Overexpression of blaOXA-58 Gene Driven by ISAba3 Is Associated with Imipenem Resistance in a Clinical Acinetobacter baumannii Isolate from Vietnam

The aim of this study was to investigate genetic structures and expression of blaOXA-58 gene in five Acinetobacter baumannii clinical isolates recovered from two hospitals in southern Vietnam during 2012-2014. A. baumannii isolates were identified by automated microbiology systems and confirmed by PCR. All isolates were characterized as multidrug resistant by antimicrobial testing using the disk diffusion method. Four imipenem susceptible and one nonsusceptible isolates (MIC > 32 μg·ml−1) were identified by E-test. PCR amplification of blaOXA-58 gene upstream and downstream sequences revealed the presence of ISAba3 at both locations in one multidrug-resistant isolate. Semiquantitation of blaOXA-51 and blaOXA-58 gene expression was performed by the 2-ΔΔCt method. The blaOXA-51 gene expression of five isolates showed little difference, but the isolate bearing ISAba3-blaOXA-58-ISAba3 exhibited significantly higher blaOXA-58 mRNA level. Higher β-lactamases activity in periplasmic than cytoplasmic fraction was found in most isolates. The isolate overexpressing blaOXA-58 gene possessed very high periplasmic enzyme activity. In conclusion, the A. baumannii isolate bearing ISAba3-blaOXA-58 gene exhibited high resistance to imipenem, corresponding to an overexpression of blaOXA-58 gene and very high periplasmic β-lactamase activity.


Introduction
Multidrug resistant A. baumannii constitutes a serious threat for nosocomial infection control [1]. Carbapenems are currently the antibiotics of choice against multidrug-resistant Acinetobacter infections [2], but an increasing rate of resistance to carbapenems was reported worldwide, seriously limiting therapeutic options [3]. Carbapenem-resistant A. baumannii has become an alarming health care problem, mainly in developing countries [4]. As a result, carbapenemresistant A. baumannii is classified into the critical priority group according to the urgency of need for new antibiotic treatment and the level of reported antibiotic resistance by the World Health Organization [5].
Multiple mechanisms of carbapenem resistance have been identified in A. baumannii including low membrane permeability, mutation in its chromosome genes, overexpression of efflux pumps, and acquisition of mobile resistance genes [6]. However, the production of carbapenemases is considered the principal resistance mechanism [7,8]. The most frequent ones are carbapenem-hydrolyzing class D βlactamases (CHDLs) and secondly metalloenzymes (MBL) such as bla NDM [9]. In addition, class A β-lactamases such as bla KPC gene has been recently also detected in A.
baumannii [10], presenting a serious threat of expanding resistance spectrum in the bacteria.
In Vietnam, bla OXA-23 is the most widely disseminated class D-carbapenemase in carbapenem-resistant Acinetobacter baumannii while bla OXA-24 is not detected [17]. Even though there is not any information of bla OXA-143 and bla OXA-235 in Vietnam up to now, these genes are believed to emerge in other parts of the world [18,19]. During 2003-2014, the majority of A. baumannii clinical isolates recovered harbored bla OXA-51 and bla OXA-23 genes. The bla OXA-58 gene was only detected in isolates recovered from 2010, after the introduction of imipenem in 2008-2009 into hospitals in Vietnam [17,20]. The bla OXA-58 -positive isolates investigated in the present study probably emerged at the same time. This recent emergence was in contrast with the striking replacement of bla OXA-58 by bla OXA-23 reported in Italy and China for the same period [21,22]. Furthermore, isolates bearing bla OXA-58 -like gene were recovered from different countries during outbreaks and showed remarkable conserved gene sequence [23][24][25]. The aim of this study was to investigate genetic structures and relative expression of bla OXA-58 gene, which lead to imipenem nonsusceptibility in clinical isolates recovered from two Vietnamese hospitals during 2012-2014.

Materials and Methods
2.1. Study Design. The study focused on A. baumannii isolates containing bla OXA-58 gene with the purpose of determining imipenem-resistance mechanism related to the gene.

