Epidemiological Investigation And Genetic Diversity Of Vibrio Parahaemolyticus Isolated From Coastal Areas Of China

Vibrio parahaemolyticus is an opportunistic pathogen that causes human foodborne diseases and acute hepatopancreas necrosis(AHPND,also known as early death syndrome)in Penaeus vannamei.It is one of the major food-borne gastroenteritis-causing pathogens which have caused disastrous losses to humans and aquaculture businesses.In this study,we first designed a rapid,PCR-based method for detecting Vibrio parahaemolyticus.Due to the high degree of genetic relatedness among strains of the V.harveyi group,employing the 16 S rDNA genes as the species-specific target is unable to differentiate V.parahaemolyticus from Vibrio sp.,nor from the V.harveyi group.Therefore,the key to the detection of Vibrio parahaemolyticus is to find the specific and effective genetic targets.However,few studies have been done to evaluate the specificity of these genetic markers,thereby increasing the possibility if obtaining false positive results during detection.To solve this problem,we found three target sequences on two chromosomes using bioinformatics analysis.Comparison of these genetic markers with those from previous reports indicated that these three target sequences possess the characteristics of interspecies specificity and intraspecies conservation.To investigate the virulence,antibiotic resistance and genetic diversity of Vibrio parahaemolyticus in aquaculture,we isolated and purified 1045 presumable Vibrio sp.strains from TCBS and LB solid medium in Tianjin(June 2016,September 2016 and April 2017),Nantong(September 2016),Hangzhou(September 2016),Zhanjiang(November 2016)and Hainan(August 2016).Based on the detection method used in this study,a total of 209 strains of Vibrio parahaemolyticus were screened.Specifically,0 was detected in Hainan,74 in Tianjin(22.2%),52 in Nantong(28.3%),42 in Hangzhou(30.7%)and 41 in Zhanjiang(12.5%).Based on this information,further studies were conducted including detection of virulence genes,resistance tests of antibiotics commonly used in aquaculture,multilocus sequence typing(MLST),pathogenicity to P.vannamei,and whole genome sequencinganalysis of Vibrio parahaemolyticus.Through the detection of major virulence genes,we found that trh,which is often found among clinical trials of human,occupied a considerably low proportion in aquaculture(only 3cases detected in Tianjin).The detection rate of another virulence factor PirAB in Penaeus vannamei was 9.57%,consisting of 18 cases in Hangzhou and 2 cases in Nantong.Subsequently immersion test challenging Penaeus vannamei with V.parahaemolyticus showed that PirAB was extremely pathogenic that the lethality rate reached 80% in 24 hours.Meanwhile,it was verified that Penaeus vannamei was not susceptible to the emerging,hypothetically virulent tc-like genes in V.parahaemolyticus.By analyzing the resistance of four commonly used antibiotics in aquaculture,we found that the resistance rate of oxytetracycline was the highest(17.22%),which was followed by fluorophenicol(10%),enrofloxacin(3.83%)and lastly amikacin(0.48%).Furthermore,five strains isolated from fish exhibited multiple drug resistance to oxytetracycline,florfenicol and enrofloxacin.The increase in ratio may be related to the frequent and excessive use of antibiotics,such as oxytetracycline and florfenicol.According to our results,the strains carrying trh and pirAB were sensitive to all four antibiotics,thereby confirming that antibiotics,especially amikacin,are effective in the prevention and treatment of Vibrio parahaemolyticus.In summary,through epidemiological surveys,Vibrio parahaemolyticus causing diseases in human and P.vannamei were present in aquaculture,and uni/multi-drug resistant strains also were detected.This study shed new light on the monitor,prevention and treatment of bacterial diseases.In addition,186 Vibrio parahaemolyticus were selected and analyzed by MLST.A total of 44 STs were obtained,of which 16 STs were respectively displayed by only one strain,while the remaining 28 STs were respectively displayed by at least two strains.On the other hand,24 new STs were detected,which is more than 50%.Combined with phylogenetic analysis,the high genetic diversity and geographical differences of Vibrio parahaemolyticus in aquaculture are one of the important reasons for this phenomenon.Through whole genome sequencing analysis,we also found that the genome changes mediated by mobile elements are universal.Analysis of the housekeeping gene recA confirmed the insertion of one genomic island carrying heterogenous recA.In addition,the loss of the housekeeping gene dtdS was caused by an insertion sequence(variant of ISVal1).This IS had a total of 27 insertions throughout one bacteria genome we sequenced and resulted in multiple deletions and acquisitions.ISVal1 was shown to have insertion activity through evolutionary experiments.We speculated ISVal1 may shape the genome by homologous recombination or other mechanisms so that the bacteria can adapt to more complex environments.The result of thisstudy suggested that mobile elements play an important role in genome diversity and provided a new idea for evolutionary genomics.