and clinical samples are mentioned in Table 2.

3.2. Antimicrobial susceptibility rate

The results of susceptibility testing showed that the colistin with 100% sensitivity rate had the highest effect, while ceftriaxone with 16.5% sensitivity rate had the least effect on A. baumannii isolates. The tigecycline with 77.6% sensitivity rate was the second most effective antibiotic after colistin. Additionally, more than 60% of the isolates were resistant against pipracillin/tazobactam,tetracycline, amikacin, cefepime, tobramycin, and ciprofloxacin.Among 121 A. baumannii strains, 96 (79.3%) and 99 (81.8%)isolates were resistant to imipenem and meropenem, respectively.Ninety-six (79.3%) isolates were simultaneously resistant to imipenem and meropenem. In total, 99 (81.8%) of A. baumannii strains were carbapenem resistant. The results of the susceptibility rate of all tested antibiotics are displayed in Table 3. The results revealed that all A. baumannii strains collected from the trachea,wound, and urine samples were resistant to all antibiotics except for colistin, but the strains isolated from the pleural fluid were susceptible to the majority of antibiotics (Figure 1).

3.3. Multidrug resistance patterns

MDR isolates were determined based on their resistance to at least one antibiotic in three different antimicrobial categories.A total of 109 isolates (90.0%) were MDR with 10 different resistotypes (Table 4). The R4 with the rate of 24.8% was the most prevalent resistotype.

3.4. Molecular characterization of oxacillinase genes

The obtained results from multiplex PCR revealed that 77 (77.7%)isolates of 99 carbapenem resistant A. baumannii had positive PCR band for at least one of blaOXA-likegenes. In total, 75 (75.7%) of carbapenem resistant isolates were positive for blaOXA-23-like, and 14(14.1%) for blaOXA-24-likegene. Meanwhile, the blaOXA-51-likegene was detected in all tested strains. The co-existence of blaOXA-23-likeand blaOXA-24-likegenes were found in 12 (12.1%) isolates. Finally, the blaOXA-58-likeand blaOXA-143-likegenes were not detected in any isolates.

Table 2. Frequency of collected Acinetobacter baumannii strains in terms of the ward and samples (n=121).

Table 3. Resistance rate of Acinetobacter baumannii strains to the tested antibiotics [n (%)].

Figure 1. Antimcrobial resistance of Acinetobacter baumannii in clinical specimens.

Table 4. Resistotypes of Acinetobacter baumannii strains based on tested antibiotics categories.

Table 5. Resistance rate of blaOXA-like producer and non-producer Acinetobacter baumannii isolates [n (%)].

3.5. Resistance patterns of blaOXA-like producer A.baumannii isolates

Antibiogram results of blaOXA-likeproducer and non-producer A.baumannii isolates are shown in Table 3. Based on the results,there was a significant difference in the resistance to imipenem,meropenem, ciprofloxacin, and pipracillin/tazobactam antibiotics between blaOXA-likeproducer and non-producer A. baumannii isolates(Table 5).

3.6. REP-PCR molecular typing

The molecular typing of 99 carbapenem resistant A. baumannii isolates by REP-PCR revealed 9 dissimilar clones (A-I). The five main clones A, B, C, D, and E were detected in 25 (25.2%),36 (36.4%), 10 (10.1%), 8 (8.0%), and 6 (6.1%) of isolates,respectively. The frequency of other clusters were as follows: 4 isolates (4.0%) for clone F, 1 isolate (1.0%) for clone G, 4 isolates(4.0%) for clone H, and 5 isolates (5.1%) for clone I.

4. Discussion

Nowadays, A. baumannii has become a global challenge in terms of therapeutic choices due to the resistance to various antibiotics.Therefore, careful screening of the resistance pattern of this bacterium is very important in health care centers and hospitals. In this study, as expected, similar to previous studies from Iran and different countries, the polymyxins and glycylcycline antibiotic groups were the most effective anti-bacterial drugs against A.baumannii isolates[19-21].

This can be due to the low prescription of these drug categories and their reserve as the last line of infection therapy in our region.Additionally, the prevalence of resistance rate of polymyxins and glycylcycline among A. baumannii isolates was similar to the global report that mentioned the pooled prevalence of 0.0-3.7 and 2.3-25.8 for colistin and tigecycline, respectively[22]. Also, similar to studies reported by Shoja et al.[19]and Sarikhani et al.[23]from Iran, a high resistance rate above 80% was seen against thirdgeneration cephalosporins in our study.

