Characteristics of 32 Acinetobacter baumanni isolates co-harboring OXA-Carbapenemases and 16 S rRNA methylase gene

Multiple drug-resistant strains of Acinetobacter have become common in hospitals worldwide. The problem becomes more acute with existence of hidden Carbapanemase -resistance genes that co-exist with other resistance determinants. Based on this, this research aims to determine the occurrence and co-existence of OXA-type carbapenemases and 16S rRNA methylase gene among clinical isolates of A. baumannii in a Jilin hospital, China. Between January 2012 to December 2013, 32 A. baumanii nonrepetitive strains were isolated from nosocomial clinical specimens. Susceptibility to antibiotics was determined by the standard microdilution method and phenotypic testing was used to detect the presence of carbapenemases. PCR was performed using specific primers for detection of class D carbapenemase genes and 16S rRNA methylase gene. Results revealed that all imipinem resistant A. baumannii isolates were resistant to all antibiotics tested except polymycin E. The isolates harbored different Oxacillin Type Carbapenemases, that is, 62.5% of the isolates harbored OXA-51, 37.5% OXA-23, 18.8% OXA-58, 3.1% OXA-24 and 59.4% harbored 16S rRNA methylase gene. Of the 19 isolates harboring 16S rRNA methylase gene, 47.4% coexisted with at least one oxacillinase gene while 40.6% of the 32 A. baumanii isolates harbored more than one different oxacillinase gene. At least one oxacillinase gene was detected in 28.1% of the isolates that were phenotypically negative for carbapenemase production. The current study therefore shows co-existence of resistance determinant genes and existence of hidden genes responsible for resistance. Multiple mechanisms are likely to work in synergism to produce this phenotype.

causing episodes of bacteremia and/or sepsis mostly result of transmission via multiple contaminated surfaces and objects and transiently colonized health care workers' hands (Perez et al., 2007).
The beta-lactam antibiotics are an important group of antibiotics that have been used to treat infections caused by various microorganisms, including A. baumannii, (Livermore and Woodford, 2006) due to their efficacy and safety and because their activity can be increased by chemical modification.This was until 1970s' where some isolates of A. baumannii were found to be resistant to a wide range of antibiotics including broad spectrum betalactams, aminoglycosides and flouroquinolones (Renu et al., 2010).However, decreased susceptibility to carbapenems has been reported worldwide (Uvizl et al., 2011;Trecarichi et al., 2011).This fact is associated with high mortality and morbidity rates, prolonged hospital stays and increased treatment-related costs.
The emergence of carbapenem resistance in A. baumannii has become a global concern since these βlactams are often the only effective treatment left against many multidrug-resistant strains.The major mechanism of carbapenem resistance in A. baumannii is the production of OXA-type genes (OXA-23,OXA-24,OXA-51 and OXA-58) and/or metallo-β-lactamase (VIM,IMP,NDM) (MBL) enzymes belonging to Ambler classes D carbapenemases or Class B Metalo--lactamase (MBL) respectively (Poirel et al., 2008).A recent development has been the discovery of a novel group of narrowspectrum OXA β-lactamases in carbapenem-resistant strains, some of which have acquired the ability to hydrolyse the carbapenems (Opazo et al., 2012).These enzymes belong to three unrelated groups of clavulanic acid-resistant -lactamases, represented by OXA-23, OXA-24 and OXA-58, that can be either plasmid-or chromosomally-encoded.A. baumannii also possesses an intrinsic carbapenem-hydrolysing oxacillinase, the expression of which may vary and may play a role in carbapenem resistance (Lee et al., 2006).
These OXA-type cabarpanemaes (OTC) have been described in different provinces of China.Wang et al. (2011) indicated that OXA-58-like, a carbapenemresistance determinant located on transferable plasmids and spread by clonal dissemination in most cases, is an emerging threat in China.OXA-51 and OXA-23 in China have been reportedly carried by almost all A. baumannii strains isolated due to their chromosomal location (Dijkshoorn et al., 1998).
The 16S rRNA methylase gene is often responsible for the phenotype of Gram-negative pathogens that show high MICs to most aminoglycosides partly due to its enzymatic modification and methylation.16S rRNA methylase gene has been isolated in A. baumannii strains from Korea, Japan, China and other parts of the world with the first gene reported in a strain of Klebsiella pneumoniae in France (Wang and Chen, 2005).
The 16S rRNA methylase genes are mostly located on transposons within transferable plasmids, which provide them with the potential to spread horizontally.Some of the A. baumannii strains have been found to coproduce extended-spectrum β-lactamases or metallo-βlactamases, contributing to their multidrug-resistant phenotypes.In addition, studies reveal that A. baumanii strains harboring more than one OXA-encoding gene are emerging (Brown and Amyes, unpublished results).
Identification of OTC carrying isolates has been a challenge due to emergency of hidden carbapenem susceptible oxacillinases which may be missed in daily Lab practice.These carbapenem susceptible organisms with hidden oxacillinase genes can spread unnoticed in hospitals.Furthermore, these resistance determinants are located on transferable plasmids consequently easily spread.
The objective of this study was to characterize A. baumannii isolates carrying Oxa-type carbapenemase (OTC) from clinical specimens collected from patients in a Jilin hospital in order to clarify the phenotypes and genotypes.The study emphasized on determination of the occurrence and co-existence of OXA-type carbapenemases with 16S rRNA methylase gene.

