Transition of bla OXA-58-like to bla OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study

Background The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital. Methods Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Results A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People’s Hospital, were investigated between 2002 and 2009. bla OXA-23-like, bla OXA-58-like, bla OXA-40-like, and ISAba1-bla OXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-ΔISAba3, ISAba3, and ISAba1 were detected upstream of the bla OXA-58-like gene in 69, 35, and 3 isolates, respectively. All bla OXA-23-like genes but one had an upstream insertion of ISAba1. bla OXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter bla OXA-23-like became rapidly prevalent and replaced bla OXA-58-like in 2009. The majority of bla OXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 μg/ml to 16 μg/ml), compared with the majority of bla OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). All 231 bla OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A–N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with bla OXA-58-like was the most prevalent before 2008. Clone H (ST229) with bla OXA-23-like became striking between 2007 and 2008. Clone J (ST381) with bla OXA-23-like rapidly spread and replaced clones A and H in 2009. Conclusion This study is the first to reveal that the distinct bla OXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent bla OXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People’s Hospital in 2009.

Clonal spread of carbapenem-resistant A. baumannii has been reported worldwide. Three epidemic lineages of A. baumannii, commonly referred to as the pan-European clonal lineages (EU I, EU II, and EU III), account for the majority of A. baumannii infections. Strains that belong to EU II (global clone 2) are widespread throughout the world, including China; many epidemiological studies reported the widespread of OXA-58 producers and OXA-23 producers within this lineage [10][11][12].
In this study, the transition of bla OXA-58-like to bla OXA-23-like in A. baumannii clinical isolates from a Chinese hospital between 2002 and 2009 was confirmed. The clonal relatedness of isolates was also investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).

Bacterial isolates and antimicrobial susceptibility testing
All nonduplicate clinical isolates of A. baumannii were recovered from various wards and clinical samples in Shenzhen People's Hospital (Shenzhen, Guangdong Province, China), a tertiary-care hospital with 1200 beds, over an 8-year period from 2002 to 2009. The isolates were initially identified using the Vitek 2 system (bioMerieux) and assigned to the Acinetobacter calcoaceticus-A. baumannii complex. Identification of A. baumannii was confirmed by the presence of bla OXA-51-like intrinsic to this species by using PCR [13][14][15]. Agar dilution was performed to detect susceptibilities to imipenem and meropenem for all A. baumannii isolates [16]. Isolates with imipenem and/or meropenem minimum inhibitory concentrations (MICs) 0.25 μg/ml were further investigated for the carbapenemase genes. MICs of other 13 antimicrobial agents were also determined by agar dilution for carbapenemase gene-carrying A. baumannii isolates. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the controls.

PFGE
PFGE determined the clonal relationships of the carbapenemase gene-carrying A. baumannii isolates. PFGE of ApaI (New England)-digested genomic DNA was conducted using the Gene-Path system (Bio-Rad) as previously described [20,21]. DNA macrorestriction patterns were interpreted according to the criteria described by Tenover et al [22] and cluster analysis was performed using Fingerprint II software (Bio-Rad). Dendrograms for similarity were constructed using the unweighted-pair group method with arithmetic averages. The Dice correlation coefficient was used to analyze any similarities between banding patterns. In brief, isolates that showed zero to three DNA fragment differences and a similarity of 85% following dendrogram analysis were considered to represent the same PFGE type.

MLST
MLST was conducted as previously described [11,23] for the representative isolates from the prevalent main clones typed by PFGE. In brief, internal fragments of seven housekeeping genes, i.e., gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, were PCR amplified, purified, and then sequenced with an ABI prism sequencer 3730 (Applied Biosystems). A new primer pair was redesigned (recA-F2, 5 0 -GCAGTTGAAGCCGTATCT-3 0 and recA-R2, 5 0 -TTGACCGATACG ACGAA-3 0 ) for both amplification and sequencing to obtain the specific PCR products and satisfactory sequencing results. The internal fragments for analysis were still identical to a previous scheme [23]. The sequence of each allele was compared by Basic Local Alignment Search Tool with existing sequences in Pubmlst database and sequence types (STs) were designated according to the allelic profiles (http://pubmlst.org/abaumannii/).

