Spread of the blaOXA–23-Containing Tn2008 in Carbapenem-Resistant Acinetobacter baumannii Isolates Grouped in CC92 from China

The rapid expansion of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a big issue. We investigated the antibiotic susceptibility, molecular epidemiology and resistance gene of A. baumannii collected at two hospitals in Shanghai, China. Besides, the A. baumannii PCR-based replicon typing method (AB-PBRT) was conducted to categorize the plasmids into homogeneous groups on the basis of replicase genes. Most CRAB isolates showed high-level resistance to almost all antibiotics but retain susceptibility to colistin and tigecycline. A total of 101 isolates carried blaOXA-51-like gene. Sequencing identified the presence of blaOXA-66 for CRAB isolates. blaOXA–23 gene were discovered in all CRAB isolates. Each CRAB isolate contained 1–3 of 19 different plasmid replicase (rep) gene homology groups (GRs) and the GR6 (repAci6) was ubiquitous. Genotyping by Multilocus Sequence Typing (MLST) showed seven defined MLST patterns and three novel STs were found. eBURST analysis indicated they were all grouped in CC92 (GCII) with the most frequent ST208 (50%). Two blaOXA–23-bearing transposons were found: Tn2006 and Tn2008. Tn2008 were detected in 54 (96.4%) isolates and Tn2006 in two remaining isolates. The blaOXA–23 carbapenem gene was vitally associated with repAci6 plasmid belong to CC92 clonal group. Our survey revealed severe drug resistance in A. baumannii isolates. Tn2008-containing CC92 A. baumannii were endemic, which may facilitate the blaoxa23 dissemination.


INTRODUCTION
Acinetobacter baumannii is a significant opportunistic pathogen responsible for numerous nosocomial infections, including respiratory infections (in particular ventilator-associated pneumonia, VAP), urinary tract infections, bacteremia, soft and skin tissue infections, burn wound infections and secondary meningitis (Roca et al., 2012;Kempf et al., 2013). Carbapenem resistance in A. baumannii is an emerging problem worldwide during the last decade (Jiang et al., 2014;Guerrero-Lozano et al., 2015). One data from Taiwan involving five major hospitals showed that resistance to imipenem in intensive care units increased from 22.0% in 2000 to 66.8% in 2010 (Hsueh et al., 2001). Studies performed by Reddy also reported a disturbing tendency of sharp increase in the rates of CRAB isolates, from 1% in 2003 to 58% in 2008 (Reddy et al., 2010). Data from Wallace also found the percentage of CRAB isolates was 31% before the year 2009 followed by 99% after the year 2009 at the University of Maryland Medical Center (UMMC; Wallace et al., 2016).
Carbapenem-resistant in A. baumannii is due to combined mechanisms including production of OXA-and metalloβ-lactamases, outer membrane impermeability, increased expression of efflux pumps, and penicillin-binding proteins modification (Zarrilli et al., 2013). OXA-type enzymes related to carbapenem resistance include the natural bla OXA−51 -like and three acquired genes: bla OXA−23 -like, bla OXA−24 -like and bla OXA−58 -like (Jiang et al., 2014). These genes were integrated in the bacterial chromosome or carried by plasmids (Poirel et al., 2010). Nowadays, AB-PBRT method provides a new tool to investigate the epidemiology of plasmids in A. baumannii (Towner et al., 2011). Numerous publications reported the spread of resistance gene via transposons (Poirel et al., 2010). Four transposons harboring bla oxa−23 gene have been reported: Tn2006, Tn2007, Tn2008, and Tn2009 (Zhou et al., 2011;Espinal et al., 2013;Guerrero-Lozano et al., 2015). These types of transposons share a common region "bla OXA−23 -ATPase." Tn2007 owns ISAba4 promoter upstream bla OXA−23 gene. The remaining three transposons own ISAba1. The two ISAba1 copies were inversely orientated in Tn2006 compared to the same direction in Tn2009 (Guerrero-Lozano et al., 2015). Tn2006 and Tn2008 are reported globally disseminated, while Tn2009 has only been discovered in China (Zhou et al., 2011). These ISAba1-associated transposons contributed to the dissemination of bla OXA−23 . Moreover, Tn2006, Tn2008, and Tn2009 all have been found in conjugative plasmids except Tn2007. It is noted that Tn2007 is immovable and is not actually considered as a transposon (Nigro and Hall, 2016).
Colistin is currently used as last-resort antibiotics against CRAB infection (Durante-Mangoni et al., 2014). It is important to analyze the local epidemiology of clinical CRAB isolates and control the dissemination. The aim of the present study was to investigate the drug-resistance and molecular characteristics of A. baumannii isolates in 101 A. baumannii clinical isolates from two hospitals in Shanghai. We also characterized the genetic environment surrounding bla OXA−23 gene. In addition, the distribution and epidemiology of plasmid replicase (rep) genes in CRAB isolates were also investigated.

