A Novel Plasmid,pSx1,Harboring a New Tn 1696 Derivative from Extensively Drug-Resistant Shewanella xiamenensis Encoding OXA-416

Bacterial strain identification, antimicrobial susceptibility testing, and extended-spectrum β-lactamase detection

Shewanella T17 was isolated on MacConkey agar supplemented with 2 μg/ml cefotaxime from a wastewater sample collected on March 2012 from the Nedir Mohamed Hospital's effluent collector in Tizi Ouzou, Algeria. Identification at the species level was based on the 16S rRNA and gyrB complete nucleotide sequences as described by Huang et al. ⁴

Antimicrobial susceptibility testing was performed using a disk diffusion assay on Mueller-Hinton agar plates, broth microdilution, and E-test according to the Clinical and Laboratory Standards Institutes (CLSI) guidelines. Extended-spectrum β-lactamase (ESBL) production was tested by the double-disk (DD) test using both cefotaxime and ceftazidime disks with or without clavulanate. ¹³

Cloning of bla_OXA-48-like and transformation in Escherichia coli

The genes bla_OXA-416 and bla_OXA-48 (Ay236073 GenBank accession number) were synthesized and cloned in pUC57-Kan-plasmid at GenScript. The two recombinant plasmids and the nonrecombinant (control) were transformed into E. coli DH10B by electroporation using a MicroPulser according to the manufacturer's instructions (Bio-Rad). Transformants were selected on LB plates containing 45 μg/ml of kanamycin according to GenScript recommendations and subjected to antimicrobial susceptibility testing.

Shotgun sequencing, sequence assembly, and bioinformatics analysis

Whole genome sequencing was performed using PacBio (Pacific Biosciences) at the Yale Center for Genome Analysis, Yale School of Medicine, and Yale University (New Haven, CT) using 9 μg of purified genomic DNA. Genomic DNA was purified with the MagAttract DNA Mini M48 Kit using the BioRobot M48 (Qiagen) according to the manufacturer's instructions. PacBio genome sequencing was performed using the PacBio SMRTbell Template Prep Kit v 1.0 (Pacific Biosciences). PacBio long reads were assembled using the Hierarchical Genome-Assembly Process (HGAP) (v3). Consensus polishing was performed with the Quiver algorithm to generate 2 high-quality unitigs. ¹⁴ The unitigs were annotated using Prokka (v 1.11) (http://vicbioinformatics.com). The annotation presented in this study was confirmed manually using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Artemis genome visualization tools (www.sanger.ac.uk/science/tools/artemis). Insertion sequences were identified using ISfinder (www-is.biotoul.fr).

The new transposon number was assigned using the Tn Number Registry webpage (www.ucl.ac.uk/eastman/research/departments/microbial-diseases/tn).

PlasmidFinder (http://cge.cbs.dtu.dk/services/PlasmidFinder) was used to search for known replicon types. traI was compared to plasmid sequences from other γ-Proteobacteria and used to design primers for MOB typing. ¹⁵

For strain identification, the S. xiamenensis T17 genome was compared with the genomes of 20 other Shewanella species using the Genome-to-Genome Distance Calculator (GGDC; http://ggdc.dsmz.de/). ¹⁶

Detection and sizing of the large plasmid by the S1 nuclease/PFGE method

To detect and confirm the size of the pSx1 plasmid, DNA-containing agarose plugs were incubated for 60 min at 37°C with 10 U Aspergillus oryzae S1 nuclease (Thermo Fisher Scientific). ¹⁷ The digested DNA plugs were electrophoresed at 200 V for 19 hr. Salmonella enterica serotype Braenderup strain H9812 was used as the size marker on the gel. ¹⁸

Plasmid isolation, electroporation, and conjugation

Plasmid DNA was extracted according to a modified QIAGEN Plasmid Mini Kit protocol. Solutions P1, P2, and P3 were used for lysis, neutralization, and precipitation, respectively. After centrifugation (15,000 × g at 4°C), the supernatant was poured into a new tube, and the nucleic acid was precipitated by adding isopropanol and centrifuged as described above. The pellet was washed with 70% ethanol (v/v), and the plasmid was resuspended in 30 μl of TE buffer. Two microliters of this extract was used for transformation into E. coli DH10B (Thermo Fisher Scientific) by electroporation according to the manufacturer's instructions using Bio-Rad MicroPulser. The transformants were selected on LB plates containing 32 μg/ml of ceftazidime and were subjected to antibiotic susceptibility testing.

