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. 2024 Jul 31:15:1437660.
doi: 10.3389/fmicb.2024.1437660. eCollection 2024.

Characterization and transmission of plasmid-mediated multidrug resistance in foodborne Vibrio parahaemolyticus

Haibo Zhou  1   2 , Zhaoxin Lu  1 , Xinmei Liu  2 , Xiaomei Bie  1 , Xinping Cui  1 , Zuwei Wang  1 , Xiaojie Sun  2 , Jun Yang  2
Affiliations

Characterization and transmission of plasmid-mediated multidrug resistance in foodborne Vibrio parahaemolyticus

Haibo Zhou et al. Front Microbiol. .

Abstract

Objectives: The purpose of this study was to determine the structural features and transferability of the multidrug-resistance (MDR) plasmid, and resistance phenotypes for the tested antimicrobials in foodborne Vibrio parahaemolyticus.

Methods: Plasmids were isolated from a V. parahaemolyticus strain of seafood origin, then sequenced using the Illumina NovaSeq 6000 and PacBio Sequel II sequencing platforms to obtain the complete genome data. Characterization of the MDR plasmid pVP52-1, including determination of antimicrobial resistance genes (ARGs), plasmid incompatibility groups, and transferability, was carried out.

Results: V. parahaemolyticus strain NJIFDCVp52 contained two circular chromosomes and two circular plasmids (pVP52-1 and pVP52-2). Plasmid typing indicated that pVP52-1 belonged to the incompatibility group IncA/C2 and the sequence type pST3. pVP52-1 carried 12 different ARGs, an IS110-composite transposon consisting of aac(6')-Ib-cr, qnrVC1, aac(6')-Ib, dfrA14, and the IS26-mphA-IS6100 unit flanked by inverted sequences of IS5075 and IS4321. pVP52-2 carried no ARGs. A plasmid elimination assay showed that only pVP52-1 and its ARGs were lost, the loss of resistance to several antimicrobials, causing a change from the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-trimethoprim/sulfamethoxazole resistance pattern to the ampicillin resistance pattern. In accordance, a conjugation transfer assay showed that only pVP52-1 and its ARGs were horizontally transferred, leading to increased antimicrobial resistance in Escherichia coli strain EC600, causing a change from the ampicillin-nalidixic acid resistance pattern to the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-nalidixic acid-chloramphenicol-tetracycline-trimethoprim/sulfamethoxazole-azithromycin resistance pattern. Further transferability experiments revealed that pVP52-1 could be transferred to other enterobacterial strains of E. coli and Salmonella.

Discussion: This study emphasizes the urgent need for continued surveillance of resistance plasmids and changes in antimicrobial resistance profiles among the V. parahaemolyticus population.

Keywords: Vibrio parahaemolyticus; antimicrobial resistance genes; horizontal transfer; multidrug resistance; plasmid; whole genome sequencing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Circle comparisons of the whole pVP52-1 sequence with similar plasmids. From the inside outwards: the innermost circle represents the genome size; the second and third circles represent GC content and GC skew; the fourth circle represents pVP52-1 from Vibrio parahaemolyticus strain NJIFDCVp52; the fifth circle represents pVb1796 from Vibrio alginolyticus strain; the sixth circle represents pWCX23_1 from Aeromonas hydrophila strain; the seventh circle represents pEC11-1b from Escherichia coli strain; the eighth circle represents KP113-OXA-10 from Klebsiella pneumoniae strain; the ninth circle represents pSL131 from Salmonella enterica subsp. enterica strain; the tenth circle represents pAR-0429-1 from Escherichia coli O157 strain; and the outermost circle represents ARGs of the reference sequence pVP52-1. Visualization of the BLAST analysis and ARGs of annotation was performed with BRIG v0.95.
Figure 2
Figure 2
Linear comparisons of pVP52-1 resistance regions with similar plasmids. Arrows represent the positions and transcriptional directions of genes, and colors represent gene functions. Shaded areas indicate positive genetic similarity (blue) and reverse genetic similarity (pink) in the corresponding region between plasmids. The alignment was performed with Easyfig_win_2.2.5.
Figure 3
Figure 3
PCR amplification products of ARGs and the plasmid-specific gene. 15 different ARGs were detected by PCR before and after plasmid elimination (A) and conjugation transfer (B). pVP52-2-mediated specific gene (vp52004910) was detected by PCR before and after plasmid elimination (C). (A,B) M, DL500 DNA marker; Lane 1, tet34; Lane 2, tet35; Lane 3, tetA; Lane 4, sul2; Lane 5, dfrA1; Lane 6, dfrA14; Lane 7, blaCARB-4; Lane 8, blaCARB-18; Lane 9, qnrVC1; Lane 10, floR; Lane 11, mphA; Lane 12, aph(3″)-Ib; Lane 13, aph(6)-Id; Lane 14, aac(6′)-Ib-cr; Lane 15, aac(6′)-Ib. (C) M, DL500 DNA marker; N, blank control; Lane 1, NJIFDCVp52; Lane 2, Vp52-Δplasmid; Lane 3, EC600; Lane 4, Vp52-EC600. The blue rectangle indicate the area of target bands.
Figure 4
Figure 4
Inhibitory zone of Vibrio parahaemolyticus strain NJIFDCVp52 before and after plasmid elimination. OFX, Ofloxacin; CHL, Chloramphenicol; CAZ, Ceftazidime; TET, Tetracycline; SXT, Trimethoprim/sulfamethoxazole; CIP, Ciprofloxacin; FOX, Cefoxitin; SAM, Ampicillin/Sulbactam; IPM, Imipenem; CFZ, Cefazolin; AZI, Azithromycin; and CTX, Cefotaxime.
Figure 5
Figure 5
Inhibitory zone of Escherichia coli strain EC600 before and after conjugation transfer. OFX, Ofloxacin; AMP, Ampicillin; CTX, Cefotaxime; AZI, Azithromycin; CAZ, Ceftazidime; CHL, Chloramphenicol; SAM, Ampicillin/Sulbactam; IPM, Imipenem; CFZ, Cefazolin; CIP, Ciprofloxacin; FOX, Cefoxitin; SXT, Trimethoprim/Sulfamethoxazole; and TET, Tetracycline.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Natural Science Foundation of China (grant number 32272295), the Science and Technology Project of Jiangsu Provincial Administration for Market Regulation (grant number KJ207544), the National Key Research and Development Program of China (grant number 2022YFF1101003), and the Science and Technology Project of State Administration for Market Regulation (grant number 2022MK158).

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