Molecular Detection of Carbapenemase-Encoding Genes in Multidrug-Resistant Acinetobacter baumannii Clinical Isolates in South Africa

Introduction Carbapenem-resistant Acinetobacter baumannii has been responsible for an increasing number of hospital-acquired infections globally. The study investigated the prevalence of carbapenemase-encoding genes in clinical multidrug-resistant A. baumannii strains. Materials and Methods A total of 100 nonduplicate multidrug-resistant A. baumannii strains were cultured from clinical samples obtained from healthcare facilities in the O. R. Tambo district. The strains were confirmed by detecting the intrinsic blaOXA-51-like gene. Antimicrobial susceptibility testing was performed by VITEK® 2 and autoSCAN-4 systems. The MIC of imipenem and meropenem was rechecked by E-test. Colistin MIC was confirmed by the broth microdilution method. Real-time PCR was performed to investigate the presence of carbapenemase-encoding genes. Results Most strains showed high resistance rates (>80%) to the antibiotics tested. Resistance to amikacin, tetracycline, and tigecycline were 50%, 64%, and 48%, respectively. All strains were fully susceptible to colistin. The blaOXA-51-like was detected in all strains whilst blaOXA-23-like, blaOXA-58-like, blaOXA-24-like, blaIMP-1, blaVIM, and blaNDM-1 were found in 70%, 8%, 5%, 4%, 3%, and 2% of strains, respectively. None of the tested strains harboured the genes blaSIM and blaAmpC. The coexistence of blaOXA-23-like, and blaIMP-1 or blaOXA-58-like was detected in 1% and 2% strains, respectively. A distinct feature of our findings was the coharbouring of the genes blaOXA-23-like, blaOXA-58-like, and blaIMP-1 in 2% strains, and this is the first report in the Eastern Cape Province, South Africa. The intI1 was carried in 80% of tested strains whilst ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like were detected in 15% and 40% of the strains, respectively. The detection of blaOXA-23-like, ISAba1/blaOXA-51-like, ISAba1/blaOXA-23-like, and blaOXA-23-like, blaOXA-58-like, and blaIMP-1 carbapenemases in strains had a significant effect on both imipenem and meropenem MICs. Conclusions Results showed a high level of oxacillinases producing A. baumannii circulating in our study setting, highlighting the need for local molecular surveillance to inform appropriate management and prevention strategies.


Introduction
Acinetobacter baumannii, an opportunistic Gram-negative bacterium, has become a predominant cause of healthcareassociated infections worldwide [1]. is microorganism has emerged to become a major concern for clinicians worldwide causing infections such as septicaemia, meningitis, endocarditis, ventilator associated pneumonia, burns, urinary tract infections, surgical site infections, and wound infections in hospitals due to its intrinsic as well as its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents [2,3]. e World Health Organization (WHO) lists A. baumannii among critical antibiotic-resistant "priority pathogens," highlighting its serious threats to public health [4]. Risk factors for A. baumannii infections include immune suppression, burns, trauma, mechanical ventilation, catheters, invasive medical procedures, previous antibiotic treatment, an extended hospital stay (>90 days), and underlying diseases such as diabetes [5].
Carbapenem has been the drug of choice used to treat MDR A. baumannii infections over the past decade because they are highly efficacious and have low toxicity [6]. However, the resistance rate against carbapenem has increased dramatically due to its frequent application in recent years and is mainly mediated by the production of carbapenem-hydrolysing enzymes [7]. e most common mechanism of carbapenem resistance in A. baumannii is the production of β-lactamases, including enzymes of Ambler Classes D OXA-type carbapenemases consisting of bla OXA-23-like (OXA-23, OXA-27 and OXA-49), bla OXA-40like , (OXA-24/40, OXA-25, OXA-26 and OXA-72), bla OXA-58-like (OXA-58 and OXA-96), bla OXA-143-like and bla OXA-51-like enzymes, and B Metallo-β -lactamases (MBLs) mainly bla VIM , bla IMP-1 , bla NDM-1 , bla SPM , bla GIM , and bla SIM types [8]. Among the multiple types of MBL genes described throughout the world, the bla IMP-1 , bla VIM , and bla SIM types are the most common [9]. e upstream of OXA-type class D carbapenemases in Acinetobacter is often associated with insertion sequence (IS), of which ISAba1 is the most commonly detected. ISAba1 and other IS may modulate the expression and transfer of OXA-type carbapenemase genes [10]. Integrons have been demonstrated to play a major role in the dissemination of multidrug-resistance among Gramnegative bacteria through the capture and expression of resistant genes embedded within cassettes [11].
Studies in Africa and the Gulf Cooperation Council (e.g., Saudi Arabia) have reported increasing carbapenem resistance among A. baumannii isolates [12][13][14]. Since nosocomial A. baumannii infection has generated an abundance of therapeutic obstacles for the treatment of hospitalized patients in South Africa, as well as other countries, the knowledge regarding the prevalence level of antibiotic resistance genes and the resistance pattern of this bacterium to the initial antibiotics is required to control, prevent, and cure A. baumannii driven infections. erefore, the aim of the current study was to investigate the rates of carbapenem resistance as well as to detect the frequency of carbapenemase-encoding genes in MDR A. baumannii isolates obtained from hospitalized patients receiving treatment in healthcare facilities in O.R. Tambo district in the rural Eastern Cape Province of South Africa.

