Introduction

In recent years, carbapenem resistance in species of the genus Acinetobacter has increased worldwide, and the rapid spread of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe threat to public health [1, 2].

Some reports identified Ambler class A carbapenemases and metallo-β-lactamases (MBLs) in Acinetobacter spp., but carbapenem resistance in these species has mostly been associated with the production of five main groups of carbapenem-hydrolyzing class D β-lactamases (CHDLs), namely, OXA-23-like, OXA-24-like, OXA-51-like, OXA-58-like, and OXA-143-like enzymes [2]. The OXA-23-like enzyme now contributes to carbapenem resistance in A. baumannii throughout the world [3–5]. The OXA-24-like and OXA-58-like enzymes are often involved in hospital outbreaks of CRAB in some European countries, such as Spain, France, Belgium, Italy, and Greece [4, 6]. The OXA-143-like enzyme is the first representative of a novel subgroup of CHDLs, and a high prevalence of OXA-143-producing Acinetobacter isolates has been reported in Brazil [7, 8].

Regarding the current situation in China, our group previously reported clonal dissemination of CRAB harboring bla OXA-23-like among different cities [9], while OXA-24 and OXA-58 were found in several carbapenem-resistant Acinetobacter spp. isolates by Wang et al. in a small-scale molecular epidemiological study in 2007 [10]. However, the distribution of bla OXA and their contribution to the high carbapenem resistance rates of Acinetobacter spp. in the mainland of China are still unclear. In this study, we collected 2,880 Acinetobacter spp. isolates across different geographical regions of China to assess the prevalence of CHDLs and studied the clonal relationship and genomic environment of these CHDLs genes.

Materials and methods

Bacterial strains and susceptibility testing

A total of 2,880 sequential clinical Acinetobacter spp. isolates (up to a maximum of 50 per hospital) were collected from 67 hospitals representing 23 provinces in China from January 2009 to December 2010 (Fig. 1). Each hospital undertook collection for three consecutive months. Isolates were obtained from bile (n = 20), blood (n = 71), catheter (n = 26), exudate (n = 192), lavage (n = 53), puncture fluid (n = 41), respiratory tract (n = 68), skin (n = 2), sputum (n = 2,276), tissue (n = 16), urine (n = 104), and wound (n = 11). All isolates were identified to the genus level using Vitek GNI+ cards (bioMérieux, Marcy l’Etoile, France). A. baumannii were confirmed by polymerase chain reaction (PCR) detection of the bla OXA-51-like gene. 16S-23S rRNA gene intergenic spacer (ITS) sequencing and partial RNA polymerase β-subunit (rpoB) sequencing were performed for the identification of the other Acinetobacter species, as previously described [11, 12]. Antimicrobial susceptibility testing was performed by the disk diffusion method and Etest strips, and the results of susceptibility testing were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2012 guidelines [13].

Fig. 1
figure 1

Distribution of carbapenem-hydrolyzing class D β-lactamases (CHDLs) in Acinetobacter spp. in China. OXA-24-like-producing isolates are denoted with squares and OXA-58-like-producing isolates are denoted with circles. The provinces covered in this study are highlighted in gray and OXA-23-like-producing isolates were detected in all these provinces

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Molecular detection of resistance genes

Isolates were screened for the presence of bla OXA genes, including bla OXA-23-like, bla OXA-24-like, bla OXA-51-like, bla OXA-58-like, and bla OXA-143-like genes, by multiplex PCR using published primers and PCR parameters [14]. PCR screening was performed for additional MBLs genes of the bla OXA-24-like and bla OXA-58-like isolates, including bla IMP-like, bla VIM-like, bla SIM-1, and bla NDM-1 [15, 16].

Plasmid analysis and Southern blot

Genomic DNA was digested with S1 nuclease and separated by pulsed-field gel electrophoresis (PFGE) with a switch time from 2.16 to 63.8 s for a 20-h runtime. Then, the DNA fragments were transferred to nylon membranes (Millipore, Billerica, MA, USA), hybridized with digoxigenin-labeled bla OXA-24-like-specific probes, and detected using an NBT/BCIP color detection kit (Roche Applied Science, Mannheim, Germany).

