Co-existence of blaOXA-23 and blaNDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance

Background Molecular analysis of carbapenem-resistant genes in Acinetobacter baumannii, an emerging pathogen, is less commonly reported from Nepal. In this study we determined the antibiotic susceptibility profile and genetic mechanism of carbapenem resistance in clinical isolates of A. baumannii. Methods A. baumannii were isolated from various clinical specimens and identified based on Gram staining, biochemical tests, and PCR amplification of organism specific 16S rRNA and bla OXA-51 genes. The antibiotic susceptibility testing was performed using disc diffusion and E-test method. Multiplex PCR assays were used to detect the following β-lactamase genes: four class D carbapenem hydrolyzing oxacillinases (bla OXA-51, bla OXA-23, bla OXA-24 and bla OXA-58). Uniplex PCRs were used to detect three class B metallo-β-lactamases genes (bla IMP, bla VIM and bla NDM-1), class C cephalosporin resistance genes (bla ADC), aminoglycoside resistance gene (aphA6), and ISAba1 of all isolates. Insertion sequence ISAba125 among NDM-1 positive strains was detected. Clonal relatedness of all isolates were analyzed using repetitive sequence-based PCR (rep-PCR). Results Of total 44 analyzed isolates, 97.7% (n = 43) were carbapenem-resistant A. baumannii (CR-AB) and 97.7% (n = 43) were multidrug resistant A. baumannii (MDR-AB). One isolate was detected to be extremely drug resistant A. baumannii (XDR-AB). All the isolates were fully susceptible to colistin (MICs < 2 μg/ml). The bla OXA-23 gene was detected in all isolates, while bla NDM-1 was detected in 6 isolates (13.6%). Insertion sequence, ISAba1 was detected in all of bla OXA-23 positive isolates. ISAba125 was detected in all bla NDM-1 positive strains. The bla ADC and aphA6 genes were detected in 90.1 and 40.1%, respectively. The rep-PCR of all isolates represented 7 different genotypes. Conclusion We found high prevalence of CR-AB and MDR-AB with bla OXA-23 gene in a tertiary care hospital in Nepal. Systemic network surveillance should be established for monitoring and controlling the spread of these resistant strains. Electronic supplementary material The online version of this article (doi:10.1186/s13756-017-0180-5) contains supplementary material, which is available to authorized users.


Background
Acinetobacter baumannii, an emerging pathogen of healthcare centers, shows intrinsic as well as acquired drug-resistance mechanisms [1]. Multidrug-resistant A. baumannii can be resistant to all of the currently available antibiotics, and in its deadliest form these are only susceptible to potentially toxic polymyxins and colistins, leaving limited options for treatment [2]. Infections with carbapenem-and colistin-resistant A. baumannii are emerging globally [3].
A. baumannii remains a critical problem in many healthcare settings throughout the world despite the implementation of infection control practices. There are limited data on carbapenem-resistant A. baumannii in Nepal. The objective of this study was to determine antibiotic susceptibility profile, antibiotic resistance genes and genetic mechanism of carbapenem resistance of A. baumannii in clinical isolates at a tertiary care hospital, Nepal.

Bacterial isolation and identification
A. baumannii isolates were collected from inpatient units of a tertiary hospital, Nepal. Forty-four nonduplicate isolates were collected (24 male and 20 female; age range between 24 to 80 years) over 9 months periods (October 2014 to June 2015). All isolates were identified by classical biochemical methods and confirmed by PCR method for detecting 16S rRNA gene and bla OXA-51 gene [15,16]. Isolates were identified as A. baumannii by PCR result of positive for both PCRs.

