Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

Genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021 / 中华预防医学杂志

Objective: To characterize the prevalence and genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021. Methods: Based on the Shenzhen Infectious Diarrhea Surveillance System, acute diarrheal patients were actively monitored in sentinel hospitals from 2013 to 2021. Whole-genome sequencing (WGS) of Vibrio parahaemolyticus isolates was performed, and the genomic population structure, serotypes, virulence genes and multilocus sequence typing were analyzed. Outbreak clusters from 2019 to 2021 were explored based on single-nucleotide polymorphism analysis. Results: A total of 48 623 acute diarrhea cases were monitored in 15 sentinel hospitals from 2013 to 2021, and 1 135 Vibrio parahaemolyticus strains were isolated, with a positive isolation rate of 2.3%. Qualified whole-genome sequencing data of 852 isolates were obtained. Eighty-nine serotypes, 21 known ST types and 5 new ST types were identified by sequence analysis, and 93.2% of strains were detected with toxin profile of tdh+trh-. 8 clonal groups (CGs) were captured, with CG3 as the absolute predominance, followed by CG189. The CG3 group was dominated by O3:K6 serotype and ST3 sequence type, while CG189 group was mainly O4:KUT, O4:K8 serotypes and ST189a and ST189 type. A total of 13 clusters were identified, containing 154 cases. About 30 outbreak clusters with 29 outbreak clusters caused by CG3 strains from 2019 to 2021. Conclusion: Vibrio parahaemolyticus is a major pathogen of acute infectious diarrhea in Shenzhen City, with diverse population structures. CG3 and CG189 have been prevalent and predominant in Shenzhen City for a long time. Scattered outbreaks and persistent sources of contamination ignored by traditional methods could be captured by WGS analysis. Tracing the source of epidemic clone groups and taking precise prevention and control measures are expected to significantly reduce the burden of diarrhea diseases caused by Vibrio parahaemolyticus infection in Shenzhen City.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification / 生物医学与环境科学（英文）

Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Epidemiological and etiological characteristics of Vibrio parahaemolyticus strains causing foodborne disease outbreaks in Guangdong Province from 2017 to 2020 / 中华预防医学杂志

Objective: To study the epidemiological and pathogenic characteristics of Vibrio parahaemolyticus isolated from outbreaks cases in Guangdong Province, 2017-2020. Methods: Epidemiological characteristics of 87 outbreak events caused by Vibrio parahaemolyticus were analyzed. Strains were serotyped, and then analyzed by pulsed-field gel electrophoresis (PFGE). Results: The food-borne disease outbreak caused by Vibrio parahaemolyticus was found in 16 cities. 44.8% (39/87) and 37.9% (33/87) of the outbreaks occurred in hotels, restaurants and school canteens, respectively. Improper food processing and storage (40.2%, 35/87) and cross contamination caused by indiscriminate raw and cooked food (25.3%, 22/87) were the main causes of food-borne disease outbreaks of Vibrio parahaemolyticus. The main serotypes of patient derived strains were O3:K6 (87.5%) and O4:KUT (22.5%). The similarity value between O3:K6 type isolates was 65.5%-100.0%, and the PFGE pattern similarity value of O4:KUT type isolates was 66.5%-100.0%. Conclusion: Outbreaks caused by Vibrio parahaemolyticus are widely distributed in Guangdong province. It is necessary to strengthen the publicity and education on the correct handling of food in hotels, restaurants, schools, and unit canteens. O3:K6 and O4:KUT serotypes are the main serotypes of the outbreak. There is genetic diversity among the epidemic strains.

Diversidad molecular de variantes patogénicas de Vibrio parahaemolyticus en el Perú / Molecular diversity in pathogenic variants of vibrio parahaemolyticus in Peru

RESUMEN Con el objetivo de determinar la diversidad de variantes patogénicas de Vibrio parahaemolyticus en el Perú durante el periodo 1995-2017, se analizaron 102 genomas peruanos (97 clínicos y 5 ambientales) empleando el esquema de tipificación multilocus y BLASTn para la búsqueda de genes de virulencia. Se identificaron 15 tipos de secuencia diferentes, encontrándose que el genotipo ST3, perteneciente al clon pandémico, fue el más abundante, con 52% (n=53); seguido por el ST120, con 23,5% (n=24); y el complejo clonal CC345, con 11,8% (n=12). Un total de 89 cepas analizadas presentaron genes que codifican la isla de patogenicidad VpaI-7 (87,3%), mientras que 96 presentaron el gen tdh (94,1%), y 6, el trh (5,9%). Durante el periodo evaluado, se resalta la predominancia del ST3, causante de un importante brote en el pasado del Perú, además de otros genotipos patógenos que representan un riesgo latente en salud pública asociado al consumo de alimentos marinos.