Study Design and Settings.
is was a prospective crosssectional study conducted at healthcare facilities in the OR Tambo  e strains were preliminarily speciated phenotypically using standard microbiological procedures: the VITEK ® 2 automated machine with Gram-negative identification (GNI) cards (BioMérieux, France) and the MicroScan autoSCAN-4 System (Dade Behring Inc., Deerfield, IL) with Gram-negative ID type 2 panel. A high percentage (≤95%) was utilized as the acceptance criterion for identification by the MicroScan autoSCAN system. Suspected colonies were also further verified using the Acinetobacter specific primer set Ac436F and Ac676r to amplify the 16S rRNA gene. Confirmation of A. baumannii strains was carried out by polymerase chain reaction analysis of the presence of inherent bla OXA-51-like genes. All of the strains were stored at −80°C in skim milk with 15% glycerol until further use.

Antimicrobial Susceptibility Testing.
A total of 18 clinically relevant antibiotics were tested using MicroScan autoSCAN-4 Gram-negative MIC panel, and results were analyzed and interpreted according to the recommended clinical breakpoints given in the clinical and laboratory standard institutes (CLSI) guidelines v27 [15].  baumannii isolates were defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories [17]. All results were within quality control ranges.

Detection of Carbapenem-Resistant Genes.
Genomic DNA from an overnight culture on tryptic soy broth of A. baumannii was extracted using the MagNaPure Compact® nucleic acid isolation kit I (ref. no. 03730964001, Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. A final elution volume of 200 µL of pure A. baumannii DNA was used. 2 µL of the DNA extract was used for each PCR analysis. e extracted DNA was stored at −80°C until required for further analysis. All MDR isolates were systematically screened by LightCycler 2.0 real-time PCR (Roche Diagnostics) instrument targeting some of the epidemiologically most relevant resistant genes. e presence of the genes encoding Amber class D Serine-Carbapenemase genes (bla OXA-51-like , bla OXA-23-like , bla OXA-24-like , and bla OXA-58-like ), Ambler class B Metallo-β-lactamases (MBLs) (bla IMP-1 , bla VIM , bla SIM , and bla NDM-1 ), and Ambler class C bla AmpC were assessed. MDR A. baumannii strains were subsequently screened for the presence or absence of IS element ISAba1 [18] and class 1 integrons by real-time PCR [19]. To determine the overexpression of bla OXA-23-like and bla OXA-51-like families due to the upstream adjacent ISAba1, PCR was firstly performed for ISAba1, and in cases where bla OXA-like is present, a combination of forward primer of ISAba1 and reverse primer of bla OXA-like family was further used. Appropriate positive and negative controls were run simultaneously (Table 1). e primers used for PCR amplification of the carbapenemase genes are listed in Table 2.

