Molecular Detection of blaOXA -type Carbapenemase Genes and Antimicrobial Resistance Patterns among Clinical Isolates of Acinetobacter baumannii

Acinetobacter baumannii is a bacterium found in most places, especially in clinics and hospitals, and an important agent of nosocomial infections. The presence of class D enzymes such as OXA-type carbapenemases in A. baumannii is proven to have a key function in resistance to carbapenem. The aim of the current study is to determine the blaOXA -type carbapenemase genes and antimicrobial resistance among clinically isolated samples of A. baumannii. We assessed 100 clinically isolated specimens of A. baumannii from patients in intensive care units of educational hospitals of Hamadan, West of Iran. The A. baumannii isolates' susceptibility to antibiotics was performed employing disk diffusion method. Multiplex polymerase chain reaction was used to identify the bla OXA-24-like , bla OXA-23-like , bla OXA-58-like , and bla OXA-51-like genes. The bla OXA-23-like , bla OXA-24-like , and bla OXA-58-like genes' prevalence were found to be 84, 58, and 3%, respectively. The highest coexistence of the genes was for bla OXA-51/23 (84%) followed by bla OXA-51/24-like (58%). The bla OXA-51/23- like pattern of genes is a sort of dominant gene in resistance in A. baumannii from Hamadan hospitals. The highest resistance to piperacillin (83%) and ciprofloxacin (81%) has been observed in positive isolates of bla OXA-23-like . The A. baumannii isolates with bla OXA-58-like genes did not show much resistance to antibiotics. Based on the results of the phylogenetic tree analysis, all isolates have shown a high degree of similarity. This study showed the high frequency of OXA -type carbapenemase genes among A. baumannii isolates from Hamadan hospitals, Iran. Thus, applying an appropriate strategy to limit the spreading of these strains and also performing new treatment regimens are necessary.


Introduction
Acinetobacter baumannii is an oxidase-negative, gram-negative, nonmotile, nonfermentative coccobacilli and it is found inside most places, especially in hospitals and other health care facilities. [1][2][3] The bacterium is mostly originating from the intensive care units (ICUs). It can cause septicemia, infections of the skin, meningitis, endocarditis, urinary tract infections, soft tissues infections, and ventilator-associated pneumonia in patients in ICU wards. [4][5][6][7] Therefore, infections caused by the bacterium need early and effective antimicrobial therapy. The carbapenems are the most effective antibacterial agents in the case of clinical isolates of A. baumannii. 8 Nowadays, due to excessive and inappropriate use of these drugs, the resistance to carbapenems has increased. 9 A. baumannii can become carbapenem resistant by the acquisition of plasmid-mediated carbapenem hydrolyzing classes of A, B, and D metallo-β-lactamase enzymes. 10 Class D enzymes such as OXA-type carbapenemases, especially the intrinsic presence of bla OXA-58-like , bla OXA-24-like , bla OXA-23-like , bla OXA-51 , and β-lactamases in A. baumannii, are proven to have a vital role in resistance to carbapenem. [11][12][13] The OXA-carbapenemase genes can be encoded by chromosome or plasmid and are mostly associated with insertion elements, particularly ISAbal. 8 With the rise of pan-drugresistant (PDR) and multidrug-resistant (MDR) strains of A. baumannii all around the world, the bacterium has become a very serious threat for health care organizations. 10,14 Thus, the current study focused to discover the blaOXA -type carbapenemase genes, which, according to previous studies, contribute to more antibiotic resistance among clinical isolates of A. baumannii.

Sequencing
Three PCR products of each gene, which was separately performed, were purified and sequenced directly by Sanger dideoxy-sequencing technology in two directions using a capillary DNA analyzer (Applied Biosystems, Waltham, Massachusetts, United States).

Phylogenetic Analysis of OXA-type Variant
The phylogenetic analysis was conducted independently for the OXA-type variant. The tree is drawn to scale, with branch lengths measuring the number of substitutions per site. The sequences were aligned and manually edited in consensus positions and compared with that of the sequences from

Statistical Analysis
Statistical analysis was done using Statistical Package for the Social Sciences 23.0 (SPSS, Chicago, Illinois, United States). The Pearson chi-squared test and Fisher's exact test were used to analyze the qualitative data. All p-values < 0.05 were considered statistically significant.

