| ObjectiveIn recent years,with the development of economy,the rising of people living standard and the changes of diet structure,food poisoning events often occur.The incidence of food poisoning disease caused by Vibrio parahaemolyticus(V.parahaemolyticus)expanded year by year,which had accounted for the first place among microorganisms food poisoning events.Antimicrobials weren't unreasonable applied to the health care industry and aquaculture,the drug resistance of strains increase gradually,and even multiple drug-resistant strains were found.Therefore,many researchers involved the study of Vibrio parahaemolyticus that was focused on the status of drug resistance and genotyping.But there were almost no data that the other researchers reported involved Vibrio parahaemolyticus strains of the surrounding area near the yellow sea at home and abroad.There were 21 clinical strains and 19 environmental strains in the study.On the one hand,the drug sensitive tests were applied to detect the drug resistance of Vibrio parahaemolyticus strains.On the other hand,gene sequence typing of Vibrio parahaemolyticus adopted molecular biology technology.In a word,the results would provide the theory reference and technical support for clinical diagnosis,hospital infection monitoring and the rational use of antibiotics.At the same time,the results would offer genetic evidences for uncovering the microevolution relationship of Vibrio parahaemolyticus.Methods1.Collect some samples of Vibrio parahaemolyticus from sediment,sediment and silt of the yellow sea waters,and the samples that were from the feces of diarrhea patients who were treated in a hospital near the yellow sea.Culture forty-two strains that were suspected to be Vibrio parahaemolyticus.The cultures were identified by VITEK 2 Compact automatic biochemical identification instrument.Save the strains that were identified to be Vibrio parahaemolyticus.2.Take out the 40 strains stored in the-80? refrigerator that were collected from 2015 to2016.The strains were inoculated in TCBS culture dishes when they were revived.Put these dishes in an incubator with a constant temperature and culture them in 37? for 12 h.VITEK 2 Compact automatic biochemical identification instrument were applied to conduct the drug susceptibility tests of Vibrio parahaemolyticus clinical and environmental isolates.The drug resistance status of Vibrio parahaemolyticus was understood by the analysis of drug sensitivity differences.3.Apply the polymerase chain reaction to amply gene sequences.After the genome DNA was extracted,the sequence of Vibrio parahaemolyticus strains was amplified by using of the specific 7 primers(recA,gyrB,dnaE,dtdS,pntA,pyrC and tnaA).However,the seven gene protocol was selected due to the availability of a public repository for the data from the international site sequence classification database(http://pubmlst.org/vparahaemolyticus),which allows comparisons to be made with isolates analyzed by others researchers.Target gene sequences were amplified,sequenced and analyzed.Vibrio parahaemolyticus clinical and environmental isolates were classified in the aspect of molecular by the multiple locus sequence typing(MLST)technique.4.Neighbor-joining method from MEGA6.06 software was used to construct phylogenetic tree.The clinical and environmental isolates were compared in the aspect of the genes and sources in order to infer the spread way of epidemic strains.Results1.Forty strains were positive by VITEK 2 Compact automatic biochemical identification instrument,and 2 strains islated from environment was Vibrio damsel who would be eliminated.in subsequent research.Vibrio parahaemolyticus strains were in good condition on the TCBS medium.The strains were circular,translucent and green,and the surface of strains were smooth,the diameters of the strains were about 2mm ~ 3mm.When the strains were touched by the inoculation loop,the texture of strains were thick like chewing gum.2.There was one kind of drug-resistant spectrum in the strains from different sources.The resistant rate of Vibrio parahaemolyticus strains to ampicillin was 100%,and the mediation rate of the Vibrio parahaemolyticus strains to cefazolin was 37.5%,but no resistance to other 15 antibiotics,including ceftazidime,imipenem and levofloxacin were detected.There weren't multiple drug-resistant strains that were detected in the study.3.There were 22 sequences types that were identified among the 40 isolates from different sources by using MLST analysis,of which 17 had not previously been reported in the Vibrio parahaemolyticus MLST database.In the study,ST3 was the most frequent sequence type.ST3 accounted for 25%(10/40).The other main sequence types were ST332,accounting for 20%(8/40),ST1533,accounting for 5%(2/40),and ST1545,accounting for 5%(2/40).17 novel sequences types were ST1533,ST1534,ST1535,ST1536,ST1537,ST1538,ST1539,ST1540,ST1541,ST1542,ST1543,ST1545,ST1546,ST1547,ST1548,ST1549 and ST1550.Ten strains of Vibrio parahaemolyticus that were clinical isolates had cloned complex,and they were for CC3.4.In system evolutionary tree,40 V.parahaemolyticus isolates from different sources were divided into two groups,VP11 isolated from the patients was in a single group,while the other 39 isolates belong to another group.Even through VP17(clinical isolate)andVP28(environmental isolate)had not the same sequence type,they showed complete similarity in genetic relationship from the evolutionary tree.What's more,VP22 and VP24,VP23 and VP26 also revealed a very high affinity in evolutionary tree(100%).Conclusion1.All the Vibrio parahaemolyticus isolates whose drug susceptibility results were same.They were only resistant to ampicillin,a part of strains were sensitive moderately for cefazolin.They were sensitive completely for the rest antibacterial drugs,including ceftazidime,imipenem and so on.In the study,the multiple drug-resistant strains weren't found.The study showed that most of antimicrobials were feasible currently on clinical treatment.Therefor,the doctors should not use the antimicrobials that included ampicillin and cefazolin by their experience.They could use the second and third generation of cephalosporin(cefaclor,ceftazidime,etc),the ?-lactam/?-lactamase inhibitor(Ampicillin/ sulbactam,piperacillin/ tazobactam,etc)compounds and quinolone(ciprofloxacin and norfloxacin)antibacterial drugs to treat the patients and animals who infected Vibrio parahaemolyticus.2.There were 22 sequences types found in the 40 isolates from different sources,the result demonstrated that a high level of population genetic diversity existed in Vibrio parahaemolyticus from the eastern coastal areas in China As many as 17 novel sequence types weren't found before.At the same time,Vibrio parahaemolyticus international bases were expanded.And 15 sequences types novel are environmental strains isolated,which showed that the research of vibrio parahaemolyticus near the yellow sea waters in China wasn't enough.3.The MLST results of 40 Vibrio parahaemolyticus strains from different sources showed that the genes of strains were diversity,this may be due to the recA that was in the mutation.The mutation of recA were very high,the phenomenon had increased the genetic polymorphisms of strains.4.According to the evolutionary tree,40 Vibrio parahaemolyticus strains from different sources were divided into two groups.But VP11 isolated from clinical patients was only in a group,which was a novel ST.Therefore,the result indicated that there were some mutations in the gene of VP11.VP28 that was from environmental isolates,and VP17 from clinical isolates had as much as 100% of genetic relationship,which confirmed the pathogenic strains were in the spread between the clinical and environmental isolates. |
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