PCR amplification of antibiotic resistance genes
PCR assays to detect bla OXA-23 , bla OXA-24 , bla OXA-51 , bla OXA-58, bla IMP, bla VIM, bla NDM, bla ADC and ahpA6 genes were performed using primers as describe previously ( Table 1). The amplification reaction was performed using A. baumannii cell lysate as DNA template. Each PCR was performed in triplicate in a thermocycler with a PCR condition as described previously [14,16,[19][20][21]. All PCR assays used 16S rRNA or bla OXA-51 genes as the internal control. The ISAba1 of bla OXA-23 gene was detected using combination of primers ISAba1-F/ISAba1-R and ISAba1-F/bla OXA-23 -R (Table 1) [22]. The ISAba125 of bla NDM-1 gene were determined in all bla NDM-1 positive strains using combination of primers ISA125-F/ISA125-R and ISA125-F/bla NDM -R (Table 1). PCR products of the bla NDM-1 genes were purified and sequenced. BLAST was used to compare the sequences of bla NDM-1 genes against the GenBank Database. PCR products were analyzed by electrophoresis in 1% agarose gel containing 0.5 μg/ml ethidium bromides.

IPM-EDTA combined disk test
All bla NDM-1 positive strains were tested for MBL production by IPM-EDTA combined disk test. The test was performed as previously described [23]. After 24 h incubation, the difference of inhibition zone diameter between IPM-EDTA disk and IPM disk alone (≥7 mm) was considered the positive criteria for the presence of MBL.

Repetitive element PCR-mediated DNA fingerprinting (rep-PCR)
Genomic DNA of each isolates was extracted from the overnight cultures using GF-1 bacterial DNA extraction kit (Vivantis, Malaysia). Rep-PCR was performed by using genomic DNA as a template for PCR amplification with the ERIC-2 primer (Table 1) using condition as describe previously [24,25]. PCR-banding patterns and rep-PCR types were analyzed and interpreted as previously described [25].

MBL production
Six A. baumannii isolates harbored bla NDM-1 gene were detected for MBL production. All of bla NDM-1 positive strains were positive for MBL production. MBL positive strains showed resistance to fluoroquinolones and βlactam.

Epidemiological typing
Clonal relationship among isolates were studied using rep-PCR typing. The fingerprinting represented 7 different DNA patterns consisting of 2 to 5 DNA fragment sizes. The amplicons size for ERIC-2 PCR was 500-4000 bp. The genotype was named A-G as shown in Fig. 2. The high prevalence genotype was type C (n = 14; 31.8%) and D (n = 12; 27.3%). Genotype A, B, C and D were disseminated in all isolated ward (ICU, general ward and post-operative ward). Among 44 isolates, one isolate of type F (2.3%) and G (2.3%) was found. Type F was obtained from a catheter tip specimen from the ICU ward. Type G was obtained from sputum of a patient from a general ward. All NDM-1 positive strains exhibited genotype A (n = 1), B (n = 1), C (n = 3) and D (n = 1).