PFGE and MLST
All bla OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A-N). Three dominant PFGE-defined clones A, J, and H comprised 100 (43.3%), 97 (42.0%), and 19 (8.2%) isolates, respectively. Clone A with bla OXA-58-like had been the most prevalent prior to 2008. Clone H with bla OXA-23-like became notable between 2007 and 2008. Clone J with bla OXA-23-like rapidly increased and became the dominant clone in place of clones A and H in 2009 (Table 5). Ten representative isolates of the three dominant clones A, J, and H, which were obtained from ten inpatients, belonged to three different sequence types ST92/ST208, ST381, and ST229, respectively. ST229 was different from ST92/ST208 and ST381 by six alleles. Only two allelic (gyrB and gpi) differences were observed between ST381 and ST92/ST208 (Fig 1), both of which belong to global clone 2. ST381 (clone J) showed apparently different resistance profiles compared with ST92/ST208 (clone A) ( Table 6). ST381 isolates were uniformly resistant to all β-lactam drugs tested. By contrast, ST92/ST208 isolates showed variable resistance to imipenem, meropenem, cefoperazone-sulbactam, ampicillin-sulbactam, and cefepime.

Discussion
bla OXA-23-like carbapenemase genes are disseminated worldwide [1]. In China, bla OXA-23-like is the most common carbapenemase gene in A. baumannii, with more than 90% of imipenem-   in 2009. Notably, the similar replacement of bla OXA carbapenemase genes in A. baumannii was reported in Italy during the same period [10,26]. We also found that the majority of bla OXA-58-like -carrying isolates showed lower level of resistance to carbapenems compared with bla OXA-23-like -carrying isolates. Only 54 (50.5%) and 24 (22.4%) of 107 bla OXA-58-like -carrying isolates were classified as resistant to imipenem and meropenem, respectively, using the current CLSI breakpoint. By contrast, all 119 bla OXA-23-like -carrying isolates were classified as resistant to both imipenem and meropenem. Less bla OXA-58-like -carrying isolates would be classified as resistant to imipenem (16/107) and meropenem (3/107) using the previous CLSI breakpoint for susceptibility of 4 μg/ml and resistance of 16 μg/ml [27]. Interestingly, bla OXA-58-likecarrying isolates showed moderate susceptibility to cefoperazone-sulbactam, ampicillin-sulbactam, and cefepime compared with bla OXA-23-like -carrying isolates, which were highly resistant to these drugs in the present study. Coelho et al. [28] examined 28 isolates of bla OXA-58-like -carrying A. baumannii collected worldwide. They found that imipenem and meropenem MICs of 1-4 μg/ml were detected in 17 and 22 isolates, respectively. The carbapenem MICs varied from 32 μg/ml to 1-4 μg/ml for the isolates from different countries. Based on these findings, we speculate that some bla OXA-58-like -carrying A. baumannii isolates may spread undetected in previous studies from China because of the relatively low imipenem and/or meropenem MICs for these organisms. The flanking IS elements ISAba1, ISAba2, ISAba3, ISAba825, IS18, and IS1008 regulate bla OXA-58-like gene expression. Meanwhile, the latter four all provide hybrid promoters, as described in the recent studies [5,6,8,29]. IS1008-ΔISAba3 was the most common IS upstream of the bla OXA-58-like gene in A. baumannii clinical isolates in this study, followed by ISAba3 and ISAba1. Chen et al. reported that a single plasmid-borne IS1008-ΔISAba3-bla OXA-58 is enough to confer a high level of resistance to carbapenem for A. baumannii. The insertion of IS1008 provided a hybrid promoter and increased the transcription level of the bla OXA-58 gene [8]. However, the present study found that IS1008-ΔISAba3-bla OXA-58-like -harboring A. baumannii isolates showed variable susceptibility to carbapenems (MICs 1 μg/ml to 32 μg/ml). Meanwhile, the similar carbapenem MIC distributions were also detected in ISAba3-bla OXA-58-like -harboring A. baumannii isolates (MICs 0.5 μg/ml to 32 μg/ml). The reasons for the variation in the resistance levels remain unknown. Several previous studies demonstrated that the overexpression of the AdeABC efflux pump and expression of OXA-23 or OXA-58 lead to higher levels of carbapenem resistance [26,[30][31][32]. In addition, Bertini et al. [33] described that the multiple copies of bla OXA-58 increase the level of resistance to carbapenems. However, the study of D'Arezzo showed the opposite conclusion; they reported that the resistance to meropenem or imipenem is not associated with bla OXA-58-like gene copy number per plasmid or to loss of integrity of the CarO porin [26]. Taken together, we speculate that the variable ISs upstream of the bla OXA-58-like gene, high copy number of bla OXA-58-like , overexpression of efflux system, and other cofactors may confer a high level of resistance to carbapenem in A. baumannii. In addition, neither ISAba1 nor ISAba4 was detected upstream of the bla OXA-23-like gene in one isolate in the current study, though several attempts were conducted. This result may be due to another unknown resistance mechanism, which confers resistance to carbapenems in this isolate.