ABSTRACT During the period from 1995 to 2017, in order to determine the diversity of Vibrio parahaemolyticus pathogenic variants in Peru, 102 Peruvian genomes (97 from a hospital setting and 5 from an out-of-hospital setting) were analyzed using the multilocus typification scheme and BLASTn in the search for virulence genes. Fifteen different sequence types were identified. It was found that the ST3 genotype, which is found in the pandemic clone, was the most abundant, with 52% (n=53); followed by ST120, with 23.5% (n=24); and the CC345 clonal complex, with 11.8% (n=12). A total of 89 analyzed strains presented genes encoding the pathogenicity island VpaI-7 (87.3%), while 96 presented the tdh gene (94.1%), and 6 the trh gene (5.9%). The ST3 genotype was the predominant one during the evaluated period, this genotype was the cause of a major outbreak in Peru's past history. Other pathogenic genotypes found represent a latent public health risk associated with seafood consumption.

Genomic basis of antibiotic resistance in Vibrio parahaemolyticus strain JPA1

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.

Amplification of tlh gene in other Vibrionaceae specie by specie-specific multiplex PCR of Vibrio parahaemolyticus

Background The surveillance of Vibrio parahaemolyticus in the Chilean coast has been mainly performed by multiplex PCR amplification of three different hemolysin genes, which are specie-specific virulence factors. These genes are also employed in the determination of V. parahaemolyticus pathogenic load in seafood and for characterization of pathogenic strains associated to diarrhea cases in human. During environmental surveillance that we performed every summer, we occasionally observed a thermolabile hemolysin (tlh) PCR product of a slightly smaller size than expected, which was coincident with low loads of V. parahaemolyticus in the environment. In order to understand this observation, we probed the specificity of tlh primers for the detection of V. parahaemolyticus at different bacterial loads and DNA concentrations. Results Primers used for the detection of V. parahaemolyticus specific tlh amplified a slightly smaller tlh gene, which is found in Vibrio alginolyticus and other related strains. These amplicons were observed when V. parahaemolyticus was absent or in undetectable loads in the environment. Conclusions Surveillance of V. parahaemolyticus using tlh primers can be imprecise because amplification of a V. parahaemolyticus specific marker in V. alginolyticus and other related strains occurs. This situation complicates potentially the estimation of bacterial load in seafood, because do not ensure the correct identification of V. parahaemolyticus when his load is low. Additionally, it could complicate the tracking of outbreaks of V. parahaemolyticus infections, considering the genetic markers used would not be specie-specific.

Antibiotic resistance of Vibrio parahaemolyticus isolated from pond-reared Litopenaeus vannamei marketed in Natal, Brazil

Ten out of fifty fresh and refrigerated samples of shrimp (Litopenaeus vannamei) collected from retailers in Natal (Rio Grande do Norte, Northeastern Brazil) tested positive for Vibrio parahaemolyticus. The Kanagawa test and multiplex PCR assays were used to detect TDH and TRH hemolysins and the tdh, trh and tlh genes, respectively. All strains were Kanagawa-negative and tlh-positive. Antibiotic susceptibility testing was done for seven antibiotics by the agar diffusion technique. Five strains (50 percent) presented multiple antibiotic resistance to ampicillin (90 percent) and amikacin (60 percent), while two strains (20 percent) displayed intermediate-level resistance to amikacin. All strains were sensitive to chloramphenicol. Intermediate-level susceptibility and/or resistance to other antibiotics ranged from 10 to 90 percent, with emphasis on the observed growing intermediate-level resistance to ciprofloxacin. Half our isolates yielded a multiple antibiotic resistance index above 0.2 (range: 0.14-0.29), indicating a considerable risk of propagation of antibiotic resistance throughout the food chain.