Discussions
A. baumannii harboring bla OXA-51 -like gene has been identified as a marker for species identification. An intrinsic bla OXA-51 -like gene detected in all isolates in this study supports the use of this gene as a surrogate marker of A. baumannii identification [8][9][10]. High prevalence of cephalosporin resistance genes, bla ADC (90.1%) was found in this study. In addition, we found a high rate of cepharosporin resistant antibiotics (cefotaxime, ceftazidime, ceftriaxone) using the disk diffusion method. These data indicated that cephalosporins no longer work to treat A. baumannii isolated from Nepal. Carbapenem resistance in A. baumannii is a major concern and is most often associated with class D Fig. 1 Distribution of A. baumannii carrying carbapenemase genes in different specimen types a and wards b  OXA-type carbapenemases are predominant in A. baumannii [6,7]. In agreement with this finding, high prevalence of bla OXA-23 carrying A. baumannii strains has been reported in Nepalese patients [26]. The acquired bla OXA-23 is the dominant genetic determinant in Asia. The bla OXA-23 gene located on plasmid can be transferred between A. baumannii through conjugation. Thus, antibiotic resistant bacteria have been rapidly increasing worldwide [27]. The bla OXA-24 and bla OXA-58 were not detected in any isolates from this study. The bla OXA-24/40 and bla OXA-58 genes were common in A. baumannii isolated from Europe [2,28]. Recently, bla OXA-143 and bla OXA-235 , which are novel class D βlactamase genes in A. baumannii have been identified. To date, these determinants were detected only in Brazil, Mexico and the USA [29,30]. ISAba1 was detected in widespread clones of A. baumannii worldwide. Our study found ISAba1 upstream of bla OXA-23 in all A. baumannii isolates. A correlation between A. baumannii clusters carrying the ISAba1/bla OXA-23 gene and increased minimal inhibitory concentrations for carbapenems was reported [31]. One isolate (AB-13) that was recovered from catheter tips of long-stay hospital patients showed an extreme drug resistance pattern (Additional file 1: Table S1). This isolate represented bla OXA-23 , bla ADC and aphA6 genes. Further molecular study to detect other antibiotic resistance genes is needed to explain what factors correlated with extreme drug resistance. We also found one isolate (AB-25) harboring bla OXA-23 , bla ADC and aphA6 genes was sensitive to all tested drugs (Additional file 1: Table S1). This may be due to the lack of promoter or mutation of ISAba1 or bla OXA-23 gene . Further study is needed to warrant the conclusion.
The bla NDM-1 carrying A. baumannii has recently been emerged in many countries, including Germany, Spain, Israel, Egypt, Switzerland, Libya, India, Pakistan and Nepal [11,26,32,33]. The bla NDM-1 gene has been identified as a chimeric gene constructed by the fusion of the aminoglycoside-resistance gene aphA6 with a mannosebinding lectin gene. This event most likely occurs in Acinetobacter spp., indicating that these bacteria are likely the origin of this gene [34]. In this study, we identified 13.6% of A. baumannii carrying bla NDM-1 gene. Previous study has identified high prevalence (24.6%) of the A. baumannii harbored the bla NDM-1 gene in Nepal in 2013-2014 [26]. Taking into consideration the relationship between India, China and Nepal, the spread of bla NDM-1 is likely to occur rapidly, mostly through A. baumannii rather than Enterobacteriaceae. A. baumannii able to transfer the bla NDM-1 gene via conjugation to the recipients and Tn125 appears to be the main vehicle for dissemination of the bla NDM-1 genes in A. baumannii [35]. Poirel et al. reported that the bla NDM-1 gene was located within the composite transposon Tn125 bracketed by two copies of a strong promoter of bla NDM-1 gene called ISAba125 [11]. This report was correlated with our finding that found ISAba125 in 100% of NDM-1 producing A. baumannii.
The previous study reported that the most of A. baumannii isolates harboring bla NDM-1 belonged to ST85 and ST25 [35][36][37]. In Libyan hospital, Libya, the main clone of imipenem-resistant NDM-1-producing A. baumannii belonged to ST2 [33]. We used rep-PCR typing to determine the clonal relationship in NDM-1 producing A. baumannii. Our study highlighted that most of NDM-1-producing A. baumannii isolates belonged to 4 genotypes using rep-PCR. Rep-PCR is a method that generates DNA fingerprints to discriminate between bacterial strains, and has been used to characterize A. baumannii isolates from hospitalized patients [38]. Our rep-PCR typing represented a high genetic diversity (A-G) among A. baumannii isolates from Nepal. Some clonally related groups (A, B, C and D) were observed in the all wards represented the disseminated of these clones in the hospital. Four genotypes (A, B, C, and D) of co-existence of bla OXA-23 and bla NDM-1 A. baumannii isolates were found. In addition, dissemination of these four genotypes into different wards also confirms as a major epidemic. Since rep-PCR is less discriminatory for molecular typing of bacterial strains, further study using multi-locus sequence typing could be useful for epidemiological investigations.

Conclusion
Antibiotic resistance in A. baumannii is considered to be a major future challenge in Nepal. Beyond OXA-type carbapenemase, there is no doubt the emergence and spreads of NDM-1 encoding A. baumannii-a superbugwill further limit chemotherapeutic options and threaten the public health of Nepal. The mechanism of hospital adaptiveness beyond antibiotic resistance will be more demanded in order to fully understand and combat MDR and XDR A. baumannii.

Additional file
Additional file 1: Table S1. Type of clinical specimen, ward, antibiotic susceptibility patterns, rep-PCR types, resistance genes and MIC of 44 A.