Prevalence of transferable OXA-1 b-lactamase Associated with Carbapenem-Resistant Klebsiella pneumoniae isolates in iraq

this study was designed to explore the incidence of blaOXA-1 amongst Klebsiella pneumoniae isolates with resistant to carbapenem. Between December 2014 and April 2015, one hundred samples were taken from two hospitals: Babylon Teaching Hospital for Maternity and Pediatric / Babylon Province (clinical, umbilical infections, n= 40; environmental, n=20) and Karbala Hospital for Pediatric / Karbala Province (40 stool samples). All patients were hospitalized or attended these hospitals, all under 1 year of age. Seventeenth (17%) isolates were identified as Klebsiella pneumoniae. The antibiotic resistance profile of isolates was tested using disk diffusion method. High-level of resistance was recorded with ampicillin (94.1%) and piperacillin (88.2%) antibiotics. Resistance to carbapenem was reported in two K.pneumoniae isolates, these were investigated for the existence of OXA-1b-lactamase using Polymerase Chain Reaction (PCR) technique. Two (100%) isolates gave positive result. Transference of this gene was studied by conjugation experiment. The blaOXA-1 gene conjugated successfully in 1 (50%) isolate only.


iNtRODuCtiON
Antimicrobial resistance is a major public health problem worldwide. Infections caused by multi-drug resistance organisms due to long hospital stay, antibiotics treatment and poor hygiene are in continuous increase and linked with high rates of mortality and morbidity 1,2 . The possible resistance mechanism in Klebsiella spp is the production of extended spectrum betalactamases (ESBLs). These enzymes are capable of hydrolyzing penicillin, cephalosporin (3 rd and 4 th generation), monobactams, but have no effect on cephamycins or carbapenem 3,4 .
The predominant mechanisms for resistance to inhibitor penicillin combinations are : class C chromosomal b-lactamase production, overproduction of TEM-1 and TEM-2 type blactamases and OXA-1 b-lactamase production 5,6,7 .
OXA-1 b-lactamase has the ability to hydrolyzes amino, ureidopenicillins (piperacillin),cloxacillin, oxacillin and methicillin in significant mean while it hydrolyzes cephalosporins (narrow -spectrum) weakly. Moreover, it hydrolyzes broad-spectrum cephalosporins, mediated diminished susceptibility to antibiotics like cefepime and cefpirome 8,9 . OXA-1 b-lactamase distributed widely among Enterobacteriaceae family and a major reason for resistance to amoxicillin /clavulanic acid combination mainly in Escherichia coli and Salmonella enterica 10,11,12 .
The present work was attempted to evaluate the frequency of Klebsiella pneumoniae among clinical and environmental specimens, characterize resistant isolates, detect bla OXA-1 gene using Polymerase Chain Reaction (PCR) technique in isolates showed resistance to carbapenem and test its transmissibility by conjugation experiment.

Sample collection
In a five months period (December,2014 to April, 2015), 100 different specimens were recovered from two hospitals namely :Babylon Teaching Hospital for Maternity and Pediatric / Babylon Province (clinical: umbilical infections, n=40; environmental: n=20) and Karbala Hospital for Pediatric / Karbala Province (40 stool samples). Collected samples were cultured on different prepared media. Suspected K.pneumoniae isolates were identified based on their colonial, morphological characteristics and microbiological procedures as mentioned previously 13,14,15 .

Antimicrobial susceptibility testing
To determine the resistance profiles of K.pneumoniae isolates, the antimicrobial susceptibility to thirteen antimicrobial agents were analyzed by Kirby -Bauer disk diffusion method on plates with Mueller-Hinton agar medium (Oxiod, England) 16