Bacterial Isolates, Microbial Identification, and
Antimicrobial Susceptibility Testing. Five A. baumannii isolates were chosen from a total of 252 nonduplicate Acinetobacter spp. isolates recovered from patients admitted to hospitals in southern Vietnam during 2012-2014 and were named DN and TN based on their source hospitals [17]. Microbial isolation and identification in source laboratories were performed using the Phoenix System (BD) and the API 20NE system (bioMérieux). Identification of A. baumannii isolates was confirmed by PCR amplification and sequencing of 16S-23S intergenic spacer (ITS) regions. The sequences were deposited in GenBank under accession numbers KY659325, KY659326, KY659327, KY659328, and KY659329. Antimicrobial susceptibility testing was performed by the disk diffusion method and interpreted accord-ing to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2014). Tested antimicrobials included ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, cefepime, piperacillin, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanic acid, and meropenem, as well as others not belonging to β-lactams such as amikacin, gentamicin, ankamycin, netilmicin, ciprofloxacin, and levofloxacin. MIC values of imipenem were determined by the E-test (bioMérieux); the CLSI-approved breakpoints for imipenem ≥ 8 μg ·ml -1 and ≤2 μg·ml -1 were considered resistant and susceptible, respectively.

PCR Mapping of bla
2.5. Analysis of bla OXA-58 and bla OXA-51 Gene Expression by Real-Time RT-PCR. The midlog phase of bacterial cultures was treated with 1 μmol·ml -1 oxacillin for 24 h and was subsequently used for RNA extraction [28]. Treatment with RNAse-free DNAse I (Sigma) was performed at 37°C for 2 h. The concentration and DNase-free quality of RNA samples were spectrophotometrically assessed and confirmed by the amplification of chromosomal bla OXA-51 and 16S rRNA. Fifteen microliters of each RNA sample was reversetranscribed in a final volume of 20 μl containing random hexamers, MMLV reverse transcriptase (Agilent) at 42 o C for 45 min.

β-Lactamase Extraction and Quantitation.
Isolates were grown on LB medium supplemented with 1 μmol·ml -1 oxa-cillin for 18-24 h at 37 o C in a shaking incubator. The supernatants (extracellular fraction) were collected after centrifugation of bacterial cultures and precipitated with absolute ethanol (1 : 4) in 20 min at -20°C [32]. Periplasmic fractions were recovered from cell pellets [33]. Protein concentration was determined by the Bradford method [34].
β-Lactamase activity was determined based on nitrocefin hydrolysis [35,36]. Briefly, 1-5 μl extracellular and periplasmic fractions obtained from each isolate were incubated with 40 nmol nitrocefin dissolved in 0.1 M phosphate buffer, pH 7.0 in a total volume of 100 μl. Samples were loaded onto microtiter plates, and the absorbance at 482 nm was measured kinetically at room temperature for 2-30 minutes using an ELISA spectrophotometer. The specific β-lactamase activity was calculated and expressed as mU·mg -1 of protein based on the quotient of β-lactamase activity (mU·ml -1 ) and protein concentration (mg·ml -1 ).

Results and Discussion
bla OXAs are prevalent in A. baumannii. We had previously performed bla OXA identification in A. baumannii isolates from three hospitals in Southern Vietnam and found bla OXA-23 was dominant [17]. Even though bla OXA-58 existed with a small number in Vietnamese population, the exact genetic context involving antimicrobial resistance elements remained unknown. Here, we uncovered the imipenemresistance mechanism of bla OXA-58 -positive A. baumannii isolates. The overexpression of bla OXA-58 gene has been seen in the isolate with high-resistance phenotype through relative quantification of mRNA of the corresponding gene. The specific possible-intact ISAba3 sequence upstream of bla OXA-58 gene could be the key factor for the high expression. In addition, the high β-lactamase activity in the periplasmic space observed in the study could be the outcome of the phenomenon.
3.1. Antimicrobial Susceptibility Testing. All five isolates (DN050, TN078, DN014, TN341, and TN345) were classified as multidrug resistant (MDR) since they were nonsusceptible to at least one agent in three or more antimicrobial categories including aminoglycosides, antipseudomonal carbapenems, antipseudomonal fluoroquinolones, antipseudomonal penicillins and β-lactamase inhibitors, extended-spectrum cephalosporins, folate pathway inhibitors, penicillins and βlactamase inhibitors, polymyxins, and tetracyclines [37]. In this study, although several antimicrobials were not tested because of their availableness at different times and hospitals, all isolates satisfied the definition to be defined as MDR. Isolate DNA050 was nonsusceptible to all antimicrobials tested. The other four were all susceptible to imipenem (there were three isolates nonsusceptible to meropenem as hospitals reported), but for other antimicrobials, their susceptibility varied. Isolate TN078 and DN014 were nonsusceptible to three categories while isolates TN341 and TN345 were nonsusceptible to five categories ( Table 2).