For conjugation, the broth mating technique was performed using S. xiamenensis T17 as the donor with E. coli J53 Az^R as the recipient. Donor and recipient cells were mixed at a ratio of 1:4, 1:1, and 4:1 donor-to-recipient. Luria-Bertani medium plates containing sodium azide (100 μg/ml), chloramphenicol (15 μg/ml), and ceftazidime (4 μg/ml) were used to select the transconjugants.

PCR detection of the dfrA12, dfrA23, and qnrVC6 genes in E. coli DH10B (pSx1)

PCR reactions were performed in a 25 μl volume according to the manufacturer's guidelines using AmpliTaq DNA polymerase (Thermo Fisher Scientific). The primers and PCR conditions for the dfrA12 and dfrA23 PCRs were described previously.^19,20 Primers to amplify qnrVC6 were designed in this study: VC-f (5′-AGTGAACTTCTCACATCAGG-3′) and VC-r (5′-TCAGGAACAATGATTACCCC-3′), and the same PCR conditions as used for dfrA23 with an annealing temperature of 50°C were used.

Accession numbers

The whole genome of S. xiamenensis, bla_OXA-416 with its genetic environment and pSx1 sequences were deposited in GenBank under accession numbers LDOA01000000, KU198597, and CP013115.

Bacterial strain identification and susceptibility testing

Shewanella isolate T17 exhibited 99.5% 16S rRNA nucleotide sequence identity with the S. xiamenensis type strain S4. ⁴ A comparison of the gyrB sequence with those available in GenBank showed similarities between 95.6% and 98.3% (accession nos. FJ589040, KC765141, KR780672, and KP264118) with 95.6% similarity with S. xiamenensis type strain S4. ⁴ In addition, a comparison between the T17 genome with the genomes of 20 other Shewanella species by GGDC showed a high similarity (80.90% ± 3.65) with S. xiamenensis BC01, confirming the identification of isolate T17 as S. xiamenensis.

Susceptibility testing showed S. xiamenensis T17 to be resistant to at least 16 antibiotics from 11 different classes, including penicillin, ampicillin, piperacillin, ticarcillin, cefotaxime, ceftazidime, cefepime, ertapenem, aztreonam, gentamicin, streptomycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tetracycline, azithromycin, and chloramphenicol. This isolate was susceptible to imipenem, meropenem, amoxicillin–clavulanate, ticarcillin–clavulanate, amikacin, and colistin. The MICs (minimum inhibitory concentration) of the antibiotics are shown in Table 1. The DD test was positive for ESBL production.

Genomic assembly and cloning and expression of bla_OXA-416

PacBio sequencing was used to produce reads larger than 10 kb, and Genome assembly yielded two unitigs, 0 (5,085,271 bp) and 1 (282,919 bp) covering the chromosome and a large plasmid, with G + C contents of 46.27% and 47.7%, respectively.

bla_OXA-416 was located on unitig 0, S. xiamenensis T17 chromosome. The genetic context of this gene was similar to that described for bla_OXA-48-like genes in other S. xiamenensis strains^11,12,21 and shared 91.5–100% nucleotide identity (Fig. 1).

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FIG. 1.

Comparison of the genes surrounding bla_OXA-416 identified in Shewanella xiamenensis T17 with those of S. xiamenensis strains S4, ¹¹ BC01 (GenBank accession number JGVI01000025), WCJ25, ¹¹ CC4-7, ²¹ IR24, IR33, and IR34. ¹² The bla_OXA-48-like genes are shown as black arrows, and the genes upstream and downstream are shown as white arrows. The acc sequences of S4 and WCJ25 that were used for the comparison were partials. The black triangles indicate the direct repeats (CCT) flanking ISShes2.