Statistical
Analyses. Data analyses were performed using the Statistical Package for Social Sciences Statistics for Windows software package version 23.0 (IBM, Armonk, NY). Results were expressed using frequency and percentages for qualitative variables and as mean (standard deviation) or median (interquartile range: IQR) for quantitative variables. e association between A. baumannii and drug resistance was analyzed by the chi-square test. All P values were two-sided. A P-value <0.05 was considered statistically significant.

Ethical Considerations.
Ethical approval for this project was obtained from the Walter Sisulu University Research Ethics and Biosafety Committee (Reference number: 019/ 2016). No written informed consent was required because each MDR A. baumannii isolate obtained was delinked with the patient's personal information. Also authors had no direct interactions with patients from whom these isolates were isolated.

Demographic Characteristics of the Study Population.
e demographic characteristics of the patients with A. baumannii infection analyzed in our study showed a slight female preponderance of 47% males versus 53% females). e age of the overall patients ranged between 1 day and 91    to tigecycline. All strains were susceptible to colistin. Findings of the antimicrobial susceptibility patterns of all A. baumannii strains in this study are shown in Figure 1.

PCR Results of Carbapenemase-Encoding Genes.
Results showed that all of the strains investigated in this study had the intrinsic bla OXA-51-like gene. Among them, 70% were positive for the bla OXA-23-like , 8% were positive for bla OXA-58-like, and 5% positive for bla OXA-24-like . For the MBLs, 4% strains were positive for bla IMP-1 , 3% carried bla VIM , 2% carried bla NDM-1, and none of the tested isolates harboured the bla SIM . Alleles encoding bla AmpC enzymes were not detected in any strain. e ISAba1 was located upstream the bla OXA-51-like and the bla OXA-23-like carrying strains 15% and 40%, respectively. Some of the strains coharboured different carbapenemase genes mostly yielding very broad-spectrum antibiotic resistance profiles to imipenem and meropenem. Both strains possessing the ISAba1/ bla OXA-23-like and bla OXA-23-like , bla OXA-58-like, and bla IMP-1 genes had the highest carbapenem resistance with these strains having imipenem MICs of 32 µg/mL or more, and having meropenem MICs of 32 µg/mL or more. is was followed by the strains carrying the bla OXA-23-like gene with 62% having imipenem MICs of 32 µg/mL or more, and 58% having meropenem MICs of 32 µg/mL or more (Figures 2  and 3). Screening for the presence of integron integrase genes showed the presence of the class 1 integrase gene in 80% MDR A. baumannii strains (Table 3).