Molecular detection of bla OXA-1 gene
Deoxyribonucleic acid (DNA) of carbapenem resistant K.pneumoniae was extracted based on the method mentioned with some modifications 18 . Conventional Polymerase Chain Reaction technique was applied to amplification bla OXA-1 gene using specific primers (Bioneer, Korea) OXA-1/F (F: ATA TCT CTA CTG TTG CAT CTC C) and OXA-1/R (R: AAA CCC TTC AAA CCATCC) (619 bp). All amplifications were implemented in a total volume of 25 µl consisted of 12.5 µl Go Taq Green Master Mix 2X (Promega, USA), 5 µl of extracted DNA, 2.5 µl forward and reverse primer (10 pmol/ µl) each and 2.5 µl nuclease-free water. The DNA template was denatured at 94°C for 5 min, followed by 30 cycles of denaturation ( 94°C for 50 sec), annealing ( 55°C for 50 sec), extension ( 72°C for 1 min) and the final extension (72°C at 10 min) 19 . The PCR reaction product was separated by gel electrophoresis (1.5% agarose gel stained with ethidium bromide solution, 0.5 mg/ml) at 70 volts for 2-3 hrs, PCR product was examined using UV-Transilluminator, and photographed with Gel documentation system. The size of DNA band was estimated using DNA Ladder, 100 bp (Bioneer, Korea).

Conjugation experiment
To test the transmissibility of bla OXA-1 gene, two carbapenem-resistant K.pneumoniae harboring OXA-1 gene (donors) and rifampicin resistant Escherichia coli MM294 (University of Kufa, College of Medicine) (recipient),were selected. Conjugation experiment was attempted by liquid mating assay 20,21 . All the transconjugants were screened for the existence of this gene using PCR assay with same primers applied in the procedure. The Minimum inhibitory concentrations (MICs) for ampicillin , cefotaxime, ceftazidime, imipenem and meropenem were detected using HiComb Minimum Inhibitory Concentration
H o w e v e r, K . p n e u m o n i a e f r o m environmental samples was not detected in this study. The reason may be related to low number of tested samples. One study in Hillah city identified the species in various clinical and environmental samples 24 . Also, Abbas 25 proved the detection of K.pneumoniae from burn unit environment of Al-Hillah teaching hospital.
In this study, all K.pneumoniae isolates presented higher resistance against penicillin antibiotics (ampicillin, piperacillin) with (94.1%) and (88.2%) resistance rates, respectively, (table -2). One work documented high level of resistance (98.6%) for ampicillin by K.pneumoniae among septicemic patients in India 9 . The higher frequency of resistance can be attributed to excessive consumption of these drugs in clinical settings. Additionally, the lower frequency was observed with imipenem (11.8%) and meropenem (11.8%), (Table 2).One report carried out by Hashemi et al. 26 documented (20%) as a rate of resistance against imipenem and meropenem for K.pneumoniae isolated from two hospitals in Tehran, Iran. Other research characterized (28.57%) resistance rate for meropenem antibiotic by clinical isolates of K.pneumoniae in Southeastern Nigeria 27 .
All carbapenem-resistant K.pneumoniae were positive for OXA-1 gene using PCR technique (Fig.1). According to Flores et al. 28 bla OXA-1 gene was   29 . Conjugation has been regarded as a very efficient method for horizontal transfer of resistance genes in bacteria with higher frequency in nature than under laboratory conditions 30, 31 . In current research, conjugative transfer of bla OXA-1 gene was successful for only 1 (50%) isolate of K.pneumoniae (K2) which was selected as a donor for conjugation (Table-3, Fig.2).Successful transfer of OXA-1 gene by conjugation was previously reported in a spanish isolates of K.pneumoniae 32 . Also, Rakotonirino et al. 33 documented the transfer of this gene in 6 isolates of K.pneumoniae obtained from four hospitals and medical centers in Antananarivo, Madagascar. The widespread of OXA-1 gene may be related to localization of this gene on variable region of integrin (class I) that also contain other resistance determinants like aac (6) Ib, CTX-M ESBL type and carbapenemases 32 .
The MIC of imipenem and meropenem for transconjugat (TCK2) was relatively lower (2 µg /ml) than the donor isolate (table -3).This finding suggest the presence of other resistant mechanism like chromosomal -mediated resistance genes in the donor that can not transferred by conjugation .

CONClusiON
The current study documents the presence of K.pneumoniae carrying ESBL of OXA-1 gene .The ability of this gene for transference pose a significant challenge for therapeutic options currently in use. Therefore, effective prevention and control strategies must be applied to prevent occurrence and dissemination of these strains.