Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla _OXA-58 in Acinetobacter spp. from Multiple Cities in China

Bacterial Strains and Antimicrobial Susceptibility Testing

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates collected from six hospitals in five different cities in China between 2005 and 2010 were analyzed in this study (Table 1). The genomic species identification was performed by sequence analysis of the 16S-23S rRNA intergenic spacer region [18].

Imipenem and ticarcillin-susceptible clinical A. baumannii strain LS0148 (imipenem MIC, 0.5 mg/L; ticarcillin MIC, 16 mg/L), deposited in our laboratory, was used as the recipient for plasmid electrotransformation (Table 1). A colistin-resistant mutant strain of A. baumannii LS0148 (colistin MIC, 64 mg/L) was used as the recipient for plasmid conjugation.

MICs were determined by the agar dilution method. Interpretation of the results was in accordance with the CLSI 2013 criteria.

All isolates present in this study were stored in the Department of Microbiology, the First Affiliated Hospital, College of Medicine, Zhejiang University. We obtained an exempt status from the Institutional Review Board of the First Affiliated Hospital, College of Medicine, Zhejiang University to use these strains to perform all experiments in this study.

PCR Experiments for the Resistance Genes

PCR assays for the presence of carbapenemase encoding genes (bla _OXA-51-like, bla _OXA-58-like, bla _OXA-23-like, bla _OXA-40-like, bla _OXA-143, bla _IMP, bla _VIM, bla _SIM and bla _NDM) and ESBL genes (bla _PER and bla _SHV) were performed as previously reported [19]–[21].

Pulsed-field Gel Electrophoresis and Multilocus Sequence Typing Analysis

The genetic relationship of the isolates was evaluated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results of PFGE were interpreted as Tenover et al. recommended [22]. MLST was carried out using the scheme developed by Bartual et al. with some modifications to the primers of the alleles of gyrB and rpoD as we previously reported [23], [24].

Plasmid Conjugation and Electrotransformation

Plasmid conjugations were performed between OXA-58 producing Acinetobacter spp. as donors and a colistin-resistant mutant strain of A. baumannii LS0148 as the recipient. The transconjugants were selected on MH agar plates containing ticarcillin (100 mg/L) and colistin (10 mg/L).

The electrical pulse setting of plasmid electrotransformation was 1.8 kV, 25 µF, 200 Ω with Bio-Rad GenePulser Xcell system (Bio-Rad, Shanghai, China). A. baumannii strain LS0148 was used as the recipient. The transformants were selected on MH agar plates containing ticarcillin (100 mg/L).

S1 Nuclease-based Plasmid Analysis

The plasmid size and the location of bla _OXA-58 were analyzed using the S1 nuclease-PFGE method as previously reported [25]. The bacterial-imbedded gel slices were incubated with 10 U S1 nuclease (Takara, Dalian, China) for 40 minutes in 37°C water bath. The digestion products were separated by PFGE using Bio-Rad CHEF Mapper XA system (Bio-Rad, Shanghai, China) with switch times of 2.16S to 63.8S for 18 hours.

The separated DNA was transferred to a positive charged Nylon membrane (Millipore, Shanghai, China) and hybridized with a digoxigenin-labled bla _OXA-58 probe. The detection of hybrids was performed using enzyme immunoassay and NBT/BCIP coloration according to the manufacturer’s instruction (Roche, Shanghai, China).

PCR-based Plasmid Replicon Typing

The plasmid replicase genes were detected by multiplex PCR scheme developed by Bertini et al [26]. The novel replicase gene repAci10 was detected by a single PCR with primers designed in this study (Forward primer: 5′-TAGGACGTCAAGCATCTTA-3′; backward primer: 5′-TCGCTATCAAGAAGATCAC-3′).