The E. coli DH10B (pUC57-Kan-OXA-416) and (pUC57-Kan-OXA-48) transformants showed MICs of 2 μg/ml for imipenem and ertapenem, 1 μg/ml for meropenem, and >128 and 32/4 μg/ml for piperacillin and piperacillin/tazobactam, respectively (Table 1). The MIC of piperacillin was decreased by tazobactam in S. xiamenensis T17 and E. coli DH10B (pUC57-OXA-416) and (pUC57-Kan-OXA-48), suggesting that OXA-48 and OXA-416 may be partially inhibited by tazobactam.

Experimental detection of a novel large plasmid

The 14,538 bp at both ends of unitig 1 showed 99% identity, indicating that it was likely a circular plasmid. In addition, the sequence coverage of unitig 1 was significantly higher than that of unitig 0 (226.36X vs. 96.92X). The read-length coverage of the plasmid was 2.35 times higher than that of the chromosome, suggesting the presence of approximately two copies of the plasmid per chromosome. The presence of this large plasmid was further confirmed by S1 nuclease digestion followed by PFGE, which showed a single band with an estimated size of 270 kb. Plasmid electroporation into E. coli showed the growth of ceftazidime-resistant strains, confirming the existence of this plasmid. This new plasmid was designated pSx1. The attempted conjugation of pSx1 into E. coli was unsuccessful.

The sequence analysis of pSx1 revealed the presence of repA encoding an initiator protein and parA and parB encoding partition proteins and transfer genes, including traC_F, traV, trbI, traK, traE, traL, traD, traF-like, traH, traG-like, traR, traG, virD2-like, traI, traF, traN, traU, and traW. No known replicon type was identified using PlasmidFinder. This plasmid was not classified into any MOB family based on the sequence analysis of relaxase.

Based on the AT content and direct/inverted repeats, the predicted replication and transfer origins oriV and oriT were located at 193,670–194,613 bp and 248,457–249,184 bp, respectively.

Identification and description of a new transposon, Tn6297

XDR-associated genes are mainly clustered in a 78,178 bp segment on pSx1 located between nucleotide positions 81,645 and 159,821 bp. The G + C content of this segment is 53.96% compared with 45.1% for the rest of the plasmid. The plasmid harbors 84 ORFs and is delimited by two 38 bp inverted repeats (IR _tnp and IR _mer ) from Tn1696 and flanked by a pair of short 5 bp direct repeats (ATGAT), suggesting a duplication event during transposition (Fig. 2). Designated Tn6297, this transposon includes three class 1 integrons and transposons associated with drug resistance genes.

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FIG. 2.

Schematic representation of the 78-kb multidrug resistance Tn6297 in S. xiamenensis T17. Black arrows with white dots correspond to the transposases, resolvases, and tni module genes. Gray arrows represent the integrase genes, and dashed arrows indicate the resistance genes. Black arrows represent the 3′-CS conserved segment of class 1 integrons, and white arrows indicate the other genes, including ORFs with unknown function. The IS are shown as gray boxes. The ovals represent the attC gene cassettes recombination sites. IS, insertion sequences.

The In27 class 1 integron carries 5′-CS, which contains the intI1, dfrA12-orfF-aadA2 gene cassette array conferring resistance to trimethoprim and aminoglycosides and the conserved segment 3′-CS (qacEΔ1, sul1, and orf5). It includes tniBΔ1 and tniA (part of the tni module), a hybrid of Tn402 and related transposons, and is flanked by inverted repeats from Tn402 (IRi and IRt-like).

The second class 1 integron harbors a qacG gene cassette conferring resistance to quaternary ammonium compounds. This integron lacks IRi and IRt, and its integrase was disrupted by the insertion of Tn-tet.

