Biological Characteristics Of Drug-resistant Strains Of Vibrio Parahaemolyticus And Analysis Of Related Drug-resistant Genes Mutation

Vibrio parahaemolyticus（VP）,is a Gram-negative halophilic bacterium,is the main microbial factor that causes foodborne diseases in humans.In this study,the MIC value of 9 antibiotics to 21 Vibrio parahaemolyticus isolates and 2 standard strains was determined by micro-broth dilution method,and difference of the biological characteristics and toleration ability of Vibrio parahaemolyticus with different drug resistance levels was studied.Non-synonymous mutations in related resistance genes was discovered by genomics high-throughput sequencing.The aim of this study was to provide data support for the monitoring and prevention of Vibrio parahaemolyticus and to conduct a preliminary study on the drug resistance mechanism of Vibrio parahaemolyticus from the perspective of gene mutation.The main research results were as follow:（1）Drug sensitivity testThe MIC value of 9 antibiotics to 21 Vibrio parahaemolyticus isolates and 2standard strains was determined by drug sensitivity test,and they were divided into 2drug-resistant strains and 3 drug-resistant strains according to the drug resistance level of the tested strain.The result showed that the resistance rate of 23 Vibrio parahaemolyticus to 9 antibiotics from high to low was:CHL（100%）,AMP（100%）,AMC（100%）,CXM（21.7%）,NAL（13%）,CIP（8.6%）,KAN（4.3%）,CZO（4.3%）,NOR（0%）,and found four 3 drug-resistant strains VP41,469,H128,H256,of which H256 multidrug resistance was the most serious.The drug resistance spectrum of H256 included:amidoalcohols（CHL）,β-lactams（AMP,AMC,CXM,CZO）,quinolones（NAL,CIP）.（2）Analysis of biological characteristics and toleration ability to environmental stressesUsing the 2 drug-resistant strains ATCC17802,F7,VP27 and 3 drug-resistant strains H256,VP41 as the tested strain.Biological characteristics（growth curve,moving ability,biofilm forming ability）and stress toleration ability（high osmotic pressure,strong oxidizing,acid）of tested strains were analyzed.The result was as follow:during the growth process,the stasis period of 3 drug-resistant strains was longer than that of 2 drug-resistant strains,adaptive cost may be reason for this phenomenon.Moving ability:VP27>F7>ATCC17802>VP41>H256,the moving ability of 3 drug-resistant strains was weak than that of 2 drug-resistant strains.Biofilm forming ability:ATCC17802>VP27>H256>F7>VP41,no obvious consistency was found between the biofilm forming ability of tested strains and drug-resistance level.Ability to tolerate high osmotic pressure environment:H256>VP41>VP27>F7>ATCC17802;Ability to tolerate strong oxidizing environment:H256>ATCC17802>VP27>VP41>F7;Ability to tolerate acidic environment:H256>ATCC17802>F7>VP41>VP27,among tested strains,H256 was the best to tolerate stresses of high osmotic pressure,strong oxidizing and acid.（3）Detection of drug resistance genes and mutation sitesIllumina Hiseq high-throughput sequencing was used to perform genomic sequencing of the strain VP41,VP44,460,469.Using the genomic data of the standard strain ATCC17802 of Vibrio parahaemolyticus in NCBI as the reference substance,genomic comparation analysis was performed.The analysis showed that the detected 13 genes related to drug resistance was cat B,bla-_CARB,gyr A,par C,qnr,str A,str B,vme A,vme B,vme C,vme D,tet R₁,tet R₂,strain VP41,VP44,460,469 occurred same non-synonymous mutation:gene catB（Pro165-Ser,Gly208-Asn）,gene vme A（Ile313-Thr）,gene vme C（Glu329-Ala）,gene vme D（Asp205-Ser）.These mutations may be related to increased drug resistance of Vibrio parahaemolyticus isolates.