Cloning Experiments

The genetic contexts of bla _OXA-58 were determined by cloning and sequencing experiments. The plasmids or total DNA were digested by EcoRI or SacI. The digested fragments were inserted into corresponding sites of pET28a, and the ligation mixture was used for transformation. Transformants were selected on MH agar containing ampicillin 50 mg/L and kanamycin 50 mg/L. The bla _OXA-58-containing inserts were fully or partially sequenced to obtain the context of bla _OXA-58.

Nucleotide Sequence Accession Numbers

The novel insertion sequence ISAba20 has been submitted to the IS Finder Database (http://www-is.biotoul.fr/). The nucleotide sequences surrounding bla _OXA-58 of AN119, AP04, WA3 and WH8144 are deposited in the GenBank database under accession no. JQ241789 to JQ241792 respectively.

Species Identification and Antimicrobial Susceptibility Profiles

The 12 OXA-58-producing Acinetobacter spp. isolates were assigned to three genomic species: A. baumannii (seven isolates), A. nosocomialis (three isolates) and A. pittii (two isolates), and showed various resistance profiles (Table 1 and 2). The five non-baumanii Acinetobacter displayed imipenem and meropenem susceptible. On the contrary, all of the A. baumannii isolates were imipenem and meropenem resistance. In general, the A. baumannii isolates were more frequently resistant to broad-spectrum cephalosporins, ampicillin/sulbactam, aminoglycosides, ciprofloxacin and minocycline than the five non-baumanii Acinetobacter.

Molecular Epidemiology of the OXA-58-producing Acinetobacter spp.

PFGE identified five pulsotypes among the 12 OXA-58-producing Acinetobacter spp. isolates (Table. 1). Two A. pittii isolates from different hospitals showed a same pulsotype. Three A. nosocomialis isolates from a single hospital belonged to two pulsotypes. Seven A. baumannii isolates were divided into two pulsotypes, corresponding to two sequence types (ST91 and ST363). A. baumannii ST91 were identified from two hospitals in different cities (Wuhan and Jinhua). Moreover, ST91 were detected in A. baumannii collected from Jinhua Center Hospital in 2005 and 2009, implying probable endemic in this hospital.

Distribution of Resistance Genes

The A. pittii and A. nosocomialis were negative for other carbapenemase genes and ESBLs. Intrinsic bla _OXA-51 was detected in all A. baumannii isolates. All A. baumannii ST91 isolates carried another CHDL gene bla _OXA-23. bla _PER-1 was detected in WA3 and WA8 (Table 1).

The Plasmid Localization of bla _OXA-58

The bla _OXA-58-probe hybridized with plasmid bands of different sizes, from ca. 52 kb to 143 kb. Isolates with same pulsotypes generally possessed same plasmid location of bla _OXA-58, except AP04 and AP25 (Table 2).

The Transferability of bla _OXA-58-carrying Plasmids

While plasmid conjugation ultimately failed, bla _OXA-58-carrying plasmids were successfully electro-transferred from all Acinetobacter spp. isolates to the recipient strain.

PCR detection of transformants found bla _PER-1 and bla _OXA-23 were not co-transferred with bla _OXA-58, suggesting these genes are not colocalized on a single plasmid.

The results of antimicrobial susceptibility testing are presented in Table 2. Electrotransformation of bla _OXA-58-harboring plasmids into recipient strain LS0148 resulted in high resistance to ticarcillin (>256 mg/L) and increased MICs of imipenem and meropenem (2 to 32 folds), but transformants retained similar MICs of cefepime, ceftazidime and cefotaxime when compared with that of the original LS0148 strain. The transformants of A. nosocomialis AN119 and all A. baumannii displayed higher MICs of imipenem and meropenem than transformants of A. pittii isolates and remaining A. nosocomialis isolates. Transformants TWH8144, TJH01, TAB212 also showed gentamicin and amikacin resistance, implying potential aminoglycosides resistant determinants are colocalized with bla _OXA-58 on the same plasmid.