Bacterial Isolates
A total of 101 non-repetitive A. baumannii clinical isolates were collected in this study from two hospitals in Shanghai, China. All subjects were anonymised in this study. Eighty-seven isolates were recovered from neurosurgery in Renji Hospital Shanghai Jiaotong University School of Medicine from July 2011 to June 2013. And the remaining 14 isolates were collected from Obstetrics and Gynecology Hospital of Fudan University from January 2015 to August 2016. All isolates were identified using the VITEK 2 Compact automatic bacteria and drug susceptibility analysis system.

Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing was conducted in the present study. Fourteen antimicrobial agents were tested including piperacillin/tazobactam, cefoxitin, ceftazidime, cefotaxime, imipenem, meropenem, gentasin, amikacin, minocycline, ciprofloxacin, trimethoprim/sulfamethoxazole, cefepime, polymyxin, and tigecycline. The Minimum inhibitory concentrations (MICs) were determined by agar dilution method except polymyxin, which was performed by broth dilution method. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as reference strains. The susceptibility results were interpreted based on the Clinical and Laboratory standards Institute (CLSI) breakpoints (CLSI, 2011). For tigecycline, we used breakpoints recommended by the British Society for Antimicrobial Chemotherapy guidelines (BSAC, 2011). CRAB isolates were defined with both imipenem and meropenem resistance (MICs > 8 mg/L).

PCR and Sequencing of Drug Resistance Genes
PCR assay was performed to screen the presence of drug resistance genes in 101 isolates, including bla OXA−23 -like, bla OXA−24 -like, bla OXA−51 -like, bla OXA−58 -like, bla IMP−1 , bla VIM−1 , bla VIM−2 , and bla AmpC . The entire products were sequenced and analyzed with the BLAST website 1 .

Multilocus Sequence Typing
Molecular typing of CRAB isolates was determined by MLST. Briefly, it was detected by sequence analysis of gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD housekeeping genes as previously described (Ying et al., 2015). Primers are available at http:// pubmlst.org/abaumannii/. The results were compared with the STs databases online at http://pubmlst.org/databases/. eBURST analysis was conducted to investigate the genetic relationships and clonal complexes (CCs) of these isolates.

Genetic Environment of the bla OXA−23 Gene
PCR was performed to detect the occurrence of bla OXA−23carrying transposons in CRAB isolates, including Tn2006, Tn2007, Tn2008, and Tn2009. Primers used in this study were shown in Table 1. Structures and primer locations were shown in Figure 1. In addition, bla oxa−23− ATPase was the common region of Tn2006 and Tn2008.

AB-PBRT
Nineteen PCR amplifications were organized into six multiplexes and used to detect 27 different plasmid rep genes as described previously (Bertini et al., 2010). Each PCRs recognized three or four different homology groups (GRs). PCR products were

Primer name
Primer sequence (5 -3 ) Replicase gene Tn2009 Int-P8 CTGTCTGCGAACACATTCAC recognized by DNA sequencing. An additional PCR was performed to identify the gene encoding the type IV secretion system protein TraC, which was found on the plasmid pACICU2 carrying the repAci6 gene (Towner et al., 2011).

Antimicrobial Susceptibility Testing
The MICs of 14 antimicrobial agents were determined for all A. baumannii isolates. Sixty-five (64.4%) and 70 (69.3%) strains were resistant to imipenem and meropenem (

Genetic Structure of bla OXA−23 Gene
The gene encoding bla OXA−23 of 56 CRAB isolates was located exclusively within the Tn2006 or Tn2008 transposons. We found Tn2008 was almost ubiquitous (54 of 56 isolates). However, the specific region (ISAba1-bla oxa−23 -ISAba1) for Tn2006 was only detected in 2 (3.6%) isolates. No ISAba4-bla oxa−23 was found, indicating there was no Tn2007 in these CRAB strains, followed by the Tn2009.