Plasmid DNA was extracted with the Qiagen Midi Kit (Qiagen, Hilden, Germany). The transformation of plasmids was performed using electroporation and Acinetobacter baylyi ADP1 as the recipient. Transformants were selected on agar plates containing meropenem (1 mg/L) and confirmed by PCR analysis.

MLST

Multilocus sequence typing (MLST) was carried out using seven standard housekeeping loci (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) as described by Fu et al. and Bartual et al. [3, 17]. The novel sequence types (STs) unassigned were numbered N1, N2, N3, etc. consecutively.

PCR mapping and primer walking for the genetic context of bla OXA-58-like

Primers (IS6F 5′-CGTAAGCCGTCTTCATGGAT-3′ and ISAba3R 5′-CTTCTGAAGCTACGCCTAAT-3′) were designed to determine the presence of IS6 family-ΔISAba3-like-bla OXA-58-like-ISAba3 structure in bla OXA-58-like-harboring isolates according to the sequences previously deposited in GenBank under nucleotide sequence accession number GU327621 [18].

The plasmids extracted from the ten plasmid-mediated bla OXA-24-like-harboring isolates were partially sequenced on both strands by primer walking.

Results

Distribution of bla OXA-like genes and antimicrobial susceptibility testing

Multiplex PCRs for the detection of the five OXA carbapenemase gene groups were performed with all 2,880 Acinetobacter spp. isolates. bla OXA-51-like genes were found in 2,197 isolates, all of which were presumptively identified as A. baumannii in this study. bla OXA-23-like, bla OXA-24-like, bla OXA-58-like, and bla OXA-143-like genes were detected in 1,316, 11, 32, and 1 isolates, respectively.

The resistance rates of these 2,880 Acinetobacter spp. isolates to the antimicrobial agents tested were about 50 %, except for minocycline (11.6 %) (Table 1). A total of 1,399 (48.6 %) and 1,442 (50.1 %) isolates were resistant to imipenem and meropenem, respectively, while 1,354 (47.0 %) isolates were resistant to both.

Table 1 Antibiotic susceptibilities of Acinetobacter spp. isolates
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OXA-51 group

Among the 2,880 Acinetobacter spp. isolates, 2,197 (76.3 %) possessed bla OXA-51-like genes and were identified presumptively as A. baumannii. The rates of resistance to β-lactams, aminoglycosides, and quinolones in bla OXA-51-like-positive isolates were significantly higher than those in bla OXA-51-like-negative isolates (Table 1). 62.6 % (1,375 isolates) of the A. baumannii isolates were non-susceptible to carbapenems.

OXA-23 group

bla OXA-23-like genes were detected in 1,316 isolates, which covered all provinces included in this study, and 98.3 % (1,294 isolates) were A. baumannii. The resistance rates of these bla OXA-23-like-harboring isolates to the 12 antimicrobial agents tested were extremely high, with a range from 86.1 to 99.2 %, except for minocycline (Table 1). In particular, the resistance rate of these bla OXA-23-like-positive isolates to imipenem and meropenem was above 95 %.

OXA-24 group

bla OXA-24-like genes were detected in 11 isolates obtained from six provinces, which were identified as A. baumannii (n = 6), A. pittii (n = 4), and A. baylyi (n = 1) (Table 2). Isolate A1429 possessed bla OXA-24 and the other isolates possessed bla OXA-72 genes. In addition, all these isolates were resistant to meropenem and no MBL gene was detected. The six bla OXA-24-like-positive A. baumannii isolates were classified into five STs. Interestingly, three isolates (A2485, A2503, and A2706) belonged to CC92, had multidrug resistance phenotypes to all β-lactams, aminoglycosides, and quinolones tested, and co-carried bla OXA-23-like genes (Table 2).

