Identification of a Novel Plasmid-Mediated Carbapenemase-Encoding Gene, bla_VMB-2, in Vibrio diabolicus

ABSTRACT

Here, we characterized a carbapenem-resistant Vibrio diabolicus strain of shrimp origin with various experiments and bioinformatics analysis. A novel metallo-β-lactamase (MBL) gene, bla_VMB-2, that confers resistance to β-lactams, including meropenem and cephalosporins, was identified on a plasmid-borne composite transposon, ISShfr9-ISCR1-bla_VMB-2-bla_CARB-12-aadA1-ISShfr9, which is capable of generating a bla_VMB-2-bearing circular intermediate. ISShfr9 was found to be disseminated in multidrug-resistant (MDR) pathogens, arousing the concern of further transmission of a bla_VMB-2-bearing circular intermediate to clinical Enterobacterales strains via such insertion sequences, which warrants further investigations.

INTRODUCTION

Carbapenems are important antimicrobials that are considered the last-resort drugs in the treatment of life-threatening infections caused by extended-spectrum β-lactamase (ESBL)-producing and other multidrug-resistant (MDR) Gram-negative pathogens (1). However, the emergence and widespread transmission of carbapenemase-producing Enterobacterales (CPE) species, which are empowered by various carbapenemases, such as NDM, KPC, etc., that are capable of hydrolyzing carbapenems, undermines the efficacy of such vital drugs in clinical setting (2, 3). Environmental and commensal bacteria have been recognized as important reservoirs of novel resistance genes, including metallo-β-lactamases (MBLs) (4). Recently, the MBL-encoding gene bla_VMB-1 was described from a foodborne Vibrio alginolyticus isolate (5). In this study, we report a new bla_VMB variant located on a transposon structure in Vibrio diabolicus recovered from shrimp, which has the potential to disseminate among Enterobacterales species.

Strain SLV18 was isolated from a shrimp (Litopenaeus vannamei) sample purchased at a supermarket in Nanjing, China, in September 2020 during a surveillance project focusing on carbapenem-resistant bacteria of seafood origin. Briefly, the sample was homogenized in a sterile homogeneous bag with 9 ml marine broth in a tissue homogenizer for 2 min, transferred to a tube, and cultured at 30°C overnight, and then the culture was streaked on marine agar plates containing 1 μg/ml meropenem and incubated at 30°C for 18 to 20 h. Colonies were purified and the bacterial species was identified by 16S rRNA gene sequencing. Antimicrobial susceptibility testing (AST) was performed by the standard agar dilution method according to the CLSI guidelines (6). Escherichia coli ATCC 25922 was used as the control. To decipher the molecular mechanism of carbapenem resistance, the complete genome sequence of SLV18 was obtained by whole-genome sequencing combining the high-accuracy short-read Illumina and error-prone long-read MinION platforms with hybrid de novo assembly using Unicycler (7–9). Different bioinformatics tools, such as RAST, ResFinder, ISfinder, and BRIG, were used to annotate and analyze the genome (10–12). Phylogenetic analysis based on the core genome sequences was performed between SLV18 and available representative Vibrio species genomes in the NCBI database using Roary and FastTree tools with default parameters (13, 14). To validate the activity of the identified bla_VMB-2 gene, it was amplified with a pair of primers, SacI-blaVMB-2 and HindIII-blaVMB-2 (see Table S1 in the supplemental material), cloned into pET23a based on homologous recombination technology with the ClonExpress Ultra one-step cloning kit (Vazyme, China), and transformed chemically into E. coli BL21(DE3), while E. coli BL21(DE3) harboring empty pET23a was used as a negative control for AST. Transconjugant/transformant recovery was attempted using the following methods. A filter mating assay with E. coli C600 (rifampin resistant) and E. coli J53 (azide resistant) as the recipient strains, with LB agar plates containing meropenem (1 μg/ml) and rifampin (200 μg/ml) or azide (250 μg/ml) as the selection medium, was performed. Similarly, electroporation was used to evaluate the transfer of the bla_VMB-2-bearing plasmid to E. coli C600 electrocompetent cells, followed by screening on LB agar plates containing 1 μg/ml meropenem.

