Etiologic Characteristics Of Vibrio Parahaemolyticus Strains Isolated From Patients In Guangdong,2007-2013

Background:Vibrio parahaemolyticus, a Gram-negative halophilic bacterium autochthonous to estuarine, marine, and coastal environments. V.parahaemolyticus is a seafood-borne pathogenic bacterium and is the leading cause of traveler’s diarrhea and gastroenteritis worldwide due to the consumption of undercooked contaminated seafood, particularly shellfish. The public health and commercial burdens associated with V.parahaemolyticus contamination are very high in Guangdong province due to the wide consumption of seafood. A serotyping scheme based on O (somatic) and K (capsular) antigens is able to differentiate isolates of V. parahaemolyticus into 13 O groups and 71 K types by using the commercial antisera, although many strains remain untypable.V.parahaemolyticus can be divided into two categories, non-pathogenic and pathogenic. All most all the environmental isolates are non-pathogenic, whereas isolates from patients are pathogenic. V.parahaemolyticus has a variety of virulence factors, but the most important virulence factors are the thermostable direct hemolysin (TDH)、TDH-related hemolysin (TRH)、urease、pathogenicity islands and T3SS.At the beginning of 1996 in Kolkata, India, during ongoing surveillance, an increase in patients with V.parahaemolyticus gastroenteritis was observed. Analysis of the strains revealed a new unique serotype, O3:K6, which accounted for 50-80% of infections during the following months. All O3:K6 isolates were tdh+trh-and shared nearly identical genotype profiles. Within a few months thereafter, strains of the same serogroup were isolated in the neighboring countries of Vietnam, Indonesia, Bangladesh, Japan, Korea, and Thailand. Specific methods to identify the new clone, based on variation in nucleotide sequence of the toxRS region, were employed and the results showed existence of strains almost indistinguishable from the "new" O3:K6 clone, even though the isolates belonged to different serovars. To date,21 serotypes have been identified, collectively referred to as serovariants of O3:K6, the most common being O4:K68, O1:K25, O1:K41, and O1:KUT. These serotypes differ from O3:K6 in altered O:K antigens, but are of clonal origin. By the end of 2006, V.parahaemolyticus O3:K6 and its serovariants were being isolated in Europe, Mozambique, the United States, Mexico, and South American countries, marking what has been claimed to be the beginning of the first pandemic of V. parahaemolyticus, bringing this pathogen to the forefront of the global public health agenda.Multilocus sequence typing (MLST) is based on sequence analysis of chosen housekeeping (HK) genes and is becoming the method of choice for determining the global epidemiology of bacterial pathogens. Being sequence-based, MLST provides a definitive characterization of bacterial isolates that is consistent from one laboratory to the next. The nuclei acid sequences are typically stored in a public database that can be readily accessed via the Internet. MLST studies have lead to better understanding of the genetic relatedness of strains within a species and have identified the relative evolutionary importance of mutations and lateral transfer events. Gonzalez-Escalona et al. developed a successful MLST scheme for V.parahaemolyticus in a study of 100 strains of global origin.In the present study, we analyze the serotypes, virulence genes and MLST genotyping results of V.parahaemolyticus strains isolated from patients in Guangdong, 2007-2013, in order to better present population structure, epidemiology and evolution of this pathogen. This study can provide data support for the surveillance and control of foodborne illness caused by V.parahaemolyticus.Objectives:1. Investigation on the distribution of serotype and virulence genes of V.parahaemolyticus strains isolated from patients was conducted in Guangdong, 2007-2013, in order to present the dominant serotype and the distribution of pathogenic V.parahaemolyticus.2. The present study was also aimed to present the distribution of "pandemic group" of V.parahaemolyticus strains isolated from patients in Guangdong,2007-2013, in order to confirm the dominant clone in Guangdong.3. Analysis on the population structure and evolutionary relationships of the V.parahaemolyticus strains isolated from patients was conducted in Guangdong, 2007-2013.Methods:1. Bacterial strains:A total of 469 V. parahaemolyticus strains isolated from patients in Guangdong, were obtained from 2007-2013, through the National Foodborne Disease Surveillance System by Guangdong Provincial Center for Disease Control and Prevention. The strains were collected from 15 cities in Guangdong. All strains were identified previously to be V. parahaemolyticus by the Institute of Pathogenic Microbiology Bacteriology Laboratory of Guangdong Provincial Center for Disease Control and Prevention. All strains were Lyophilized and stored under -70℃.2. Serotyping:Strains were plated onto 3% NaCl thiosulfate-citrate-bile salts-sucrose agar at 37℃ for 24 h. Lipopolysaccharide (O) and capsular (K) serotypes of the V.parahaemolyticus isolates were identified by performing agglutination tests with specific antisera, according to the manufacturer’s instructions.3. Detection of virulence genes:PCR was performed to detect the existence of tdh, trh, GS-PCR and orf8 genes. The criteria of "pandemic group" in this study is:① isolated after 1996;②TDH positive;③TRH negative;④GS-PCR (group-specific PCR) positive and/or orf8 positive.4. MLST:150 strains were chosen and PCR amplification and sequenceing were performed for seven HK genes, the HK genes chosen were dnaE (DNA polymerase III, alpha subunit), recA (RecA protein), gyrB (DNA gyrase, subunit B), dtdS (threonine 3-dehydrogenase), pntA (transhydrogenase alpha subunit), pyrC (dihydro-orotase), and tnaA (tryptophanase). Numbers for alleles and STs were assigned according to the database created for V. parahaemolyticus. eBURST was conducted to identify the different clonal complexes. Minimum-evolution (ME) trees for the concatenated sequences of sevev STs were constructed by Mega 5.1 software to analyze the genetic relationships within strains. START 2 (Sequence Type Analysis and Recombinational Tests Version 2) was used to analyze the MLST data, and to calculate dN/dS ratio, the "standardized" index of association IAS, the number of silent nucleotide sites. These consequences can present天the nucleotide diversity at each locus and recombination.Results:1. Serotypes:In the present study, a total of 469 V. parahaemolyticus strains isolated from patients in Guangdong,2007-2013, were obtained.7 O serotypes,16 K serotypes and 24 combinations of serotypes were identified among 469 strains.03:K6 (330,70.36%) was the dominant serotype, then followed by 04:K8 (54,11.51%) and 01:KUT (28株,5.97%). There were 44 K untypeable strains and only 2 O untypeable strains. At the same time,01:K36 strains which were few reported previously were also identified in this study.2. The distribution of virulence genes:Among 469 strains,95.74%(449/469) were tdh+trh-,1.28%(6/469) were tdh-trh+ and 3 were tdh-trh-. Only one tdh+trh+ strain was found. There were 64.39%(302/469) contained GS-PCR and/or orf8 genes. It is noteworthy that there were 8.61%(26/302) only carried orf8 gene, while 8.61% (26/302) only carried GS-PCR gene. Both of them were lack of one genetic mark.3. The distribution of "pandemic group":According to the criteria of "pandemic group",63.75%(299/469) of the strains were classified as belonging to the V.parahaemolyticus "pandemic group". These strains were identified into 7 serotypes: 03:K6, O4:K8, O1:KUT, O1:K38, O3:K29,O4:K68 and O5:K68. And most of these were of the O3:K6 serotype.4. MLST:① High levels of nucleotide and allelic diversity:150 V. parahaemolyticus strains were analyzed by MLST using the sequences generated from internal fragments of seven HK genes. The number of alleles observed for each locus ranged from 16 (pntA) to 20 (gyrB). The number of polymorphic sites observed varied per locus from 23 (dnaE) to 161 (recA). The most frequently found alleles per locus analyzed were dnaE-3 (96), gyrB-4 (101), recA-19 (95), dtdS-4 (95), pntA-29 (94), pyrC-4 (94), and tnaA-22 (95). The ratio of nonsynonymous to synonymous substitutions (dN/dS) was lower than 1 in dnaE, recA, pntA, pyrC and tnaA, while there was no nonsynonymous substitutions in gyrB and dtdS. The mean G+C% of seven housekeeping (HK) genes ranged from 43.92%(pntA) to 50.27%(dtdS). These data were similar to 45.4% of V. parahaemolyticus genome.② STs generated by MLST:A total of 30 STs were identified among the 150 isolates, and 3 of these were novel, since they had not previously been recorded in the MLST database. They were ST-1117, ST-1118 and ST-1119.21 of the STs contained single isolates, while 9 STs included between 2 and 89 isolates. ST-3 (89,59.33%) was most frequent and was mostly composed of strains belonging to O3:K6 serotype (79, 88.78%).③ Clonal Complexes:The application of eBURST to the data resolved the 30 STs into one clonal complex (CC3), three doublets (Dl, D2 and D3), and 19 singletons. Clonal Complexes 3 consists of 5 STs (ST-3、ST-431、ST-661、ST-435和ST-787),95 isolates, ST-3 was defined by eBURST as the ancestral type or founder for this clonal complex.④ Phylogenetic analysis:A ME tree (minimum evolution tree) representing the concatenated sequences of the seven housekeeping genes in 150 isolates. In this study, the phylogenetic analysis consisted of two major lineages, which were separated by a relatively large genetic distance. eBURST and ME tree analysis were consistent with one another, but in ME tree ST-189 and ST-265 belonging to D1 were divided into two separate parts because they differed at recA locus. The ME tree definitively established the relationship between some singletons., which indicated that ME tree gave better resolution and uncovered some phylogenetic relationships among groups or singletons not observed or resolved by eBURST.⑤ Test for recombination:The START version 2.0 software was used to analyze the DNA sequences of seven housekeeping genes. The results showed there were 126 silent nucleotide sites at recA locus, which was significantly higher than other housekeeping genes. The START version was also used to analyze the linkage equilibrium among seven housekeeping genes by calculating the "standardized" index of association (IAS), a value of 0.9024 was obtained, indicating that the alleles are in linkage disequilibrium. After eliminating the "pandemic group" isolates from the data set, the IAS decreased (IAS= 0.8063), but significant linkage disequilibrium still was detectedConclusions:1.03:K6 was the dominant serotype of V. parahaemolyticus strains isolated from patients in Guangdong,2007-2013. And most of the V. parahaemolyticus strains isolated from patients were pathogenic.2. The "pandemic group" accounted for over 50% of infections caused by V.parahaemolyticus in Guangdong,2007-2013, which indicated that the "pandemic group" was still the dominant clone in Guangdong. Therefore, we should strengthen the research of growth environment and transmission of "pandemic group" in order to prevent and control the foodborne illness caused by Vibrio parahaemolyticus.3. There was only one clonal complexes (CC3) identified in Guangdong,2007-2013. ST-3 was defined as the ancestral type or founder for this clonal complex. The CC3 consisted of most strains belonging to "pandemic group", while a small part of "pandemic group" was classified into other STs.4. There was genetic recombination existing in V.parahaemolyticus in Guangdong, especially at recA locus. The frequent recombination at recA locus may make it not an ideal molecular marker for MLST.