Table 2 Characteristics of 11 bla OXA-24-like-harboring Acinetobacter spp. isolates, one bla OXA-143-like-harboring Acinetobacter spp. isolates, and transformants
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Southern blot hybridization indicated that the bla OXA-24-like genes were located on different small plasmids (<20.5 kb), except A2706 (data not shown). The plasmids carrying bla OXA-24-like genes from the ten isolates were successfully transferred to A. baylyi ADP1. The transformants exhibited at least 30-fold higher minimum inhibitory concentration (MIC) values for carbapenems compared with those for ADP1, and there were minor discrepancies in the susceptibility to minocycline, aminoglycosides, fluoroquinolones, and the other β-lactams between transformants and the recipient (Table 2). After performing PCR detection for the transformants, we found that no bla OXA-23-like gene was co-transferred to the recipient from donor strains A2485 and A2503. The electrotransformations of the bla OXA-24-like gene in A2706 to the recipient failed in repeated attempts. Of note, analysis of the plasmid sequences showed that the bla OXA-24-like genes were flanked by XerC/XerD-like recombination sites in all isolates (GenBank accession no. JX968505).

OXA-58 group

Thirty-two Acinetobacter spp. isolates obtained from ten provinces had the bla OXA-58-like genes (Table 3). Among these bla OXA-58-like-gene-positive isolates, five A. baumannii, one A. haemolyticus, and one A. pittii were non-susceptible to carbapenems and showed variable susceptibilities to other β-lactams. The six bla OXA-58-like-positive A. baumannii isolates were classified into three STs. Four isolates from one hospital in Henan had the same sequence type (ST91) and co-harbored bla OXA-23-like genes. No MBL gene was detected in these 32 isolates.

Table 3 Characteristics of 32 bla OXA-58-harboring Acinetobacter spp. isolates and transformants
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The seven carbapenem-non-susceptible bla OXA-58-like-harboring Acinetobacter spp. isolates were selected to evaluate the transferability of bla OXA-58-like genes. The bla OXA-58-like-harboring plasmids extracted from these isolates were successfully transferred to the recipient strain ADP1 using electrotransformation, except isolate A1323. The transformants exhibited increased MIC values (16- to >340-fold) for carbapenems compared to the ADP1 strain (Table 3). The PCR performed for transformants confirmed that no other CHDLs genes were co-transferred with bla OXA-58 to the recipient, suggesting that they did not co-exist with bla OXA-58 in the same plasmid.

The structure of IS6 family-ΔISAba3-like-bla OXA-58-like, which could increase the transcription level of the bla OXA-58-like gene, has been described in different Acinetobacter spp. isolates [18, 19]. In this study, PCR mapping was performed to identify the genetic sequences surrounding the bla OXA-58-like genes in the 32 bla OXA-58-like-harboring isolates. The seven carbapenem-non-susceptible bla OXA-58-like-harboring isolates gave positive results in PCR detection based on primers located on sequences of IS6 family-ΔISAba3-like-bla OXA-58-like structure (GenBank accession no. JX968506). ISAba3 elements were identified downstream of the bla OXA-58-like genes in all isolates.

OXA-143 group

Only one isolate named A1254 was positive for the bla OXA-143-like gene. Sequencing of the amplicon (564 bp) obtained from A1254 identified a fragment that shared 96 % nucleotide identity with bla OXA-143 (GenBank accession no. JX968504). Isolate A1254 was identified as A. pittii using rpoB sequencing and was susceptible to carbapenems, cephalosporins, aminoglycosides, and quinolones. The S1-digested plasmid DNA and Southern blot hybridization with bla OXA-143-like-specific probe gave a negative result, indicating that this homologous gene might be located on the chromosome.

