Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus is a major foodborne pathogen, particularly in Asian countries. Increased occurrence of outbreaks of V. parahaemolyticus gastroenteritis in China indicates the need to evaluation of the prevalence of this pathogenic species. V. parahaemolyticus distribution in shellfish from the eastern coast of China has been reported previously. However, to date, the prevalence of V. parahaemolyticus in retail aquatic products in North China has not been determined. To investigate the prevalence of V. parahaemolyticus in aquatic products in North China, 260 aquatic product samples were obtained from retail markets in 6 provinces of North China from November to December in 2012 and July to August in 2013. V. parahaemolyticus was detected in 94 (36.2 %) of the samples by the most probable number method. The density of V. parahaemolyticus ranged from 1.50 to 1100 MPN/g. V. parahaemolyticus was detected at a rate of 50.0 % and 22.7 % in summer and in winter, respectively. The density of V. parahaemolyticus was significantly higher in summer than in winter, with mean levels of 16.5 MPN/g and 5.0 MPN/g, respectively. Among 145 V. parahaemolyticus isolates examined, none of the isolates possessed tdh and trh. In multiplex PCR-based O-antigen serotyping of these 145 isolates, all serotypes, other than O6, O7, and O9, were detected, and serotype O2 was found to be the most prevalent (detected in 54 isolates). The 145 isolates were grouped into 7 clusters by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) at a similarity coefficient of 0.66. The antimicrobial resistance patterns of these 145 isolates to 12 antimicrobial agents revealed that most of the isolates were resistant to streptomycin (86.2 %), while fewer were resistant to ampicillin (49.6 %), cefazolin (43.5 %), cephalothin (35.9 %), and kanamycin (22.1 %). All of the examined isolates were susceptible to azithromycin and chloramphenicol. The findings of this study will help in defining appropriate monitoring programs, understanding of the dissemination of antibiotic resistant strains, and providing information for the assessment of exposure to this microorganism at the consumption level.

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Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China

Vibrio parahaemolyticus is a causative agent of human serious seafood-borne gastroenteritis disease and even death. Shrimps, often eaten raw or undercooked, are an important reservoir of the bacterium. In this study, we isolated and characterized a total of 400 V. parahaemolyticus strains from commonly consumed fresh shrimps (Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Exopalaemon carinicauda) in Shanghai fish markets, China in 2013–2014. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying two major toxic genes (tdh and trh, 0.0 and 0.5 %). However, high incidences of antibiotic resistance were observed among the strains against ampicillin (99 %), streptomycin (45.25 %), rifampicin (38.25 %), and spectinomycin (25.50 %). Approximately 24 % of the strains derived from the P. monodon sample displayed multidrug resistant (MDR) phenotypes, followed by 19, 12, and 6 % from the E. carinicauda, L. vannamei, and M. rosenbergii samples, respectively. Moreover, tolerance to heavy metals of Cr3+ and Zn2+ was observed in 90 antibiotic resistant strains, the majority of which also displayed resistance to Cu2+ (93.3 %), Pb2+ (87.8 %), and Cd2+(73.3 %). The pulsed-field gel electrophoresis (PFGE)-based genotyping of these strains revealed a total of 71 distinct pulsotypes, demonstrating a large degree of genomic variation among the isolates. The wide distribution of MDR and heavy-metal resistance isolates in the PFGE clusters suggested the co-existence of a number of resistant determinants in V. parahaemolyticus population in the detected samples. This study provided data in support of aquatic animal health management and food safety risk assessment in aquaculture industry.

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Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China

Comparison of Vibrio parahaemolyticus isolates from aquatic products and clinical by antibiotic susceptibility, virulence, and molecular characterisation

Molecular characterization, antibiotic resistance pattern and biofilm formation of Vibrio parahaemolyticus and V. cholerae isolated from crustaceans and humans.

Jingye Xu

Papers

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China.