Strain SLV18 conferred high-level resistance to tested β-lactam antibiotics, including ampicillin, amoxicillin, ceftriaxone, ceftazidime, and meropenem (MIC = 4 μg/ml) (see Table S2 in the supplemental material). Meropenem, but not imipenem, is hydrolyzed very well by VMB-2. This is consistent with previous work analyzing VMB-1 (5). With 16S RNA gene sequence analysis and confirmed by phylogenetic analysis based on core genomes of available V. diabolicus and other representative Vibrio species consisting of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus in the NCBI genome database (see Fig. S1 in the supplemental material), strain SLV18 was clustered together with and identified as Vibrio diabolicus, a marine Vibrio species related to members of the Harveyi clade (15). SLV18 contained two chromosomes (chromosome 1 [chr1], 3,208,522 bp with 45.10% GC content; chr2, 1,769,354 bp with 44.78% GC content) and two plasmids, termed pSLV18-111K and pSLV18-213K. No resistance genes were found on the two chromosomes. pSLV18-111K was 111,069 bp in length and encoded 114 hypothetical proteins without any identifiable antibiotic resistance genes, and no typeable replicon gene was observed. pSLV18-213K was an untypeable plasmid with a size of 213,874 bp and harbored several antibiotic resistance genes, including bla_CARB-12, aadA, sul2, strA, strB, tet(A), floR, and catA, in three resistance regions. Interestingly, a β-lactamase-encoding gene (741 bp) was found between bla_CARB-12 and ISCR1. An online BLASTn search against the NCBI nonredundant (nr) database indicated that this novel gene was most phylogenetically related to bla_VMB-1, with 74% identity at 88% coverage, and which was recently identified encoding a novel metallo-β-lactamase in V. alginolyticus (see Fig. S2 in the supplemental material). The next-greatest similarity was to the bla_IMP-67 (GenBank accession number NG_055271; 81% identity at 13% coverage) gene, encoding B1 metallo-β-lactamase. Amino acid sequence alignments demonstrated that VMB-2 and VMB-1 were clustered together with GIM-1, DIM-1, and TMB-1 (Fig. 1a). bla_VMB-1 lies within the integron within the int-bla_VMB-1-dfrA1-bla_CARB-4 structure. However, bla_VMB-2 was embedded among different mobile elements, namely ISShfr9-ISCR1 and bla_CARB-12-aadA1-ISShfr9. We propose that ISShfr9-ISCR1-bla_VMB-2-bla_CARB-12-aadA1-ISShfr9 is a novel composite transposon. We found that pSLV18-213K shared 99% identity at 90% query coverage with pVCGX2 (GenBank accession number CP020079), hosted by Vibrio campbellii. However, comparative sequence analysis showed that pVCGX2 does not encode either bla_VMB-2 or the elements flanking bla_VMB-2. Notably, it was found that pC1579 (GenBank accession number MN865127) from V. alginolyticus harbored the core region of bla_VMB-2 in four copies of the unit ISShfr9-ISCR1-bla_VMB-2-bla_CARB-12-aadA1-ISShfr9, which was identical the one in pSLV18-213K, but the backbone structure of pC1579 was totally different from that of pSLV18-213K. This implied that the bla_VMB-2-bearing transposon was mobilizable. To further test whether ISShfr9 has any role in the dissemination of bla_VMB-2, PCR was performed using primer pairs P1/P4 and P2/P3 (Table S1). Results showed that the transposon region could generate a circular intermediate, ISShfr9-ISCR1-bla_VMB-2-bla_CARB-12-aadA1, by copy-out mechanism (Fig. 1c), which may facilitate the dissemination of bla_VMB-2 to other genomic loci harboring ISShfr9. The insertion sequence ISShfr9, encoding a transposase with 52% identity to IS3000, was first identified in Shewanella frigidimarina (GenBank accession number NC_008345) and expanded to other species, including Vibrio spp., Aeromonas spp., Proteus spp., etc. Significantly, we found that ISShfr9 existed in an E. coli plasmid, pRF14-1, in which ISShfr9 was close to tet(X4), and in Providencia rettgeri ICEPreChnRF14-2, harboring tet(X6) (16); this demonstrates that ISShfr9 may has the potential to transfer bla_VMB-2 to other MDR elements among different bacterial species. This requires investigation and performance of surveillance, especially regarding the potential to spread to Enterobacterales species. Apart from the bla_VMB-2-bearing structure, another two ISShfr9-bounded MDR elements were identified on pSLV18-213K, namely ISShfr9-sul2-strAB-tet(A)-strB-ΔISCR2-floR-virD2-ΔISCR2-ISShfr9 and ISShfr9-mexEF-ISShfr9. The mexEF gene cluster was a derivative of the complete RND efflux pump operon mexEF-oprN, with most similarity (70% identity at 70% coverage) to homologs in Vibrio spp. and Shewanella spp. in the NCBI database. It was also similar to the homologous genes found in Pseudomonas aeruginosa (63% identity at 68.83% coverage) (17), and the functions of the mexEF gene cluster warrant further research. A resistance gene, catA3, was found surrounded by reverse repeats of ISPrst2.

FIG 1.

(a) Phylogenetic analysis of novel VMB-2 and other similar metallo-β-lactamases. The tree was constructed by the neighbor-joining maximum-likelihood method with 100 replicates. (b) Circular comparison between the bla_VMB-2-bearing novel plasmid pSLV18-213K and other similar plasmids in the NCBI nonredundant (nr) database. The outermost circle indicates the reference plasmid investigated in this study. (c) Linear comparison of the bla_VMB-2-bearing multidrug resistance segments in both pSLV18-213K and pC1579 with the potential ancestor plasmid pVCGX2. The circular intermediate was detected by reverse PCR (P2/P3). Homologous regions are highlighted in gray parallelograms.

The bla_VMB-2-bearing plasmid cannot be conjugated into E. coli C600 or E. coli J53. Electroporation of pSLV18-213K to E. coli C600 was also unsuccessful. Online BLASTn analysis retrieved similar plasmids only in V. campbellii and V. alginolyticus (Fig. 1b). Cloning and antimicrobial susceptibility testing demonstrated that bla_VMB-2 in E. coli BL21 can confer resistance to various β-lactams, including ceftazidime and meropenem, with at least 32-fold increase in MICs compared with that against BL21 with empty vector. This is consistent with the antimicrobial resistance phenotype of bla_VMB-1 in V. alginolyticus (5).

Emergence of the novel plasmid-mediated metallo-β-lactamase gene bla_VMB-2 undermines the antimicrobial arsenal. To the best of our knowledge, this is the first report of a plasmid- and transposon-mediated metallo-β-lactamase gene of V. diabolicus origin. The transmission of such novel resistance gene to other Vibrio strains and to clinical Enterobacterales strains must be investigated immediately.

Data availability.

The complete genome sequences of SLV18 were submitted to the NCBI GenBank database with the following accession numbers: CP069194 to CP069197.