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

Weiyuan Wu; Yi He; Jian Lu; Yuemei Lu; Jinsong Wu; Yingxia Liu

doi:10.1371/journal.pone.0137174

Abstract

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.

Citation: Wu W, He Y, Lu J, Lu Y, Wu J, Liu Y (2015) Transition of bla_OXA-58-like to bla_OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study. PLoS ONE 10(9): e0137174. https://doi.org/10.1371/journal.pone.0137174

Editor: Ruth Hall, The University of Sydney, AUSTRALIA

Received: March 23, 2015; Accepted: August 14, 2015; Published: September 4, 2015

Copyright: © 2015 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper.

Funding: This work was supported by Natural Science Foundation of Guangdong Province, China. (5009113) (http://www.gdstc.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Over the past decade, increasing resistance to carbapenems in Acinetobacter baumannii has been observed worldwide [1]. This increasing resistance is mainly mediated by production of class D (carbapenem-hydrolyzing oxacillinases [CHDLs]) β-lactamases (OXAs) with carbapenemase activity. Currently, six OXAs with carbapenemase activity gene clusters have been described in A. baumannii, including bla_OXA-23-like, bla_OXA-40-like, bla_OXA-51-like, bla_OXA-58-like, bla_OXA-143-like, and bla_OXA-235-like genes [2, 3]. Although the hydrolytic efficiencies of these OXA carbapenemases for carbapenems are relatively low [4], various insertion sequences (ISs) upstream of the bla_OXA carbapenemase genes, including ISAba1, ISAba2, ISAba3, IS18, IS125, IS1008, and ISAba4, provide promoters for the expression of bla_OXA carbapenemase genes, except for bla_OXA-40-like and bla_OXA-143-like genes, and mediate resistance to carbapenems [5–9].

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 [10–12].

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).

Material and Methods

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 [13–15]. 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.

Detection of carbapenemase genes and ISs upstream of CHDL genes

PCR assays for genes coding for known carbapenemases (i.e., bla_IMP, bla_VIM, bla_SPM, bla_SIM, bla_GIM, bla_OXA-23-like, bla_OXA-40-like, bla_OXA-58-like, bla_OXA-143-like, and bla_KPC) were performed as previously described [2, 17–19]. PCR with primers within the ISs (i.e., ISAba1, ISAba2, ISAba3, IS18, IS125, IS1008, and ISAba4) and reverse primers within the CHDL genes [5–9] mapped the upstream locations of ISs.

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 GenePath 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′-GCAGTTGAAGCCGTATCT-3′ and recA-R2, 5′-TTGACCGATACGACGAA-3′) 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/).

Results

Distribution of carbapenemase genes

During the study period, 393 nonduplicate clinical isolates of A. baumannii, with imipenem and/or meropenem MICs ≥ 0.25 μg/ml, were recovered from 367 colonized or infected inpatients in Shenzhen People’s Hospital. A total of 231 isolates of bla_OXA carbapenemase gene-harboring A. baumannii were detected among of them. 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 single isolates, respectively. bla_OXA-143-like, bla_KPC genes, and metallo-β-lactamase genes undetected in any isolates identified in this study. bla_OXA-58-like had been the most common carbapenemase gene in A. baumannii prior to 2008; thereafter, bla_OXA-23-like remarkably increased and became rapidly prevalent in A. baumannii in 2009 (Table 1). IS1008-ΔISAba3, ISAba3, and ISAba1 were found 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.

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Table 1. A. baumannii (Ab) isolates with imipenem and/or meropenem MICs ≥ 0.25 μg/ml from 2002 to 2009.

https://doi.org/10.1371/journal.pone.0137174.t001

Antibiotic resistance profiles

Table 2 shows the MIC distributions for both imipenem and meropenem and bla_OXA carbapenemase gene-harboring A. baumannii. The majority of bla_OXA-58-like-carrying isolates showed lower level of resistance to imipenem (MICs, 1 μg/ml to 16 μg/ml) and meropenem (MICs, 1 μg/ml to 8 μg/ml), compared with bla_OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). Notably, 26 (24.3%) and 27 (25.2%) isolates with bla_OXA-58-like were classified as “susceptible” (MICs, 0.5 μg/ml to 2 μg/ml) and “intermediate” to imipenem, respectively, using the current Clinical Laboratory Standard Institute (CLSI) breakpoint for susceptibility of ≤ 2μg/ml and resistance of ≥ 8 μg/ml. Furthermore, higher susceptible rate of 35.5% (38/107) and intermediate rate of 42.1% (45/107) were observed to meropenem against bla_OXA-58-like-carrying A. baumannii 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. By contrast, 119 bla_OXA-23-like-carrying isolates were classified as resistant to both imipenem and meropenem. One bla_OXA-40-like-carrying isolate and four ISAba1-bla_OXA-51-like-carrying isolates were classified as intermediate or resistant to imipenem and meropenem. Majority of A. baumannii isolates without carbapenemase gene were classified as susceptible to imipenem and meropenem, except for the two (1.2%) and four (2.5%) isolates classified as intermediate to imipenem and meropenem, respectively.

