Study On Antibiotic Resistance And Pathogenicity Of Foodborne Vibrio Spp.in China

Vibrio spp.is a group of halophilic,thermophilic and acid-susceptible gram-negative bacteria,named for its short and curved shape.There are many species of bacteria in this genus,which are widely distributed in estuaries,bays,coastal waters and marine animals.As an important pathogen,Vibrio not only causes serious economic losses in aquaculture industry,but also poses a serious threat to human health.When people eat or wound contact with aquatic products contaminated by Vibrio,it is easy to cause acute gastroenteritis,sepsis,and death in severe cases.In this study,the southern coastal city of Shenzhen in China,which has a large demand for aquatic products,was selected as the survey site.The contamination of Vibrio in fresh meat and shrimp samples sold in Nanshan District of Shenzhen City was continuously monitored from August 2015 to April 2017 and the genetic characteristics of Vibrio isolates,such as antibiotic resistance,pathogenicity and molecular typing were described.Based on that,the complete genetic information of main representative strains was obtained by using the second and third generation genome sequencing techniques in order to analyze the antibiotic-resistant genes and their transmission mechanism in Vibrio.Finally,the virulence of Vibrio vulnificus isolates was evaluated by establishing animal models and measuring the biofilm forming ability of Vibrio vulnificus to reveal the potential relationship between the biofilm and the pathogenicity of Vibrio vulnificus.The main contents and results of this study are as follows:1.In this study,a total of 2,051 samples were collected randomly in Nanshan District of Shenzhen City from August 2015 to April 2017.Among them,593 samples were contaminated with Vibrio bacteria and the contamination rate reached 28.9%.The contamination rate of Vibrio in shrimp samples?91.9%?was significantly higher than that in beef,chicken and pork samples.Meanwhile,the contamination of Vibrio was more serious in markets?31.1%?,but the prevalence rate in supermarkets also reached 25%.The contamination rate in summer was 37.1%,which was higher than that in winter?22.6%?.2.A total of 1856 Vibrio spp.isolates were collected in this study.Among them,1340isolates?72.2%?were identified as Vibrio parahaemolyticus,482 isolates?26.0%?as Vibrio alginolyticus,22 isolates?1.2%?as Vibrio cholerae and the other 12 isolates?0.6%?as Vibrio vulnificus.Antimicrobial susceptibility tests showed that during the survey period of nearly two years,the antibiotic resistance rate of all Vibrio isolates to penicillin antibiotics remained at a very high level,with the resistance rate of 92%.The resistance rate to cephalosporins,tetracyclines and quinolones showed an increasing trend year by year,with an average increase of 37.9%.No Vibrio spp.isolates resistant to carbapenems were found.3.In this study,150 isolates of cephalosporin-resistant Vibrio parahaemolyticus were classified by MLST.The results showed that 61 isolates?40.7%?belonged to unknown ST type.The remaining 89 isolates had 17 ST types,among which ST163 has the highest proportion?31.5%?,suggesting ST163 is the dominant population in cephalosporin-resistant Vibrio parahaemolyticus.4.A total of 270 cephalosporin-resistant Vibrio spp.isolates were screened for carriage of?-lactamase genes.The results showed that 20 known?-lactamase genes and two novel metallo-?-lactamases were detected.Among them,VEB type had the highest positive rate?48%?.TEM,VIM and IMP types accounted for the least.The conjugation assay showed that the cephalosporin-resistant phenotype of 71 Vibrio spp.isolates?26.3%?could be transferred to E.coli J53 by conjugation transfer.S1-PFGE and hybridization experiments confirmed that conjugative plasmids were the major vehicles of these antibiotic-resistant genes.5.Four novel?-lactamase genes were identified,which were named bla_CMY-2.1,bla_VIM-55.1,bla_VMB-1 and bla_VMB-2,respectively.In addition,these four novel?-lactamase genes were cloned and expressed successfully by genetic engineering technology,and their functions were qualitatively and quantitatively analyzed by antimicrobial susceptibility tests and enzyme kinetic parameters.The results showed that,except bla_CMY-2.1,the other three novel genes all had different degrees of ability to mediate the resistance of receptor bacteria to cefotaxime and meropenem.6.In this study,the complete genome sequences of 270 cephalosporin-resistant Vibrio isolates were obtained.The genetic environment of six?-lactamase genes was confirmed and their transmission mechanism in Vibrio was revealed.Among them,bla_CTX-M-15,bla_CTX-M-55,bla_NDM-1DM-1 and bla_VMB-1 were all located on the conjugative IncC plasmids.bla_VIM-1IM-1 gene was located on an unknown type of conjugative plasmid pVb1978?MG456577?.The transmission of the bla_CTX-M-14 gene is related to the insertion sequence ISEcp1 and IS903B,the novel IncP-1 plasmid pVb1636?MH548371?,and the novel integrative conjugative element ICEVpaChn0574?MN199028?.7.In this study,the pathogenicity of Vibrio vulnificus strains isolated from seafood was analyzed.Virulence gene detection results showed that most of food-borne Vibrio vulnificus isolates had characteristic genes of clinical strains,which indicated that most of Vibrio vulnificus isolates in this region had potential infective ability and would pose a threat to human health.By comparing the lethal rates of Vibrio vulnificus in Galleria mellonella larval model and mouse model,it was found that the same isolate had different lethal rates in the two models.Therefore,the larval model may be not suitable for the pathogenicity study of Vibrio vulnificus.Combining the biofilm-forming ability of Vibrio vulnificus with its lethality in mice,the results showed that the isolates with strong biofilm-forming ability also showed strong virulence.