Resistance to carbapenems in A. baumannii strains is an increasingly remarkable phenomenon leading to limitation of treatment choices. Several reports from different regions of Iran indicated a trend toward increasing resistance to carbapenems in clinical isolates of A. baumannii[24]. In the current study, the results of antibiogram revealed that 18.2% of A. baumannii isolates were susceptible to carbapenems which was in line with other studies from different geographic regions of the world that reported an 8%-26% susceptibility rate for imipenem among A. baumannii isolates[25].

The high resistance rate of A. baumannii isolates against meropenem (81.8%) and imipenem (79.3%) in current study was consistent with previous studies from Iran[18,22]. In a recent study by Mobasseri et al.[26]from Iran a resistance rate of 96% were reported against both imipenem and meropenem antibiotics in clinical isolates of A. baumannii.

These findings reflect the fact that the prescription of aforementioned antibiotics for the treatment of infections caused by the A. baumannii should be done with caution and based on the results of in vitro antimicrobial susceptibility testing. Another finding of the current study was the high percentage (90%)of multi-drug resistance among A. baumannii isolates, which confirmed the results of other studies from around the world[27,28].The carbapenem resistance in A. baumannii is caused by various mechanisms, including class D beta-lactamase enzymes. After discovering the OXA-23 the first class D beta-lactamase in a strain of A. baumannii, other oxacillinases with carbapenemase activity have been characterized[29]. The present study demonstrated the presence of intrinsic OXA-51 oxacillinase in all isolates. Despite its poor anti-carbapenem activity, when this enzyme is overproduced,it is able to increase the minimum inhibitory concentration of carbapenems[30].

The OXA-23, OXA-24, OXA-58, OXA-143, and OXA-72 are most abundant acquired oxacillinases that have been observed in A. baumannii isolates[30]. In this study, the multiplex PCR method was performed to investigate the frequency of OXA-23, OXA-24, OXA-58, and OXA-143 oxacillinases in carbapenem resistant A. baumannii isolates. Not surprisingly, the evidence from this research showed the highest frequency of OXA-23 oxacillinase(75.7%) in studied isolates that was in good agreement with earlier studies from Iran[19,23].

However, in this study, 14.1% of A. baumannii isolates were positive for OXA-24 oxacillinase which was in contrast to the findings of Shoja et al.[19]and Karmostaji et al.[31]that reported lower occurrence rate for this oxacillinase. Furthermore, in another study from Brazil, this oxacillinase was not found in any carbapenem resistant A. baumannii isolates that was contrary to present study[21]. Unlike a research carried out by Sarikhani et al.[23]in this area in Iran, we did not detect the OXA-58 and OXA-143 oxacillinases in A. baumannii isolates. They reported an occurrence rate of 55.6% and 14.4% for OXA-58 and OXA-143,respectively.

However, our results were consistent with that of Shoja et al.[19]and Mohajeri et al.[32]that did not detect the OXA-58 in their isolates. Meanwhile, consistent with previous reports from Algeria[33] that mentioned the co-existence of oxacillinases in A. baumannii isolates; this research found that 12.1% of A.baumannii isolates have co-existence of two OXA-23 and OXA-24 oxacillinases.

It should be noted that in this study, 22 isolates from all 99 carbapenem resistant A. baumannii lacked the acquired oxacillinases. The mechanisms of carbapenem resistance in these isolates may be raised from porin loss, specific efflux pump, or presence of metallo-β-lactamases such as the NDM, IMP and VIM types[34,35].

5. Conclusion

The main aim of this study was to achieve a baseline background to determine the resistant pattern of oxacillinase producing A.baumannii in clinical samples. Result from this study affords valuable knowledge to perform further large prospective study to identify the other mechanisms involved in carbapenem resistant A.baumannii isolates.

The OXA-23 was the main oxacillinase that contributed to carbapenem resistance in our study. Observing the high frequency of multiple drug resistant A. baumannii isolates in this study,highlighted the need for continuous and regularly monitoring of antibiotic resistance patterns of these bacteria in our region to control the spread of MDR pathogens in the healthcare facilities.

Conflict of interest statement

The authors report no conflict of interest.

Acknowledgement

The authors would like to appreciate the staff of the Clinical Research Development Unit at Imam Hossein hospital, affiliated to Shahid Beheshti University of Medical Sciences, Tehran, Iran for their kind services to develop and release of the results of this research.

Authors’ contribution

M.A.M., M.D., A.J., B.G.S., M.K.Z.: Conceived and designed the experiments; performed the experiments; contributed reagents,materials, analysis tools or data and interpreted. M.D., A.J., B.G.S.and S.K.: Analyzed and interpreted the data; contributed reagents,materials and wrote the paper.