Results
The highest resistance to piperacillin (83%) and ciprofloxacin (81%) has been observed in blaOXA23-like, and the highest resistance and sensitivity were observed for ciprofloxacin (57%) and ampicillin/sulbactam (28%) in blaOXA-24-like positive A. baumannii isolates, respectively. The A. baumannii isolates with blaOXA-58-like genes did not show much resistance to antibiotics. Considering the presence of the studied genes, 3, 58, and 84% of the 100 samples were carrying blaOXA-58-like, blaOXA-24-like, and blaOXA-23like genes, respectively (►Fig. 1 and ►Table 2). Among the 100 A. baumannii isolates, 97.9% of the imipenem-resistant isolates were positive for at least one OXA-type gene. Moreover, 95.9% and 1% of the meropenem and colistin sulfate resistant isolates were carrying OXA-type genes.

Results of Phylogenetic Tree Analysis of OXA-type Variant
The sequences received from the isolates were confirmed as the OXA-type variants and were deposited in the GenBank under the accession numbers as follows: bla OXA-51-like : KJ451411, KJ451412, and KJ451413; bla OXA-23-like : KJ451414 and KJ451415; and bla OXA-24-like : KJ451416, KJ451417, and KJ451418 (►Fig. 2). Analysis of the phylogenic tree demonstrates that all isolates have shown a high degree of similarity.

Correlation between Contributions of bla OXA Genes with Antibiotic Resistance
The relationship between blaOXA-types and resistance to different antibiotics was determined by statistical analysis.
The prevalence of oxacillinases enzymes encoding genes in antibiotic-resistant isolates was higher than the susceptible strains, but there was no statistically significant relationship (►Table 3).

Discussion
Currently, carbapenem resistance in A. baumannii isolates has become a worldwide problem. This study's results demonstrated that significant numbers of clinical isolates of A. baumannii, especially resistant to imipenem and meropenem isolates, were affirmative for the variety of OXA gene expressions. Commonly, the carbapenem antibiotics are highly active against A. baumannii isolates and resistant to β-lactamase enzymes. However, the resistance to these compounds is a problematic issue and is not a result of the presence of a distinct mechanism, while a combination of diverse mechanisms such as enzymatic and nonenzymatic ones are involved. The most significant mechanism of resistance to carbapenems is the enzymatic hydrolysis, arbitrated by the carbapenemases. 11,20 Recently, the number of βlactamase enzymes in class D has increased significantly. Some of the OXA-type carbapenemases are widely found in A. baumannii isolates. However, many of the OXA-type    carbapenemases illustrate simply weak catalytic activity, but resistance to carbapenems may be caused by a combined action of different OXA-type carbapenemase. 21 Feizabadi et al in 2008 studied the susceptibility to antimicrobial and blaOXA genes distribution among Acinetobacter spp. in Tehran hospitals. They reported the coexistence of bla OXA-51/23 and bla OXA-51/24-like in 25% (n ¼ 32) and 17.9% (n ¼ 23) of their studied isolates, respectively. Over 70% of their studied carbapenem-resistant isolates had at least two genes encoding OXA-type carbapenemase. 22 In our study, the highest coexistence was for bla OXA-51/23 (84%) followed by bla OXA-51/24-like (58%), which is considerably higher than their report from Tehran and indicates an elevation in the prevalence of A. baumannii isolates carrying these genes over the time.

Conclusion
This study's results demonstrated that carbapenem resistance is increasing among A. baumannii clinical isolates in our area and is often associated with multidrug resistance. Moreover, the diversity of blaOXA genes was high among these isolates and OXA-23 is the most important carbapenemase mechanism responsible for the resistance phenotype.

Consent for Publication
All authors consented for publication of the above paper.

Availability of Data and Material
All data generated or analyzed during this study are included in this published article.

Funding
The present study was supported financially by the Vice-Chancellor of Research and Technology, Hamadan University of Medical Sciences, Hamadan, Iran.