Discussion
Understanding the fundamental mechanism that underlines A. baumannii infections, including original source of infecting strains, resistance patterns, and genes responsible for the development of resistance is critical for the development of appropriate infection control measures and more efficient treatment strategies [5]. Data from countries that lack routine surveillance, including South Africa, are necessary in order to understand and prevent the dissemination of carbapenem-resistant strains of species that are in epidemiological expansion, like A. baumannii. Results from the present study revealed a 100% resistant rate of MDR A. baumannii to third-generation cephalosporins (ceftazidime, cefotaxime, and ceftriaxone). Resistance to cefepime was 87%. All strains were resistant to piperacillin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and ampicillin/sulbactam. e percentage rates of resistance to remaining antibiotics range from 48% to 89% with the exception of colistin which had no resistant strain detected.
Carbapenem resistance of A. baumannii is a major concern since it is the drug of choice in the treatment of A. baumannii infections [26]. e results showed that the majority of A. baumannii strains tested were resistant to both imipenem 81% and meropenem 83%. Lowings et al. found all MDR A. baumannii isolates showed high level resistance (cefotaxime 100%; cefotaxime 100%; ceftazidime 89%; cefepime 90%; imipenem 86%; and meropenem 86%) in a study conducted at the Department of Medical Microbiology, Prinshof Campus, University of Pretoria/National Health Laboratory Service (NHLS) in 2013 [27]. A previous study by Kock et al. in the same setting showed that the overall percentage of resistance to the cephalosporin antibiotics was cefepime (62%) and ceftazidime (45%) [28]. A report on antimicrobial resistance surveillance from sentinel public hospitals in South Africa in 2013 showed a resistance rate of 80% to cefepime and 73% to ceftazidime [29]. Comparing the results of the present study with these earlier studies indicates that the resistant rate of MDR A. baumannii to various antibiotics has not decreased. High levels of resistance to third and fourth generation cephalosporins in this study indicated that cephalosporins are no longer efficacious in the treatment of MDR A. baumannii isolated in South Africa. e full susceptibility of all MDR A. baumannii to colistin in this study indicated that colistin is still a better option of the drug for the treatment of infections caused by A. baumannii in South African hospitals. However, the issue of nephrotoxicity, neurotoxicity, colistin-resistance, and heteroresistance shown by colistin monotherapy is a challenge in the management of this infection [30]. e high resistance rate to other antibiotic categories may probably be due to the heavy selection pressure from overuse of these antibiotics.
Although A. baumannii infections are observed in all body parts, they are mostly observed in the respiratory system. Our study showed that A. baumannii isolates were predominantly found in the respiratory samples (tracheal aspirate) and the most frequent setting for MDR A. baumannii was found in the ICUs (41%). ICUs (Neonatal and general intensive care) are settings where most patients have indwelling devices and invasive procedures. Guckan    International Journal of Microbiology Molecular detection of carbapenemase-encoding genes has shown that the production of OXA-type carbapenemases is predominant in A. baumannii [30,32]. e chromosomally encoded bla OXA-51-like gene was detected in all the strains supporting those of other studies demonstrating that the detection of the bla OXA-51-like gene can be used as a supplementary tool to identify A. baumannii at the species level together with additional methods [33]. It was observed that the expression of bla OXA-23-like was the second dominant carbapenem resistance mechanism (70%) in MDR A. baumannii among the strains investigated aside the intrinsic bla OXA-51-like gene. Its prevalence among isolates was higher than this in previous reports in South Africa. Its prevalence among strains was higher in this study than in the previous report (59%) by Kock et al., whilst Lowings et al. reported a prevalence of 77%.
e bla OXA-23-like gene is considered a virulence biomarker and a significant cause of carbapenem resistance worldwide [34]. is gene was first reported in 1985 in Scotland and since then outbreaks of OXA-23 carbapenemase-producing A. baumannii have been reported all over the world and is a dominant genetic determinant in the Asia subcontinent [35]. In Latin America, Brazil was the first country to report OXA-23-like-producing isolates in 2003, followed by Colombia, Argentina, and Mexico some years later [36].
is study also detected bla OXA-24-like and bla OXA-58-like genes in 5% and 8% isolates, respectively. Although the acquired bla OXA-23-like is the dominant genetic determinant reported in most studies in South Africa, the detection of bla OXA-58-like and bla OXA-24-like genes in our study shows that these genes are becoming significant in carbapenem resistance in A. baumannii and may be responsible for the increase in carbapenem resistance in A. baumannii. It also suggested that these oxacillinases may locally spread in Mthatha, South Africa.
ese findings concur with a study by Ji et al. in China, who reported that bla OXA-23-like are the most frequent carbapenemresistant A. baumannii, whereas bla OXA-24-like and bla OXA-58-like genes pose potential threats of hospital outbreaks of multidrugresistant A. baumannii [37]. For class B metallo-β-lactamases, we found that 4%, 3%, and 2% of the isolates had bla IMP-1 , bla VIM, and bla NDM-1, respectively, and no isolate was found to produce bla SIM gene. None of the clinical A. baumannii isolates harboured bla AmpC . IMP (primarily detected in South Korea) and NDM-1 (recently reported in many countries, such as India, Israel, Egypt, Germany, Spain, Switzerland, the United Arab Emirates, and China) are the two MBL genes most frequently detected in A. baumannii isolates [7,33]. e bla NDM-1 producing bacteria are mainly reported in Enterobacteriaceae [38]. is gene was first identified from Klebsiella pneumoniae and E. coli strains from a patient previously hospitalized in India and has recently emerged in A. baumannii isolates [32]. Taking into consideration the diversity of the South African community (prominence of Asian and European communities where bla NDM-I have been reported) and the frequency of travels made by individuals between these countries, it is believed that the spread of bla NDM-1 was likely to occur mostly through A. baumannii rather than Enterobacteriaceae. However, these findings need further investigation.
Interestingly, this study showed that some isolates were found to coharbour carbapenemase genes. Among these isolates include the coharbouring of carbapenemase genes bla OXA-23-like and bla IMP-1 in 1% isolate, bla OXA-23-like and bla OXA-58-like in 2% isolates, and bla OXA-23-like , bla OXA-58-like , and bla IMP-1 in 2% isolates. To the best of our knowledge, this is the first report of the coharbouring of bla OXA-23-like , bla OXA-58-like , and bla IMP-1 in the Eastern Cape province of South Africa. We believe that coharbouring of these resistance genes is due to the presence of different genes in the same strain. is warrants the use of molecular typing methods to determine the clonal types of the isolates. e coharbouring of these major resistance mechanisms seriously limits therapeutic options, raising concerns regarding their transmission to other organisms. For example, we found that all isolates that coharboured bla OXA-23-like , bla OXA-58-like, and bla IMP-1 had imipenem MICs of 32 μg/mL or more and meropenem MICs of 64 μg/mL. e study showed that carbapenem susceptibility phenotypes of our A. baumannii isolates clearly correlated with the presence or absence, in the genomes, of ISAba1-linked bla OXA-23-like or bla OXA-51-like genes (Figures 2 and 3). For instance, the 100% MDR isolates with ISAba1 elements upstream of the bla OXA-23-like had imipenem and meropenem MICs 32 μg/mL or more, whereas 83.34 and 77.78% of MDR isolates with ISAba1 elements upstream of the bla OXA-51-like had imipenem and meropenem MICs 32 μg/mL, respectively. Presumably, the ISAba1 element likely provides exogenous promoter functions to overexpress the linked genes. Evidence showed that carbapenem resistance mediated by overexpression of bla OXA-like families due to upstream IS elements is more prevalent [39][40][41][42]. Our data are consistent with the findings of previous studies in that acquisition ISAba1 are the main mechanism of carbapenem resistance among A. baumannii by acting as a promoter to increase oxacillinase expression [9,39,43]. However, there are limited studies in South Africa as most of the studies evaluated the prevalence of OXA-like families in detail. ere is, therefore, the need for further investigations to find the contribution of other IS elements (ISAba2, ISAba3) upstream to OXA-like families in different parts of the country. e results suggested that class 1 integrons did not contribute resistance to carbapenems since its presence did not have an effect on the MICs of carbapenems.

