Identification of blaOXA23 and blaNDM1 from Carbapenem- resistant Acinetobacter baumannii at a Private Hospital in Thailand

1 Internal Medicine Unit, Phyathai II International Hospital, Bangkok, Thailand. 2 Department of Pharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand. 3 Antibiotic Resistance Knowledge Project by Pharmaceutical Initiative for Resistant Bacteria and Infectious Diseases Working Group [PIRBIG]. 4 Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand.

ISSN 2287-0237 (online)/ 2228-9674 (print) In Thailand, several reports have revealed that bla OXA23 constituted the majority of carbapenemase genes 6,7 but the rare prevalence of OXA-40 8 and IMP-1 9 among CRAB clinical isolates was observed. However, previous studies were conducted in university hospitals and a general hospital. The resistant mechanisms in CRAB from a private hospital might be different regarding the patterns of antibiotic use, patient characteristics and infection prevention and control methods. Thus, our study aimed to identify the presence of carbapenemase genes and clonal relationship among CRAB strains isolated from patients admitted to a private hospital.

Bacterial strains
All clinical A. baumannii strains were obtained from patients admitted to Phyathai II International Hospital, a 550-bed private hospital between August 2014 and April 2015. A carbapenem resistant strain was defined as isolates that were phenotypically resistant to imipenem (10 µg) and meropenem (10 µg) using the disk diffusion method based on The Clinical and Laboratory Standards Institute (CLSI). 10 Only the first CRAB isolate from each patient was kept in tryptic soy broth containing 20% glycerol at -80°C until studied. The research protocols were approved by the Ethic Committee [No. ID0014/59].

Clonal relationships
The clonal relationships of CRAB were evaluated using the REP-PCR method. The 15 µl PCR mixture was composed of 1 µl of DNA, 0.4 µl of 20 µM each forward and reverse primers, 7.5 µl of PCR master mix kit (JumpStart Red Taq ®

Carbapenemase genes
Each DNA sample of CRAB strains was extracted using a commercial kit (RBC Bioscience, California, USA). The primers of genes (bla OXA23 , bla OXA40 , and bla OXA58 ) and conditions that were used, are described in Table 1. Thermocycler was performed as follows: 94ºC for 5 minutes; 30 cycles of 94ºC for 45 seconds, annealing temperature specific for each primer pair for 45 seconds, and 72ºC for 1 minute; with a final heating at 72ºC for 10 minutes. 8 The detection of carbapenemase genes including bla IMP , bla VIM , bla KPC , bla OXA48 and bla NDM was performed, however, with multiplex PCR (Table 1). Amplification was carried out with the following thermal cycling conditions: at 94°C for 10 minutes and 36 cycles of amplification consisting of 30 seconds at 94°C, 40 seconds at 52°C, and 50 seconds at 72°C, with 5 minutes at 72°C for the final extension. 11 All amplicons were separated by agarose gel electrophoresis, stained with ethidium bromide, and compared with those of known carbapenemase genes. Finally, their identities were confirmed by nucleotide sequencing (Ward Medic, Ltd, Bangkok, Thailand) and were compared with known sequences in the GenBank database.
Leelasupasri S, et al.  1 minute, 72ºC for 2 minutes and finally at 72ºC for 16 minutes 8 . The REP-PCR products were performed using agarose gel electrophoresis and were stained with ethidium bromide. The criterion for classifying the different clones was a pattern that differed from the at least three bands or more of REP-PCR. 12

Results
Over the nine-month study period, only the first CRAB strain isolated from each patient, for a total of 15 clinical isolates were determined. The 15 samples were isolated from sputum (n = 9), blood (n = 2), urine (n = 1), pus (n = 2) and tissue (n = 1) specimens. All CRAB strains resisted to ceftazidime and were susceptible to ciprofloxacin, amikacin, and ampicillin/sulbactam at 6.7%, 26.7%, and 26.7%, respectively.
According to the clonal relationship study, the CRAB isolates were categorized by REP-PCR in 8 groups [A-H], with 53.3% belonging to group A, whereas the remaining 7 clones were in each member of B-H, respectively. (Figure 1). Of the OXA and MBL genes identified, most CRAB carried only bla OXA23 (86.7%) whereas only two isolates harbored both bla NDM1 and bla OXA23 (13.3%) ( Figure 2). However, no bla OXA40 , bla OXA58 , bla IMP , bla VIM , and bla KPC were identified in the study.

Discussion
Currently, with the mechanisms of resistance in A. baumannii especially, carbapenemases has been reported from various parts of the world. Although six studies detected carbapenemase genes in Thailand only five were from the clinical isolates in the university hospitals and the remaining were from a general hospital. [6][7][8][9]13,14 Actually, the diversity of resistant mechanisms among A. baumannii clones might be possible in different hospitals. 15 Moreover, at the same hospital but in different wards, the distribution of clones and mechanism of resistance also varied in different clinical departments. 16 Thus, our study, performed in a private hospital, revealed the same bla OXA23 as related studies but reported two patients carrying two carbapenemase genes (bla OXA23 and bla NDM1 ) simultaneously.
With the NDM-1, the Amber class B, MBL group, is one of the most commonly reported among Enterobacteriaceae, being firstly identified in a patient who had returned from New Delhi. 17 Bla NDM -carrying Enterobacteriaceae remains on the Indian subcontinent, but to date, has been found in various parts of the world. 17 Of bla NDM -bla OXA23 carrying A. baumannii, the co-carbapenemase genes found in our study, this phenomena was similar to a related study showing the coexistence of bla OXA -bla NDM1 among three isolates of CRAB in India and two isolates of CRAB in Thailand. 18,19 However, we could not explain how these genes coexisted. However, we hypothesize that the co-genes might have been transferred by mobile genetic elements within ISAba1. 18 However, no other carbapenemase genes (bla OXA48 , bla KPC , bla IMP , or bla VIM ) were detected in the present study. This might be due to the small sample size, limited period of sample collection, or their extremely low prevalence in the hospital setting. This limitation should be corrected by further studies with a larger sample size and a longer study period.
At the time of writing, avibactam and vaborbactam are diazabicyclo-octane and cyclic boronic acid respectively, having an inhibitor activity against class A, class C and some enzymes in class D beta-lactamases. Whereas class B metallo-beta lactamases (such as NDM, IMP, VIM) have proven to have less inhibition by avibactam and vaborbactam. Thus, the presence of a pathogen carrying co-carbapenemase gene (bla OXA23 and bla NDM1 ) is challenging treatment for finding a novel β-lactamase inhibitor with high affinity to all classes of beta-lactamases. 20 Regarding the clonal relationships in this study, clone A (53.3%) was predominant. This prevalence of the majority clone was less than that reported in a related study (93.0%). 8 The lower prevalence of the predominant clone and the numerous types of clone might have stemmed from well-controlled multiple factors including strict infection control guidelines, appropriate use of antibiotics and notification of infected patients. 21 However, as clone A exhibited the highest prevalence, we suggested that the infectious control program could continue minimizing the reservoir for bacterial transmission in the hospital. 22

Conclusion
This research comprised a study to confirm the most common type of bla OXA23 found in Thailand beyond academic medical centers. Additionally, this study firmly showed bla NDM1 coexisted with bla OXA23 in clinical CRAB isolates in Thailand.