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Table 2. MIC distributions of imipenem and meropenem against A. baumannii (Ab) isolates with or without carbapenemase gene.

https://doi.org/10.1371/journal.pone.0137174.t002

bla_OXA-58-like-carrying isolates showed moderate susceptibility to a few noncarbapenems (Table 3). More than half of bla_OXA-58-like-carrying isolates were still susceptible or intermediate to cefoperazone-sulbactam, ampicillin-sulbactam, and cefepime, compared with less than 5% of bla_OXA-23-like-carrying isolates. Both bla_OXA-58-like-carrying isolates and bla_OXA-23-like-carrying isolates were highly resistant to piperacillin-tazobactam, ceftazidime, ceftriazone, amikacin, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole (resistance rates, 75.7% to 100%). However, these isolates all exhibited low resistance to polymixin B, minocycline, and tigecycline (resistance rate of less than 15%). The MIC distributions of imipenem and meropenem for IS1008-ΔISAba3-bla_OXA-58-like-carrying A. baumannii were similar to those for ISAba3-bla_OXA-58-like-carrying A. baumannii (Table 4).

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Table 3. Susceptibilities of 15 antimicrobial agents against bla_OXA-58-like-carrying and bla_OXA-23-like-carrying A. baumannii (Ab).

https://doi.org/10.1371/journal.pone.0137174.t003

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Table 4. MIC distributions of imipenem and meropenem against A. baumannii (Ab) isolates with various ISs upstream of the bla_OXA-58-like.

https://doi.org/10.1371/journal.pone.0137174.t004

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.

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Table 5. PFGE types of carbapenemase gene-carrying A. baumannii (Ab) from 2002 to 2009.

https://doi.org/10.1371/journal.pone.0137174.t005

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Fig 1. PFGE dendrogram of 10 representative isolates from the three dominant clones.

Allelic profile: seven loci in the order gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD; MIC μg/ml; IPM, imipenem; MEM, meropenem.

https://doi.org/10.1371/journal.pone.0137174.g001

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.

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Table 6. Antibiotic resistance profiles of the three main carbapenemase gene-harboring A. baumannii clones (MIC μg/ml).

https://doi.org/10.1371/journal.pone.0137174.t006

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-nonsusceptible A. baumannii-harbored bla_OXA-23 [11, 24, 25]. In the present study, 119 (30.1%) and 107 (27.2%) of 393 A. baumannii isolates with imipenem and/or meropenem MICs ≥ 0.25 μg/ml carried bla_OXA-23-like and bla_OXA-58-like, respectively. Surprisingly, bla_OXA-58-like had been the most common carbapenemase gene in A. baumannii in Shenzhen People’s Hospital until 2008. bla_OXA-23-like occurred in a sporadic clone I for the first time in the hospital in 2005 and then remarkably increased and became rapidly prevalent in A. baumannii clone J 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-like-carrying 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, 30–32]. 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.

Supporting Information

S1 Fig. DDDs of imipenem and meropenem from 2004 to 2009.

https://doi.org/10.1371/journal.pone.0137174.s001

(TIF)

Author Contributions

Conceived and designed the experiments: WYW JL YXL. Performed the experiments: WYW YH. Analyzed the data: WYW YH YML JSW JL. Contributed reagents/materials/analysis tools: JL WYW YML JSW YXL. Wrote the paper: WYW YH.

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

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

Figures

Abstract

Background

Methods

Results

Conclusion

Introduction

Material and Methods

Bacterial isolates and antimicrobial susceptibility testing

Detection of carbapenemase genes and ISs upstream of CHDL genes

PFGE

MLST

Results

Distribution of carbapenemase genes

Antibiotic resistance profiles

PFGE and MLST

Discussion

Conclusion

Supporting Information

S1 Fig. DDDs of imipenem and meropenem from 2004 to 2009.

Author Contributions

References