Bacteria strains
A total of 32 non-repetitive imipenem-resistant isolates of A. baumannii w ere recovered from clinical respiratory infections in hospitalized patients from January 2012 to December 2013 in a Jilin hospital, China.The identification of strains w as performed by standard microbiology procedures including grow th on MacConkey agar and simple biochemical tests like Oxidase test, grow th on TSI and Eosin Methylene Blue Agar and ability to grow at 44°C.Analytical profile index (API) system procedure w as used to biochemically identify the strains.

Determ ination of MIC
In all Acinetobacter spp.isolates, susceptibility to antibiotics w as determined by a standard micro-dilution method according to the Clinical Laboratory Standards Unit (CLSI, 2010).

Phenotypic determ ination of carbapenem ase production
Carbapenemase production in Acinetobacter spp.Isolates w ith a MIC for meropenem of >2 mg/L w as phenotypically determined by the Modified Hodge Test (MHT) for carbapenemases production (Amjad et al., 2011).

PCR detection of OXA-genes and 16S rRNA genes
The boiling method w as used to extract DNA from the bacteria as described by Vaneechoutte et al. (1995).Briefly, one colony of a pure culture grow n on TSI slanting agar w as re-suspended in 50 µl of LB broth and incubated at 37°C in an orbital shaker for 14 h.50 µl of the grow th w as centrifuged and resultant pellet re-suspended in 50 µl of distilled w ater and heated at 100°C for 10 min.After centrifugation in a micro-centrifuge, at 6000 x g for 3 min, the supernatant w as kept at -20°C for further use.PCR w as carried out in a 25 µl reaction volumes w ith 1 µl of extracted DNA, 12.5 µm of the PCR master Mix containing 1.25 U of Taq Polymerase, 1ｘPCR buffer containing 0.1 mM MgCl2 and 240 µM of each dNTP.Specific primers used are show n in Table 1.PCR conditions w ere as follow s: 94°C for 5 min and then for 35 cycles, 94°C for 30 s, 57°C for 45 s, 72°C for 30 s and then final extension at 72°C for 10 min.Amplified products from the isolates w ere analyzed by electrophoresis on 1% (w /v) agarose gel stained w ith Gel Red.
Competent cells w ere prepared from protocol given by Joe (2005).The DNA band of interest w as excised, purified w ith a QIAqick PCR purification kit, ligated to pGEM-T Easy vector (Promega), transformed into E. coliDH5α competent cells, and the w hite colonies w hich w ere successfully transformed and selected w ere screened.Nucleotide sequencing w as performed directly on cloned fragments using an ABI Prism 377 DNA sequencer as a control and to rule out non-specificity in case of novel gene sequences.Sequence similarity searcher w as carried out w ith the BLAST program available at the w ebsite of the National Center of Biotechnology Information (w w w.ncbi.nlm.nih.gov).