DISCUSSION
The increasing drug resistance of A. baumannii has raised a big challenge, especially CRAB strains reported worldwide (Durante-Mangoni et al., 2014). As observed in other literature, here we reported a high prevalence of carbapenem resistance (55.4%) in clinical A. baumannii isolates recovered from two hospitals. Most CRAB strains always exhibited high resistance to other antimicrobial agents tested. OXA-type carbapenems resistance hydrolytic enzymes are common in A. baumannii isolates. bla OXA−51 -like gene, which shares less than 60% amino acid identity with other OXAtypes, was identified in all A. baumannii isolates, supporting that bla OXA−51 -like are natural genes in A. baumannii (Table 3). bla OXA−66 variant might be the most prevalent and found in all 56 CRAB isolates. In general, CRAB strains contained more resistance genes. This probably explains why CRAB strains show much higher MICs than non-CRAB isolates. bla OXA−24like β-lactamases was first identified in A. baumannii in 1997, FIGURE 2 | eBURST analysis results of tested CRAB isolates. One clonal complexes (CC92) were identified. The size of the dot indicates the number of CRAB isolates. which resulted in an outbreak in Spain (Bou et al., 2000). Andbla OXA−58 -like gene was first found in France in 2003 (Poirel et al., 2005). Isolates carrying bla OXA−24/58 -like genes were typically resistant to carbapenems. Fortunately, they were barely identified in China. Our results also indicated no bla OXA−24/58like nor bla IMP−1 genes were found. However, due to their plasmid location, the distribution of these genes in A. baumannii should be early monitored. Multilocus Sequence Typing is frequently used for strain phylogeny and global epidemiology. Fifty-six CRAB isolates with occurrence of bla OXA−23 in our study were typed. The most common ST was ST208 and CC92 was the unique CC clonal group tested. CC92 (GCII) clone outbreaks due to bla OXA−23producing A. baumannii strains have been reported frequently (Fu et al., 2010;He et al., 2011). The discovered of three novel STs indicated that A. baumannii isolates were diverse and still clonal expansion. Our e-BURST analysis revealed clonal spread at Renji hospital during a certain period. Hence the carbapenems resistance in A. baumannii is increasing. While isolates of the same ST clone reported owning different resistance patterns, the vital spreading of CRAB isolates may have other approaches.
Acinetobacter baumannii PCR-based replicon typing assay revealed 52 CRAB isolates owning repAci6, and the gene encoding TraC was strongly linked with repAci6 plasmid. No rep genes associated with bla OXA−24/58 were found. RepAci6 was identified on plasmid pACICU2 in general and TraC protein make pACICU2 transferable (Iacono et al., 2008). In some cases (e.g., isolates Ab18 and Ab64), different MLST genotypes were found to have identical plasmid rep gene profiles. In contrast (e.g., isolates Ab18 and Ab23), isolates with the same MLST type were found to have different plasmid rep gene profiles (Table 3). Hence the plasmid rep genes were variable in the process of strain epidemics. bla OXA−23 -like gene was associated with carriage of repAci6 plasmids as previously described (Towner et al., 2011). With few exceptions, strains grouped in ST208 were associated with pACICU2, pMAC02, and p4AYE. Strains grouped in ST191 and ST540 were associated with carriage of pACICU2 and p4AYE. These removable plasmids carrying CHDLs genes led to spreading among A. baumannii strains.
As we mentioned, the spread of carbapenems resistance gene in A. baumannii are of great importance. One of the most popular gene bla OXA−23 was discovered here. Of four transposons reported, Tn2006 and Tn2008 were identified in CRAB isolates, both of which were flanked by ISAba1. However, Tn2007 and Tn2009 are not seen in agreement with previous researches. It was noted that ISAba1 facilitated the mobilization of bla OXA−23 gene hence allowed Tn2006 and Tn2008 to move. These four transposons were observed in plasmids as well as in the chromosome of A. baumannii (Nigro and Hall, 2016). Besides, Tn2006, Tn2007, and Tn2008 were found in different locations of conjugative repAci6 group plasmids (Nigro and Hall, 2015). It seemed that the bla OXA−23 dissemination might due to transposition of mentioned transposons.
Our survey suggested that both clonal spread of a GC2 strain carrying carbapenems resistance genes and the spread of conjugative plasmid among A. baumannii strains are responsible for the serious increasing carbapenems resistance. Further understanding of related plasmids may help determine their acquisition, dissemination and regulation among A. baumannii.

AUTHOR CONTRIBUTIONS
CY conceived the work. YC performed all experiments, analyzed the results, and wrote the manuscript. JG and HZ assisted in antimicrobial sensitivity testing. All authors read and approved the final paper.

FUNDING
This study was supported by National Nature Science Foundation of China (Grant 81371874).