| OBJECTIVE:Vibrio parahaemolyticus(VP)is a gram-negative bacterium,which is widely distributed in the oceans,estuaries and seafood.Human's consumption the seafood like clams and other shellfish contaminated by the vibrio parahaemolyticus may cause diarrhea or food poisoning.Outbreaks of vibrio parahaemolyticus has been reported in many countries and regions,becoming one of the major worldwide causes of the traveler's diarrhea.Statistics by foodborne disease surveillance websites show that vibrio parahaemolyticus has become a major food-borne disease in China.Hangzhou is located in the southeast coast of China,the climate is warm and humid and rainy,the residents like to eat clams and other seafood,so it is a high incidence area of vibrio parahaemolyticus induced diarrhea.In this study,we collected stool specimens of patients with acute diarrhea in Hangzhou district for the identification of vibrio parahaemolyticus,so as to find out the epidemiological characteristics of vibrio parahaemolyticus more clearly in Hangzhou,and provide an experimental basis for the clinical treatment of vibrio parahaemolyticus infections.We conducted the O and K antigen serum agglutination test and susceptibility test,to gain the serotype distribution and characteristic of anti-microbial resistance of vibrio parahaemolyticus in Hangzhou district.And we performed the genetic test and molecular typing of the typical and special strains of vibrio parahaemolyticus to investigate the virulence characteristics of vibrio parahaemolyticus in Hangzhou district in a molecular level,study the homology and genetic relationships among strains as well as provide reference for the prevention and control of vibrio parahaemolyticus and pave a way for further studies.METHODS:We collected 120 vibrio parahaemolyticus strains separated from outpatients with acute diarrhea in the First Affiliated Hospital of Zhejiang University from 2013 to 2015,by aspects from serotype identification with serum agglutination,drug susceptibility test using the K-B method to the detection of its virulence genes and multi locus sequence analysis,to analyze the epidemiological characteristics of vibrio parahaemolyticus.RESULTS:1.From June 2013 to September 2015,we collected a total of 120 vibrio parahaemolyticus.strains,males accounted for 42.5%(51 strains),women accounted for 57.5%(69 strains);Distribution between different age groups varies,young adults aged 18-44 is the highest(64.2%);the detection rate in summer was as higher as 78.3%(94strains).2.112(93.3%)Vibrio parahaemolyticus have been detected O antigen,and 5 serogroups were found:01,02,03,04 and 05,with the 03 the mainly serogroup(71.7%,80strains).K antigen was detected in 110(91.7%)strains,and was divided into 17 serogroups.K6 were the most common serogroup,80 strains(66.7%).Also K8 has 8 strains(6.7%),K60 has four(3.3%).There were 21 combinations of O and K serogroups(not include OUT:KUT type),among which 03:K6 type is the most important serotype(80 strains,66.7%),04:K8(8 strains,6.7%).8 strains(6.7%)failed to be identifed the O and K serogroups,and were classified into the OUT:KUT type.3.Vibrio parahaemolyticus maintained high sensitivity to most of the 13 antibiotics used in the study,and the rate of sensitivity to imipenme and piperacillin/tazobactam was up to 100%.But there were 70.8%strains were sensitivity to amikacin in this study.However,cefazolin,cefuroxime,and ampicillin sensitive rates were only 45%,55%and 13.4%..In our study,14strains were found resistant to two or more antibiotics,seven strains resistant to more than 3 kinds of antibiotics,and there are two strains were resistant to seven and eight kinds of antibiotics.4 The 120 Vibrio parahaemolyticus strains were positive for tlh,112 strains(93.3%)carried tdh,and only six strains carried trh.In our study,pathogenic strains were detected in 114 strains(95.0%),including 108 tdh+trh-,4 tdh+trh+ and 2 tdh-trh+;6 strains(5.0%)were tdh-trh-.There were 77 strains(64.1%)of Vibrio parahaemolyticus belonged to the pandemic group strains(tdh+,trh-,and toxRS/new+),and non-pandemic group strainsand 43(35.6%).83.1%(64 strains)of them carried orf8 gene.The 77 pandemic strains included 9 serotypes,with 03:K6(59 strains,76.6%)the most.5.T3SS1 gene was present in all Vibrio parahaemolyticus strains,and 116(96.7%)positive for all the four T3SS1 genes,Other 4 strains(3.3%)were missing one or two of the four T3SS1 genes.T3SS2a genes were found in most isolates that were tdh positive and trh negative,but does not exist in tdh-trh+ strains,but tdh-trh-strains can also detected.108 strains of tdh+and trh-strains,of which 107 strains(99.1%)contain all the four gene of T3SS2a,only 1 strains were missing the VP 1362[vscB2]gene.T3SS2?genes were detected in all trh positive strains(tdh+ trh+ and tdh-trh+ strains),but did not found in tdh+ trh-and tdh-trh-strains.6.21 sequence type(STs)were found in the MLST analysis of 115 Vibrio parahaemolyticus,among which,ST3 up to 80 strains(69.6%),followed by ST332 is 10 strains(8.7%),ST88 accounting for 5 strains(4.3%),ST 120 accounting for 3 strains(2.6%),the other 19 kinds of STs each one.eBURsT classified these STs into eight Clone Complex(CC3,CC345,CC8,CC332 and so on),5 Groups and 5 Singletons.ST3 and ST572 belongs to CC3,and ST3 as CC3 ancestor(Founder);ST345,ST88 and ST189 belongs to CC345,ST88 belongs to CC345.Besides ST8?ST332?ST199?ST120?ST1569?ST69 belongs to a clonal population CC8?CC332?CC199?CC120?CC654?CC527.ST479?ST486?ST635?ST1570 in eBURST respectively belong to different groups.ST545\ST712\ST1031\ST1551\ST1571 each monomer?7.03:K6 was the most common serotypes in ST3 and ST332 strains respectively,which were two STs that most prevalent in Hangzhou.We found that most ST88 belong to 04:K8,and most ST120 belongs to 03:K29.CONCLUSION:1.The serotype distribution of Vibrio parahaemolyticus in this region is extensive and variable,03:K6 and 04:K8 are the major serotypes.2.Though Vibrio parahaemolyticus maintains high sensitivities to most of the anti-microbial drugs in this region,but due to the abuse of antibiotics,strains resistant to multiple antibiotics were detected.3 TDH were the major pathogenic factor of Vibrio parahaemolyticus strains in Hangzhou.Pandemic strains are the most prevalent group in this area.4 ST3 and ST332 were two STs that are most prevalent in Hangzhou.The distribution of STs is concentrated in the pandemic group strains. |
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