Minimum inhibitory concentrations (MICs)
The MICs of the 32 A.baumanii isolates were determined.All isolates (100%) were resistant to all drugs tested, that is, Cephalosporines (Cefetamet, Ceftazidime, Cefotaxime, Cefuroxime and Ceftriaxone) as their MICs were > 2 mg/L.These isolates were resistant to three or more classes of antibiotics.According to the definition given by A. P Magiorakos et al. (2011), we described these isolates as MDR.

Phenotypic determination of carbapenemase production in A. baumannii
Imipenem-resistance by disc diffusion method was found in all 32 isolates of Acinetobacter spp.Of the 32 imipenem-resistant Acinetobacter spp.18 (56.3%)were carbapenemase producers when tested by MHT.Whereas, remaining 14 (43.8%)isolates did not show evidence of carbapenemase production.

16SrRNA gene
Thirty two Imipinem resistant A. baumannii strains were tested for 16S rRNA methylase genes.Of the 32 A. baumanii isolates, 19 (59.4%) harbored 16S rRNA methylase gene of which 11 (61.1%)tested positive for carbapanemase and also 8 (57.1%) out of 14 of the noncarbapenemase producing isolates harbored 16S rRNA methylase gene.We also found that 47.4% of the isolates harboring 16S rRNA methylase gene co-existed at least with one oxacillinase gene (Table 2 and Figure 1).

Carbapenem-resistant genes
Thirty-two imipinem resistant A. baumannii strains were   Co-existence of different bla OXA genes among 32 isolates of A. baumannii are indicated in Tables 3 and 4. None of the isolates carrying bla OXA-24 associated with bla OXA-23 or bla OXA-58 genes.A big percentage (72.7%) of A. baumannii isolates containing bla OXA-51 -like gene coexisted with all other OXA-like genes, because this gene is intrinsic to A. baumannii isolates (Figure 2).
Cloning and sequencing confirmed that the PCR products were 100% identical to the OXA-51 genes (GenBank accession nos AY795964 and AY949204).).

