| Vibrio harveyi is widely present in the marine environment and is an important pathogen causing disease in aquatic animals.With the misuse of antibiotics,the level of resistance of V.harveyi is increasing,creating difficulties for antibiotic treatment of infected aquatic animals.Polymyxin,a surface-acting amphiphilic agent,is known as the last line of defense against carbapenem-resistant gram-negative bacteria.However,with the report of a plasmid-mediated polymyxin resistance gene mcr-1 by Chinese scholars in 2016,the problem of bacterial polymyxin resistance gradually received attention from various countries,and mcr-2-mcr-10 were reported one after another.We isolated a multi-drug resistant strain of V.harveyi WHSS0915 from Sebastes schlegelii,and obtained the mcrWHSS0915 gene by whole genome sequencing screening.We investigated whether mcrWHSS0915 mediated bacterial resistance to polymyxin B by protein secondary prediction,homology modeling,protein 3D structure comparison,molecular cloning and prokaryotic expression,and the main experiments were as follows:(1)V.harveyi WHSS0915 was isolated from diseased S.schlegelii.It was found that V.harveyi WHSS0915 was resistant to nine antibiotics;The virulence phenotype of V.harveyi WHSS0915 was identified by crystalline violet staining,challenge assay,LD50 determination,and inoculation of sheep blood agar plates.The results showed that V.harveyi WHSS0915 had a mean swimming distance of 33.83±3.50 mm,and crystalline violet staining demonstrated that V.harveyi WHSS0915 had a moderate biofilm forming ability and an LD50 of 4.37×108 CFU/tail for S.schlegelii.It did not have hemolytic activity.(2)Whole genome sequencing was performed on V.harveyi WHSS0915.Wholegenome sequence annotation using the CARD database showed that resistance genes present in V.WHSS0915 could mediate resistance to a variety of antibiotics including tetracyclines,fluoroquinolones,aminoglycosides,and cephalosporins,with one gene annotated as mcr-9.1 with Identity value of 53.4%;In addition,a comparative genomic analysis of the V.harveyi WHSS0915 and the V.harveyi genome published in NCBI showed that the nine included V.harveyi strains shared 4055 genes with each other,among which V.harveyi WHSS0915 had 348 unique genes,and mcr-WHSS0915 was not only present in V.harveyi WHSS0915.(3)Protein secondary structure prediction,homology modeling,protein 3D structure comparison,phylogenetic tree construction,molecular docking,gene cloning and prokaryotic expression were performed for MCR-WHSS0915 protein.The results showed that MCR-WHSS0915 was more similar to MCR-3,MCR-7,MCR-9 and MCR-10.mcr-WHSS0915 gene expression in E.coli BL21 showed that mcrWHSS0915 gene could increase the MIC value of polymyxin B on E.coli BL21 from 8 μLg/mL to 64 μg/mL.The molecular docking model showed that the mcrWHSS0915 protein had good binding ability to lipid A.In this study,we initially investigated the mechanism of polymyxin B resistance formation in V.harveyi WHSS0915 at the genetic level.Our results showed that mcrWHSS0915 gene could mediate higher levels of polymyxin B resistance.The evolutionary level of MCR-WHSS0915 was elaborated based on the primary structure,secondary structure and tertiary structure of MCR-WHSS0915 protein.This study provides a reference for the subsequent exploration of the origin of polymyxin resistance genes,the development of novel targeted drugs,antibiotic alternatives,and the prevention and control of multi-drug resistant pathogens in mariculture animals. |