Identification a Novel Plasmid Replicase Gene

Further investigation of the bla _OXA-58-containing clone fragment of strain WA3 identified a novel plasmid replication protein gene (Figure. 1). This replication protein belonged to Rep-3 superfamily group. It shared similarity with two replication proteins deposited in GenBank database: Acinetobacter sp. RUH2624 (ZP_05826577; 100% amino acid identity) and A. radioresistens SH164 (ZP_06073941; 73% amino acid identity). We have designated this novel replicase gene as repAci10 herein. Of the available A. baumannii replicase genes in the current replicon typing scheme [26], repAci5 was most similar to repAci10 (66% nucleotide identity). Therefore, repAci10 should be assigned as a novel homolog group (GR20). No iteron was identified upstream of repAci10.

Using the current PCR-based replicon typing scheme of A. baumannii [26], only GR8 was detected in strain JH01 and JH02 from the 12 OXA-58 producing Acinetobacter spp. (Table 2). GR3 and GR7 are the intrinsic plasmid rep genes of recipient strain LS0148. No other replicase genes were detected in the transformants except for the intrinsic plasmid replicase genes of LS0148 (GR3 and GR7), suggesting the bla _OXA-58-carrying plasmids do not belong to any previously known replicon group. The novel replicase gene repAci10 was detected in A. pittii (AP04, AP25), A. nosocomialis (AN113, AN116 and AN119), A. baumannii (WA3, WA8) and their transformants (Table 2).

Genetic Contexts of bla _OXA-58

Four kinds of genetic contexts of bla _OXA-58 were identified among 12 Acinetobacter spp. (Figure. 1). Structure A included two A. pittii isolates (AP04, AP25) and two A. nosocomialis isolates (AN113 and AN116). Structure B encompassed all A. baumannii ST91 isolates of WH8144, JH01, JH02, AB212 and AB222. Structure C encompassed A. baumannii isolates WA3 and WA8. Structure D included A. nosocomialis isolate AN119. The most notable difference was the IS elements located upstream of bla _OXA-58. In structure A, an intact ISAba3-like element was exclusively present upstream of bla _OXA-58. However, in structure B, C and D, the ISAba3-like element upstream of bla _OXA-58 was truncated at a same position (58 bp downstream of the start codon of the transposase gene of ISAba3-like element) by ISOur1, IS1008 and IS15 respectively. All of the latter three IS elements belong to the IS6 family. The transformants of the plasmids with the structure of IS6 family-ΔISAba3-like-bla _OXA58 displayed a much higher increase in imipenem MICs (16–32 folds) than those with intact ISAba3-like-bla _OXA58 (2–4 folds) (Table 2).

ISAba3, araC1 (putative transcriptional regulator gene) and lysE (putative threonine efflux protein gene) were identified downstream of bla _OXA-58 in all isolates. However, the araC1 and lysE in structure B were disrupted by ISAba20 and TnaphA6 respectively. ISAba20 is a novel insertion sequence of IS3 family, and is 1199 bp long with two ORFs. The insertion of ISAba20 into araC1 generated two 4-bp direct repeats (CTTA). TnaphA6 was a composite transposon, comprising an aminoglycoside O-phosphotransferase gene, aphA6, and two flanked ISAba125 of same orientation. TnaphA6 was inserted into lysE and generated two 3-bp target site duplications (CTG).

It has been reported that the acquisition of bla _OXA-58 is usually associated with recombination events characterized by the presence of two 27-bp sequences named Re27-1 and Re27-2 [9]. In structure A, we identified a similar Re27-1 sequence located 8 bp downstream of the intact ISAba3-like element (5′-ATTTAACATAATGGCTGTTATACGAAA-3′), and an imperfect Re27-2 sequence (5′-ATTTAACATAATGGTGGTTATACGCAA-3′) was just adjacent to the downstream of lysE. In structure D, a pair of 29-bp imperfect probable recombination points were identified 748 bp downstream of IS15 element (5′-ATTTAACATAATGGTGGTTATGCGAAGTC-3′) and adjacent to lysE (5′-ATTTAACATAATGGGCGTTATGCGAAGTC-3′). In structure B and C, we failed to find pairs of Re27-like regions.