DISCUSSION
Carbapenem resistance in A. baumannii has been increasingly detected in Asian countries, which is the same case with China (Zong et al., 2008;Wang and Chen, 2005).In China, national resistance surveillance data from intensive care units (ICU) at 19 teaching hospitals (1996 to 2002) showed that 5% of Acinetobacter isolates were resistant to imipenem (Wang and Chen, 2005).However, another national surveillance program involving 10 geographically disparate hospitals found that resistance to carbapenems increased from 4.5% in 2003 to 18.2% in 2004 (Wang and Chen, 2005).
Carbapenems (e.g., imipenem and meropenem) and aminoglycosides are the drugs of choice in the treatment of Acinetobacter infections in almost all medical centers, but are being compromised by the emergence of carbapenem-hydrolyzing-lactamase (carbapenemase) of molecular classes D, especially OXA-type and methylasation of 16S rRNA methylase gene respectively.Furthermore, several studies have indicated that imipenem may be hydrolyzed by the extended-spectrumlactamases of Gram-negative bacteria.
In this study, A. baumannii isolates were described as Multi-Drug Resistant (MDR) strains as the isolates were resistant to all antibiotics tested with exception of polymyxin E. This was based on the definition of MDR as given by Magiorakos et al. (2011).These findings also agree with Muthusamy and Boppe's claim that strains of A. baumannii are emerging as MRD (Muthusamy and Boppe, 2012).These results agree with the findings of Dalin et al. (2015) who observed that, all the isolates of A. baumanni isolated in an affiliated hospital of Jilin, China were resistant to all antibiotics.
By using the modified Hodge test as a screening test for carbapanemase production (Amjad et al., 2011), we found that 19 (56.25%) of 32 A. baumannii isolates were carpanemase producing isolates.This agrees with Irfan et al. (2008) observation suggesting the presence of OTC enzymes since they are considered to be the mechanism of resistance in these organisms.This finding is also consistent with reports from other tertiary care hospitals around the world and even China (Zong et al., 2008;Wang and Chen, 2005).The resistance of A. baumanni to antimicrobial agents is mediated by almost all resistance mechanisms found in bacteria.Methylation of 16S rRNA methylase gene has emerged as a mechanism of highlevel resistance to aminoglycosides among Gramnegative bacteria.In this study, the 16S rRNA methylase genes were amplified and detected in 19 (59.38%) of the isolates and 61.1% were carbapenemase producers.These findings are consistent with other published reports suggesting that A. baumannii carries 16S rRNA methylase gene (Kyungwon et al., 2011).As 16S rRNA methylse gene confer high-level resistance to aminoglycosides, its occurrence in A. baumannii limits the clinical use of aminoglycosides.
Our study reveals that 50% of the of 32 A. baumannii isolates studied harboring 16S rRNA methylase gene coexisted with at least one oxacillinase genes.This finding agree with that of Strateva et al. (2012) which showed here that A. baumannii isolates producing 16S rRNA methylase gene harbored both bla OXA-23 -like and bla OXA-51like genes.This is because these genes are linked with mobile genetic elements and usually confirmed as located on large conjugative plasmids, allowing potential spread among bacterial populations.
Beta-lactamases are the most prevalent group of enzymes that are responsible for resistance in A. baumanniii and more than 50 different types have been identified so far in this strain.Several reports from around the world indicate a large increase in the rates of carbapenem-resistant A. baumannii from 8% in 2003 to 52% and 74% in 2005 and finally to 96% in 2007 (Mendes et al., 2009) due to these group of hydrolytic enzymes.
As shown in our study, 20 (62.5%), 13 (40.63%), 6 (18.75%), and 1 (3.13%) of the 32 multidrug-resistant strains studied carried OXA-51, OXA-23, OXA-58 and OXA-24 genes respectively.Moreover, 10 (31.25%) isolates were positive for both OXA-51, OXA-23genes.Our data support those of other studies that demonstrated that OXA-51 may be used as a marker to identify A. baumannii (Qi et al., 2008).The OXA-23 genes have been documented in strains associated with outbreaks of carbapenem resistant A. baumannii in Asia, Europe and South America (Merkier and Centrón, 2006).Four strains were OXA-58 positive and only 1 (3.4%) strain was positive for OXA-24.This is consistent with studies from other hospitals in Jilin and entire China which suggests that the most widespread oxacillinase gene in A. baumannii is OXA-51, OXA-23, OXA 58 and OXA-24 in the order (Su-ying et al., 2015).This study also reveals that 9 (28.1%)out of 32 A. baumannii isolates carried at least one oxacillinase gene though they were negative for carbapenemase production.This suggests the existence of hidden carbapenemase genes.These hidden genes can easily be missed in daily laboratory practice hence posing as a diagnostic and therapeutic challenge and can easily spread unnoticed in the hospital.We also found that 13 (40.63%)out of 32 isolates carried more than two oxacillinase genes, and were MDR.
The main limitation of this study is that it was confined to a single centre and it would be valuable to extend the origin of the strains.Another limitation is the small sample size which led to a lack of power to determine the individual effects of each broad spectrum antibiotics.

Conclusion
The emergence of broad-spectrum antibiotic resistance profiles in A. baumannii clinical isolates is worrying in the hospital.The current study shows co-existence of resistance determinant genes and also existence of hidden genes responsible for resistance.Multiple mechanisms are likely to work in synergism to produce this phenotype.The co-existence and the multiple mechanisms working in synergy led us to an insight of defining A. baumannii strains in this study as extensive drug resistant A. baumannii (XDRAB), as there was still hope with the use of the highly toxic polymyxin E. The results here highlight that enhanced surveillance and health policies for the detection and control of these MDR pathogens are urgently needed to avoid the emergence and spreading of such organism.

Figure 1 .
Figure 1.Agarose gel electrophoresis of PCR patterns of genomic DNA from clinical isolates of A. baumannii harboring 16srRNA methylase gene.Lane M: 100bp DNA size marker; lane 1~11: are 1450bp positive for 16SrRNA ；Lane 6 and 9 are negative for 16SrRNA; Lane 12：negative control.

Table 1 .
Different genes/enzymes responsible for resistance in Acinetobacter baumannii (John Roberts Table).

Table 4 .
Distribution and co-existence of different blaOXA genes among clinical isolates of carbapenem resistance Acinetobacter baumannii (John Roberts Table