Isolate
Genotyping and Profiling. All isolates were identified as A. baumannii based on 16S-23S intergenic spacer (ITS) region sequencing. Based on MLVA profiling, four different MLVA types within the five isolates reflected substantial genetic diversity in the sampled Vietnamese A. baumannii isolates, as previously described [17].
No isolate with bla KPC gene was detected, while two isolates contained bla NDM gene (DN050 and TN078). Even though the two isolates were singletons (based on MLVA types from previous study [17]) with different phenotypes, they had close relatedness with just difference in 3/8 loci surveyed and very similar resistance determinants, especially the bla NDM gene. Therefore, the difference in resistance pheno-type was mostly because of the distinguished genotype with ISAba3_bla OXA-58 in isolate DN050, compared to isolate TN078. It might be necessary for bla NDM gene located in a specific genetic context to be expressed as one of the important and strong resistance determinants. The mechanism should be explored further.
Regardless of the genetic diversity of the isolates, the bla OXA-58 gene sequence analysis (data not shown) of all isolates was identical with the reported bla OXA-58 gene [38]. This was in agreement with a previous work showing a lack of diversity in this gene, probably due to its recent acquisition by A. baumannii from other species [3].
All isolates were bla OXA-58 -and bla OXA-51 -positive and bla OXA-23 -and bla OXA-24 -negative ( Table 2). The analysis of insertion sequences revealed the presence of ISAba1 and ISAba2, but they were not located upstream of bla OXA-51 nor bla OXA-58 genes in all isolates. ISAba4 and IS18 were not detected. ISAba3 was detected in all isolates (Table 2). However, only isolate DN050 possessed a bla OXA-58 gene bracketed by two ISAba3 elements (Figures 1 and 2). The promoter region of bla OXA-58 gene in this isolate (Figure 2) was similar to sequences described by Poirel and Nordmann [38]. The genetic structure of bla OXA-58 upstream sequences which led to overexpression of this gene displayed a remarkable variability [38][39][40]. Hybrid promoters constituting an ISAba3 sequence truncated by other insertion sequences were generally considered strong promoters [22,41]. However, in this study, isolate DN050 bearing possible-intact ISAba3 sequence upstream of bla OXA-58 gene was not interrupted by inserted sequences, provided -35 and -10 promoter sequences as already described [38]. This structure probably drove high level carbapenemase production. The acquisition of insertion sequences by an imipenemsusceptible bla OXA-58 harboring isolate can lead to carbapenem resistance in A. baumannii [38]. Our results highlighted the threat of undetected reservoirs of carbapenem-resistant determinants and mechanisms in Vietnamese A. baumannii isolates.  and bla OXA-51 mRNA Level. We chose three isolates (DN050, TN341, and TN345) to study the relative expression of bla OXA-51 and bla OXA-58 under condition with oxacillin as an inducer and without oxacillin induction. They all had high β-lactamase activity in periplasmic fractions as shown in the following experiment ( Table 3). The mRNA level of bla OXA-58 and bla OXA-51 genes in all isolates was determined by quantitative real-time RT-PCR. Under oxacillin induction, DN050 showed a significantly higher level of bla OXA-58 mRNA expression than isolates TN341 and TN345 (Figure 3). bla OXA-51 expression was also upregulated, but not comparable to that of bla OXA-58 . Interestingly, the high expression level of bla OXA-58 from DN050 could be associated with the presence of an upstream ISAba3 sequence as previously suggested [38]. Furthermore, in this study, the possible intact ISAba3 sequence might be customized to bla OXA-58 gene to drive a very strong gene expression, as seen in ISAba1 for bla OXA-23 and AmpC genes [42]. The other isolates lacked upstream ISAba3 sequence. 4 BioMed Research International
All isolates expressed a low level of bla OXA-51 , confirming that the presence of bla OXA-51 , without an upstream ISAba1, did not confer a resistance phenotype [16]. Furthermore, in variants harboring bla OXA-51 and bla OXA-58 genes, carbapenem resistance only correlated with bla OXA-58 [43], which is in agreement with the results of this study. The enzyme activity of extracellular fractions was not significantly different (p = 0:2187) while one of the periplasmic fractions exhibited a significant difference among isolates (p < 0:0001). Extracellular fractions possessed lower enzyme activity than periplasmic fractions (p = 0:0355) in most cases. The periplasmic fraction recovered from all isolates exhibited variable β-lactamase activity, with very high activity corresponding to isolate DN050. Isolates TN341 displayed the highest β-lactamase activity though weakly expressed bla OXA-58 gene. This high enzyme activity probably corresponded to other β-lactamases responsible for the multidrug resistance phenotypes of the isolate, such as Vertical arrows were for the truncated sites previously reported that did not exist in this isolate. Positions of primers were indicated as referred to Table 1 with short thin arrows. The figure is not to scale.  5 BioMed Research International extended-spectrum AmpCs [44]. The presence of other βlactamases could explain the high enzyme activity in periplasmic fractions of the other isolates. Particularly, bla NDM gene detected in both isolates DN050 and TN078, but the corresponding β-lactamase activities as well as the antimicrobial susceptibilities were different between the two isolates. The mechanism that a strain carrying a bla NDM gene is not resistant to carbapenems needs to be discovered further in A. baumannii. It might need a unique genetic structure for bla NDM gene to be expressed as seen in K. pneumoniae [45].
In a transformed A. baumannii strain with a bla OXA-58 plasmid-borne vector, this carbapenemase is selectively released via outer membrane vesicles (OMV) after periplasmic   [32]. Furthermore, overexpression of bla OXA-58 gene increases its periplasmic enzyme concentration and extracellular release leading to efficient carbapenem hydrolysis [32]. The bla OXA-58 high mRNA level and high periplasmic β-lactamase activity of the DN050 isolate in this study suggested a similar overexpression, periplasmic translocation, and release mechanism of bla OXA-58 carbapenemase, even though our experimental work did not directly show the selection of OMV after being translocated to a periplasmic space. The high periplasmic βlactamase activity of the isolates, especially TN341 in this study, also suggested a possible translocation and release of other β-lactamases with a mechanism similar to that identified with bla OXA-58 . Further studies should be carried out to prove the suggested mechanism in clinical isolate similar to the transformed A. baumannii strain. To the best of our knowledge, our study is the first report on the overexpression of bla OXA-58 gene of A. baumannii clinical isolates from Vietnam. This study had some limitations. The first limitation involved the small sample size due to the low prevalence of clinical isolates harboring bla OXA-58 gene in the population surveyed. The screening has been done in previous studies [17]. Secondly, we did not characterize other resistance mechanisms in A. baumannii such as the overexpression of efflux pump genes or existence of multicopy bla OXA-58 gene [7,11,46]. In addition, the presence of other β-lactamase genes such as bla IMP , bla VIM , bla GES , bla OXA-143 , and bla OXA-235 was not excluded. Furthermore, we did not carry out an alternative experimental approach, such as western blotting against bla OXA-58 to unequivocally determine if the increase in β-lactamase activity is mainly due to this protein.