The third class 1 integron is 26,640 bp with an In4-like backbone. It is delimited by a pair of inverted repeats, IRi and IRt, contains three 3′-CS (3′-CS1, 3′-CS2, and 3′-CS3), ISCR1, and ISCR3. It also includes the aacC1-orfP-orfQ-aadA1 array, which has been reported in MDR Acinetobacter baumannii from several countries. ²² 3′-CS1 is followed by a tetracycline resistance gene derived from transposon Tn1721 (tetA(A)Δ5′) and a truncated bla_VEB-16 gene cassette, which confers high-level resistance to broad-spectrum cephalosporins and aztreonam. bla_VEB-16 is flanked by 135 bp repeat elements (Re2 and Re1), which could play a role in its movement by homologous recombination.^23,24 Downstream of 3′-CS2, this integron contains catA2 (encoding chloramphenicol acetyltransferase), sul1Δ (which confers resistance to sulfamethoxazole), cmlA9 (encoding chloramphenicol efflux protein), tetR and tetA(G) (which confer resistance to tetracycline), lysR (transcriptional regulator), groEL/intI1 (chaperonin/integrase), aac(6′)-II gene cassette (which confers resistance to aminoglycosides), and bla_PSE-1 gene cassette (conferring resistance to β-lactams (ampicillin)). The bla_PSE-1 gene cassette is followed by 3′-CS3 (qacEΔ1, sul1, orf5, and orf6). At the right hand, a region similar to that found in the standard In4-type integron is present. This includes the last 123 bp from the IRt end of Tn402 in inverse orientation, followed by IS600, and the last 152 bp from the IRt end of the Tn402 tni module. Mercury resistance genes (merE, merD, merB, merA, merP, merT, and merR) were also detected. The segment covering sul1Δ, cmlA9, tetR, tetA(G), lysR, ISCR3, and groEL/intI1 has 99% identity to SGI2 of S. enterica subsp. enterica serovar Emek. ²⁵

A composite transposon harboring a set of genes encoding multidrug efflux pumps (acrA, acrB, and oep) and a transcriptional regulator (acrR) was identified. This transposon is delimited by two ISEc29 and contains tnpMut encoding a mutator transposase. The second ISEc29 of the efflux system composite transposon lacks the IRR; this could be due to the insertion of Tn-tet delimited by two IS26. These insertion sequences have been shown to cause adjacent deletions excluding the evidence of insertion provided by DR.^26,27 No similar organization was found in other described microorganisms. This region exhibits a G + C content of 45.16%, close to the G + C content of S. xiamenensis (46.27%), and different from the G + C content of Tn6297 (53.96%). This suggests that this region is native and unique to S. xiamenensis. The second composite transposon Tn-tet is delimited by two IS26 and contains tetA(C) and its regulator tetR. Additional genes, including res (resolvase) and xre (antitoxin), were detected.

The msr(E) encoding a macrolide efflux pump protein and mph(E) encoding macrolide 2′-phosphotransferase protein were located downstream of In27 and associated with IS26.

A truncated qnrVC6 gene cassette and dfrA23 conferring resistance to quinolone and trimethoprim, respectively, were associated with ISCR1. The qnrVC6 gene cassette was followed by orfc372-2 (unknown function) and gcn5 (encoding related N-acetyltransferase). The qnrVC6 was previously described in a Vibrio parahaemolyticus plasmid. ²⁸ This is the first report of the qnrVC6 gene in the Shewanella genus. The fragment covering the last 8 bp at the 3′ end of gcn5, dfrA23, the second copy of Tn1696, and up to the 114 bp upstream of intI1 from the In4-like complex class 1 integron shares 99% identity with a homologous region from the virulence plasmid t-ST4 of an S. enterica serotype Typhimurium isolate. ²⁹

Because the antimicrobial susceptibilities of ciprofloxacin and trimethoprim/sulfamethoxazole for the E. coli DH10B (pSx1) transformants were not consistent with the antimicrobial susceptibilities obtained from S. xiamenensis T17 (Table 1), dfrA12, dfrA23, and qnrVC6 were amplified. The results showed that these genes were absent from E. coli DH10B (pSx1) transformants and present in the positive control S. xiamenensis T17. This could be explained by possible genetic rearrangements in E. coli, resulting in the deletion of these genes.