Limitations.
e study has some limitations. Firstly, the study was conducted in a single centre, NMAH in the Tambo district municipality, so our findings may not be generalized to other settings. Secondly, the study did not include molecular typing epidemiological study reference methods such as PFGE, MLST, REP-PCR, etc. which would have better evaluated the dissemination of a carbapenemase or some clones to understand CRAB isolates in the clinical setting. ese studies are necessary to help highlight the need for area-specific surveillance to inform appropriate management.

Conclusion
In conclusion, the results of this study provide direct evidence of the spread of carbapenemase-encoding genes International Journal of Microbiology especially bla OXA-23-like in our setting. High presence of the ISAba1 element upstream of the bla OXA-51-like and bla OXA-23like increases expression of these carbapenemases and presents an emerging threat in this study setting. e results also provided clinically relevant insights into antimicrobial susceptibility and knowledge of the prevalent resistance mechanism among CRAB.
is information will help in preventing the development of MDR A. baumannii in hospitals and also helps clinicians in the empirical treatment of patients. To prevent the development of Carbapenemresistant A. baumannii, routine implementation of simple and cost-effective screening methods to detect carbapenemase production in hospitals is crucial in enhancing infection control practices and finally establishing antimicrobial stewardship programs. Contribution and interplay of different mechanisms to the rapid increase in antibiotic resistance of A. baumannii in South Africa warrant further investigation.

Data Availability
All data generated or analyzed during this study are included in this published article (and its supplementary information file).

Ethical Approval
Ethical approval for this project was obtained from the Walter Sisulu University Research Ethics Committee (Human) (reference number: 019/2016).

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.