A. baumannii clonal complex 92, corresponding to the global clone 2, has been found worldwide [34], which comprises more than 100 STs, including ST75, ST92, ST92/ST208, and ST381. To the best of our knowledge, ST75, ST92, and ST92/ST208 were the most common STs in China, and ST381 was first identified as sporadic clone in two hospitals in Sichuan, Southwest China in 2011. All of these STs harbored bla OXA-23 gene [11,12]. Notably, the present study demonstrated the prevalence of ST92/ST208 with bla OXA-58-like in Shenzhen People's Hospital prior to 2008. Surprisingly, ST381 with bla OXA-23-like first emerged in this hospital on December 30, 2008; thereafter, it rapidly spread and replaced the ST92/ST208 and ST229 with bla OXA-23-like in 2009. ST229 occurred for the first time in this hospital in 2006 and became one of the main clones in 2007 and 2008, which is genetically completely unrelated to ST92/ST208 and ST381. The reasons for the prevalence of clone J (ST381) are still unknown. Clone J was first isolated from the sputum of a 74-year-old male diabetes inpatient, who had been artificially ventilated for seven days in intensive care unit (ICU) for severe community-acquired pneumonia. The reinfection of A. baumannii was confirmed by infectious-disease physicians with subsequent several positive sputum cultures, clinical symptoms and signs, and effective responses to antibiotic therapy against A. baumannii with cefoperazone-sulbactam. This patient impossibly introduced the ST381 strain with bla OXA-23-like in the hospital, because both of his two sputum cultures obtained on the first and fifth days of his hospitalization in the ICU showed negative results. This strain possibly survived in the ICU environment prior to this infection. By investigating the usage of carbapenems in the inpatients of Shenzhen People's Hospital from 2004 to 2009, 1.8 and 2.2-fold increase of defined daily doses (DDDs) of imipenem (1521.25 to 2777.5) and meropenem (1464.75 to 3232.5) were observed in 2009, respectively. In particular, the DDDs of meropenem had been maintained higher than those of imipenem since 2006 (S1 Fig). Neither a change in the hospital policy nor the introduction of a new antibiotic was observed during this period. We hypothesized that the increasing selective pressure in this hospital environment screened the clone J with bla OXA-23-like , which subsequently caused the huge outbreak in 2009. Minandri et al. [10] investigated the transition of bla OXA-58 to bla OXA-23 gene carriage from 2005 to 2009 among A. baumannii isolates responsible for ICU outbreaks in the main hospitals of central Italy. They found that all isolates from the transition period demonstrate extensive genetic similarity, all belonging to ST2 determined by the scheme of Daincourt et al [35]. Interestingly, the present study also indicates the occurrence of clone replacement between genetically similar ST381 and ST92/ST208. We speculate that the higher carbapenemase activity of OXA-23-like compared with OXA-58-like, may provide bla OXA-23-like -carrying ST 381with a selective advantage over bla OXA-58-like -carrying ST92/ ST208 by increasing the resistance to both imipenem and meropenem. However, the dominant role of ST 381 remains unknown among the A. baumannii population in the short period other than ST229, although the latter occurred earlier. Further study is needed to elucidate this question.

Conclusion
We first reported the distinct bla OXA-23-like -carrying A. baumannii ST381 with high level of resistance to carbapenems, which rapidly spread and replaced the previously prevalent bla OXA-58-like -carrying ST92/ST208 with variable susceptibility to carbapenems, resulting in the increased resistance to carbapenems in A. baumannii in a Chinese hospital in 2009.