Identification of Imipenem-Resistant Genes in Acinetobacter baumannii Isolated from Baghdad Hospitals

During the last two decades A. baumannii became clinically important pathogen due to its capability for outbreaks and resistance to most antibiotics including carbapenems [1]. The outbreak of A. baumannii associated with United States military operations in Iraq generated special interest in this organism [2]. The features of A. bumanii for resistance to most available antibiotics and environmental stress enable the bacteria to persist in hospitals and epidemic spread [3]. Several studies reported that carbapenem-resistant A. baumannii strains have been now emerged around the world [4-6]. This resistance is principally caused by the production of carbapenemases [7]. Carbapenem-hydrolysing class D β–lactamases (CHDLs) are the most often reported mechanisms of carbapenem resistance in A. baumannii and four groups of CHDLs have been identified inimipenem-resistant A. baumannii, including intrinsic and chromosomally located OXA51-like β-lactamases and acquired OXA-23-like, OXA-24-like and OXA-58-like β-lactamases [8,9].


Introduction
During the last two decades A. baumannii became clinically important pathogen due to its capability for outbreaks and resistance to most antibiotics including carbapenems [1]. The outbreak of A. baumannii associated with United States military operations in Iraq generated special interest in this organism [2]. The features of A. bumanii for resistance to most available antibiotics and environmental stress enable the bacteria to persist in hospitals and epidemic spread [3]. Several studies reported that carbapenem-resistant A. baumannii strains have been now emerged around the world [4][5][6]. This resistance is principally caused by the production of carbapenemases [7]. Carbapenem-hydrolysing class D β-lactamases (CHDLs) are the most often reported mechanisms of carbapenem resistance in A. baumannii and four groups of CHDLs have been identified inimipenem-resistant A. baumannii, including intrinsic and chromosomally located OXA-51-like β-lactamases and acquired OXA-23-like, OXA-24-like and OXA-58-like β-lactamases [8,9].
Till now, no study has been performed in Iraq identification of Imipenem-Resistant A. baumannii genes among Iraqi patients. Therefore, this study aimed to investigate the Imipenem-Resistant bla-OXA β-lactamases genes in A. baumannii among patients in four hospitals in Baghdad, Iraq.

Isolation and processing of samples
One thousand and nine hundreds specimens comprising, urine, wounds, burns, blood and sputum, were collected in sterilized containers from patients attending four hospitals in Baghdad-Medical city including: Baghdad Teaching Hospital, the Martyr Gazi Al-Hariry Hospital, Welfare Teaching Hospital and the Burn Specialist Hospital.
All bacterial isolates were identified by routine morphological, biochemical carried out according to Forbes et al. [10]. Additionally, identification was confirmed by api20E (bioMerieux, France).
Furthermore, species identification of Acinetobacter baumannii isolates was performed by Polymerase chain reaction (PCR) to detect blaOXA-51-Like genes [11,12]. The results of bacterial identification were published separately [13].
Antibiotic susceptibility was done for 20 antibiotics available in the market. Disc agar diffusion test was performed according to the Kirby-Bauer standardized antimicrobial susceptibility single disc method [14]. Serial dilution agar method was applied for the determination of minimal inhibitory concentrations (MICs) to Imipenem and Meropenem [15]. Escherichia coli (E. coli ATCC 25922) was used as a quality control in susceptibility determination.
An isolate was interpreted as susceptible, intermediate, or resistant to a particular antibiotic by comparison with standards inhibition zones or MIC break point according to Clinical Laboratories Standards Institute (CLSI) [16].

Extraction of genomic DNA
DNA was extracted from A. baumannii isolates using a commercial purification system (Genomic DNA Mini Kit, Geneaid, Thailand), then the DNA concentration and purity were determined [17].

Sequencing of PCR products
Multiplex PCR products for blaOXA-23 like, blaOXA-24like, blaOXA-51 like and blaOXA-58 like genes were detected by agarose gel electrophoresis and purified by Gel/ PCR DNA Fragment Extraction Kit (Geneaid, Thailand), and then the sequencing was carried out using the ABI capillary system (Macrogen, Korea). PCR products were subjected to direct sequencing. DNA sequences were analysed and similarity searches were carried out with the Basic Local Alignment Search Tool (BLAST) in National Centre for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).

Antibiotic susceptibility
Data published previously [14] indicated a high level resistance of A. baumannii clinical isolates to most of the antibiotics under test. The MIC values for imipenem and meropenem resistant isolates ranged from 16 μg/ml to 512 μg/ml.

Detection of carbapenem resistance genes (oxacillinases) by multiplex polymerase chain reaction (PCR)
The results of resistance genes amplification presented in Figure 1 and Table 2 illustrate the presence of blaOXA-51-like genes in all 128 (100%) A. baumannii clinical and environmental isolates (regardless to imipenem suceptibility). The blaOXA-51-like genes were reported to be highly specific for the identification of A. baumannii at the species level [18,19]. The blaOXA-23-like genes present in 91.03% of IRAB which indicated its responsibility for the dominant carbapenem resistance gene in the local A. baumannii isolates. Nevertheless, similar findings were reported in countries other than Iraq, such as Bulgaria, China, Brazil, Afghanistan [20], Korea, Singapore and Thailand [21].
A study carried out by Hujer et al. [22] demonstrated that 97% of A. baumannii strains (isolated from military and civilian personnel injured in the Iraq/Kuwait region during Operations in Iraq have blaOXA-69-like gene (a member of blaOXA-51-like genes). blaOXA-23-like genes detected in 11% of those strains and the blaOXA-58-like genes were detected in 12% of the isolates; whereas blaOXA-24-like genes were not detected at all. This may specify the non-relatedness between Hujer's isolates and those isolated in the present study.
It is markedly noticed that two (4%) out of fifty imipenem sensitive A. baumannii environmental isolates harboured blaOXA-58-like genes in addition to blaOXA-51-like genes. Although these two isolates have blaOXA-58-like genes, no resistance was developed phenotypically. Such finding could be assigned to the absence of the insertion sequence ISAba1 upstream blaOXA-58-like genes. Corvec et al. [23] and Zhong et al. [24] stated that in A. baumannii the OXA genes are expressed when the insertion sequence ISAba1 inserts upstream of the gene where it provides a promoter for gene expression.

Sequencing of multiplex PCR products
Results revealed that GenBank accession numbers for the nucleotide sequences of the blaOXA-51-like, blaOXA-58-like, blaOXA-23-like and blaOXA-24-like genes fragments are JX305928, JQ409994, JQ343841 and JX310716, respectively. In OXA-51-like genes sequencing we found some differences in the nucleotides of our query and the subject in position 5,11,21,35,40,111,247,268,289  Interestingly, OXA-207 gene was found among the OXA-24-like genes in local A. baumannii isolates; this gene (OXA-207) has not been previously reported in A. baumannii strains. Nevertheless, it was mentioned to be found in other species of the genus Acinetobacter.
In this study we identify the sequences of Imipenem-resistance genes in our local A. bumannii isolates; this will improve the management and control of this pathogen in Baghdad hospitals.