Isla de patogenicidad de Vibrio parahaemolyticus en cepas chilenas clínicas y ambientales / Pathogenicity island region of clinical and environmental strains of Vibrio parahaemolyticus, isolated in Chile

Background: Most clinical isolates of Vibrio parahaemolyticus produce a major virulence factor known as the thermostable direct hemolysin (TDH). TDH is encoded by the tdh gene which is located in a genomic pathogenicity island (PAI). Most environmental isolates are described as tdh negative. Aim: To assess if environmental strains lack the full pathogenicity island or if only the tdh gene is deleted. Material and methods: Thirty eight clinical and 66 environmental strains of Vibrio parahaemolyticus were studied. PAI was characterized by polymerase chain reaction (PCR). The presence of tdhA and tdhS genes, was determined by Southern blot. Results: Fifty three environmental strains (80%) lacked a full PAI when compared with clinical strains. In environmental strains, Southern blot and sequence analysis showed that a genetic región of 80 kilobase pairs including genes from VPA1310 to VPA1396 was missing. Conclusions: These results highlight the genetic dynamism of Vibrio parahaemolyticus pathogenecity island región and suggest that new pathogenic strains could appear by horizontal transfer of the island between toxigenic and non-toxigenic strains.

Estudo de Vibrio ssp. potencialmente patogênicos através de métodos moleculares / Study of virulence factors in Vibrio ssp through molecular methods

De forma similar ao V. cholerae, outros vibrios potencialmente patogênicos podem causar doença no homem em uma variedade de formas de quadros auto-limitantes até diarréia severa a cólera, septicemia, celulite e infecções de pele. Com o advento da biologia molecular, novas técnicas vêm sendo empregadas para o estudo do gênero Vibrio com o intuito de determinar presença de fatores de virulência, traçar rotas de transmissão, ou ainda como ferramenta no estudo epidemiológico destes organismos. O presente estudo visou pesquisar a presença de genes codificadores de virulência em cepas de V. cholerae provenientes de vários países, e ainda estudar do polimorfismo genético, com o emprego de iniciadores para seqüências ERIC e BOX e eletroforese em campo pulsado (PFGE), para determinar a relação entre cepas de V. cholerae, V. mimicus, V. parahemolyticus, V. fluviais e V. alginolyticus isolados de amostras de ostras e mexilhões no Brasil, e V. metschnikovii isolados de amostras de peixe isolados de outros países bem como com o Padrão ATCC de cada espécie. Foi realizada também, a confirmação da posição taxonômica de cepas de V. metschnikovii isolados de amostras de peixes, e a comparação dos padrões de bandas com isolados de outros países e cepa padrão da espécie. A posição taxonômica de cepas de V. metschnikovii foi confirmada através da Hibridização DNA/DNA, e o estudo do polimorfismo genético através de ERIC PCR, BOX PCR e eletroforese em campo pulsado demonstrou que essas são poderosas ferramentas para estudos epidemiológicos do gênero Vibrio.

Haemolysin production by environmental isolates of Vibrio parahaemolyticus from Andamans.

Sixty marine water samples were collected from various coastal sites around Port Blair at different times during August 1996 to July 1997. The specimens were subjected to standard procedure for isolation and identification of V. parahaemolyticus. Forty four V. parahaemolyticus isolates were detected from these specimens and all showed clear haemolysis on Wagatsuma agar plates. The haemolytic activity was abolished by heating the culture supernatants at 60 degrees C for 10 min and enhanced when plates were kept at 4 degrees C. When isolates were subjected to PCR assay for tdh gene, only one showed the presence of the gene. The results indicate the existence of pathogenic V. parahaemolyticus in the marine environment of these islands.

Hemolysins and plasmid profiles of Vibrio parahaemolyticus.

Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh. The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates. Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb. Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.

Plasmid-determined degradative metabolism and halophilism of Vibrio parahaemolyticus.

A set of 25 Kanagawa(+) and Kanagawa(-) strains of V. parahaemolyticus was studied for their ability to degrade hydrocarbons in minimal media. All strains gave positive results with respect to crystal violet (CV), methyl violet, liquid paraffin, benzene, naphthalene and phenol. The CV double ring (CVDR) response had earlier appeared to be a significant pathogenic marker [Chakrabarti et al, Indian J Med Res, 85 (1987) 508]. The CVDR response was found also to be a biodegradative marker, and correlates perfectly well with polymyxin resistance and low level of halophilism (4% NaCl). All these markers (characters) were found to be controlled by a single plasmid in the wild type. Elimination of the plasmid, as confirmed by gel electrophoresis studies, resulted in loss of CVDR response, polymyxin resistance, and acquisition of halophilism at a higher level (> 7%). The massive drainage of industrial effluents, rich in hydrocarbons, in the estuarine areas in many countries might have altered the ecosystem in favour of V. parahaemolyticus and its emergence as a new biodegradative and enterotoxigenic pathogen, contaminating fauna and flora in the littoral sea regions, with increased changes of communicability to humans.