Molecular Typing And Characterization Of Vibrio Parahaemolyticus From Acute Diarrhea Patients

Objective1Study the bacterial pathogens from patients of acute diarrhea in order to get the surveillance data and the characteristics of distribution in acute diarrhea patients.2In order to provide the reference in clinical diagnosis、treatment and prevention, clinical datas of Vibrio parahaemolyticus have been analyzed and antibiotic susceptibility tests were performed for Vibrio parahaemolyticus isolates from acute diarrhea patients.3To understand the molecular epidemiological characteristics of Vibrio parahaemolyticus and lay the foundation for future research, parts of Vibrio parahaemolyticus were analyzed by MLST and PFGE.Methods1Detection of pathogens:stool specimens from acute diarrhea patients were collected from January to December2011in the First Affiliated Hospital of Zhejiang University School of Medicine and the children’s Hospital of Zhejiang University School of Medicine and processed for bacterial culture and identification.2Vibrio parahaemolyticus clinical statistics datas were analyzed and antibiotic susceptibility tests of Vibrio parahaemolyticus were performed to screen20kinds of commonly used antibiotics sensitivity by using K-B agar diffusion method.3Vibrio parahaemolyticus MLST classification:7housekeeping genes:recA gene, dnaE gene, gyrB gene, dtdS gene, pntA, pyrC gene and tnaA gene were amplified and sequenced. DNA sequencing was performed after splicing, the sequence are stored in a database of sequence alignment, determine Vibrio parahaemolyticus7housekeeping gene alleles and isolates sequence type.The program eBURST v3.0was used to identify groups cluster analysis.4Vibrio parahaemolyticus PFGE typing:the restriction endonuclease SfiI was used in Vibrio parahaemolyticus PFGE molecular typing, clustering analysis.was used BioNumerics Version4software.Resuslts11074stool specimens were detected and the positive samples were303(28.2%). The top5bacterias were:diarrheagenic Escherichia coli, Vibrio parahaemolyticus, Shigelloides Plesiomonas, Aeromonas and Vibrio cholerae.2Pathogen mixed infection:39specimens were detected with two kinds of bacteria and3specimens with three kinds of bacteria.3Positive cases feature of Vibrio parahaemolyticus:in July and August the positive rate was17.9%and12%respectively, while in the other months the rate in the average was0.76%; the positive rate in male and female was9.6%and10.1%respectively.In the age group of19-60years old, the positive rate was15.3%,60and6-18years old group was7.6%and5.6%respectively; the highest positive rate was in bloody fecal sample, followed by watery fecal sample.4Vibrio parahaemolyticus susceptibility results:more than95%of all strains were susceptible to the following antibiotics:cefepime,hydrocarbon enzyme alkenes,piperacillin/tazobactam,levofloxacin,amoxicillin/clavulanic acid,ceftazidime, cotrimoxazole, chloramphenicol, ampicillin Sulbactam.on the other hand, more than95%of the isolates were resistant to the ampicillin and cefazolin.5MLST typing results:52strains of Vibrio parahaemolyticus were identified by MLST analysis, the genotyping results were3kinds of STs, including ST3type contaied49strains, ST120type2strains and ST8type1strain.9ST3type strains were identified by PFGE analysis, the similarity level is above60%. Conclusion1The main bacterial pathogens from acute diarrhea patients were diarrheagenic Escherichia coli, followed by Vibrio parahaemolyticus, clinical laboratories should strengthen their monitoring.2The diarrhea associated with Vibrio parahaemolyticus had a certain distribution characteristics in different seasons and age groups distribution.3Vibrio parahaemolyticu was sensitive to the majority of the antibiotics, but there was also a resistance trend.we should strengthen the monitoring of the resistant strains.4ST3was the main sequence type of Vibrio parahaemolyticus from local acute diarrhea patients by MLST.This method may not be suitable for the local detection in the outbreak of Vibrio parahaemolyticus, but it can be combined with the PFGE method in order to increase the detection sensitivity.