Discussion

Carbapenem-resistant A. baumannii has become one of most troublesome pathogens throughout the world in the past decade [1, 2, 20]. According to the antimicrobial susceptibility testing results, the carbapenem resistance rates of Acinetobacter spp. were high in China, with 48.6 % (1,399/2,880) and 50.1 % (1,442/2,880) being resistant to imipenem and meropenem, respectively. The results of multiplex PCR showed that 76.3 and 45.7 % of these isolates were positive for bla OXA-51-like and bla OXA-23-like genes, respectively, while 44.9 % (1,294/2,880) of the isolates also harbored bla OXA-51-like and bla OXA-23-like genes. Moreover, more than 95 % of bla OXA-23-like-harboring isolates were resistant to carbapenems, which indicated that the high prevalence of OXA-23-producing A. baumannii was the predominant reason for high carbapenem resistance rates of Acinetobacter spp. in China. According to the results of the MLST analysis for the carbapenem-resistant A. baumannii we previously reported, it is determined that OXA-23-producing CC92 isolates have disseminated throughout hospitals in China and played an important role contributing to the high prevalence of carbapenem-resistant Acinetobacter spp. in this country [3, 21].

Comparing with the bla OXA-23-like genes, the bla OXA-24-like genes were less frequently identified in Acinetobacter spp. in China, as only 11 bla OXA-24-like-positive isolates were detected. These bla OXA-24-like-harboring isolates were resistant to carbapenems and most had multidrug-resistance phenotypes. Furthermore, the bla OXA-24-like genes were located on small plasmids in most isolates. Moreover, analysis of the genetic environment showed that the bla OXA-24-like genes were flanked by XerC/XerD-like sites, which were considered to be responsible for the mobilization of the bla OXA-24 gene [22]. Outbreaks of multidrug-resistant A. baumannii harboring bla OXA-24-like genes were reported in USA, Spain, and sporadic isolates were reported in China [10, 23–25]. Although our surveillance showed that the bla OXA-24-like-harboring Acinetobacter spp. were less prevalent in China, the plasmid location and bla OXA-24 mobilization cassette (Xer system) identified in these isolates were similar to the outbreak isolates in Europe and USA, indicating that these bla OXA-24-like genes have the capacity to disseminate among different Acinetobacter species in hospital environments and have the potential to cause outbreaks of carbapenem-resistant Acinetobacter spp. in China.

In contrast to the resistant phenotypes observed in bla OXA-24-like-harboring isolates, 25 out of 32 bla OXA-58-like-harboring Acinetobacter spp. isolates remained susceptible to carbapenems. This might be explained by the low-level expression of bla OXA-58-like genes in these isolates, due to the insertions of the IS6 family, which enhance the transcription of the bla OXA-58 gene and mediate resistance to carbapenem, and were identified upstream of the bla OXA-58-like genes in all carbapenem-resistant isolates [18]. OXA-58 is often associated with hospitals outbreaks in European countries and the United States [4]. It appears from our surveillance that the rate of carbapenem-non-susceptible Acinetobacter spp. associated with bla OXA-58-like genes is still rather low in China. However, the dissemination of this group of CHDLs is also worrisome because of the widespread distribution of these genes in Acinetobacter species and the genetic structure we found in the carbapenem-non-susceptible isolates, which contribute to the mobility and expression of bla OXA-58-like genes.

Some Acinetobacter species intrinsically possess chromosomal genes encoding CHDLs [26, 27]. For example, A. baumannii carries bla OXA-51-like, A. radioresistens bla OXA-23-like, and A. lwoffii bla OXA-134-like genes. We identified a bla OXA-143 homologue in a susceptible A. pittii isolate. Though the entire DNA sequence of this bla OXA-143-like gene has not yet been obtained, this finding indicates that the distribution of bla OXA genes is much wider than previously assumed.

In conclusion, this is a large-scale study to characterize the distribution of CHDLs in Acinetobacter spp. in China. Our results indicate that the bla OXA-23-harboring A. baumannii isolates are the most frequent carbapenem-resistant Acinetobacter spp. in China, predominantly CC92 isolates, and the bla OXA-24-like and bla OXA-58-like genes have emerged as potential threats of hospital outbreaks of multidrug-resistant Acinetobacter spp.