Conclusions
This study identified a mechanism of imipenem resistance related to the overexpression of bla OXA-58 gene preceded by ISAba3 and its corresponding periplasmic enzyme present at high concentration in a multidrug-resistant clinical isolate recovered from a hospital in Vietnam.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper. Figure S1: result of imipenem E-test for five clinical isolates of A. baumannii (blaOXA-58). Figure S2: electrophoresis results of PCR screening for the presence/absence of ISAba1, ISAba2, ISAba3, ISAba4, and IS18 in five clinical isolates of A. baumannii (blaOXA-58). Figure S3: electrophoresis results of PCR for the presence/absence of insertion sequence (IS) upstream of blaOXA-58 gene. Figure S4: duplex realtime RT-PCR analysis of the blaOXA-51 and blaOXA-58 mRNA relative expression compared with 16S rRNA in five A. baumannii isolates. Figure S5: Bradford assay standard curve of concentration versus absorbance for protein  Figure S6: nitrocefin standard curve. Table S1: duplex real-time RT-PCR analysis of the blaOXA-51 and blaOXA-58 mRNA relative expression in three A. baumannii isolates under conditions with oxacillin as an inducer or without oxacillin induction. Table S2: results for protein quantification of supernatant and periplasmic fractions. Table S3: results for β-lactamase activity of supernatant and periplasmic fractions. (Supplementary Materials)