Vibrio Parahaemolyticus: Molecular Subtyping And Duplex PCR Assays For Detection Of Major Antibiotics Resistance Genes

Vibrio parahaemolyticus (VP), a halophilic bacterium, is widely distributed in marine and seafood products. It can cause food poisoning with such symptoms as diarrhea, nausea, vomiting, fever, even dehydration and coma.Thermostable direct hemolysin(TDH) and thermostable direct hemolysin-related hemolysin (TRH) are recognized as the major virulence factors of V. parahaemolyticus. In recent years, urease and typeâ…¢secretion systems have attracted more attention as potential virulence factors.â…¤. parahaemolyticus also cause many diseases in aquatic species such as oysters enteritis, shrimp red leg disease, crab mass deaths, vibriosis of large yellow croaker. Currently, general treament in aquaculture is antibiotics. Excess use and abuse of drugs have been a serious threat to trade, aquaculture and human health. The study was attempted (1) to validate a multilocus sequence typing system (MLST) for studies on genetic diversity ofâ…¤. parahaemolyticus and on source-tracking of the organisms along the food chain, and (2) to established a rapid method to detect the major antibiotic resistance genes ofâ…¤. parahaemolyticus isolates.To establish the system of molecular subtyping of Vibrio parahaemolyticus,we used the MLST technology to subtype 71 strains (clinical,seafood and environmental strains) isolated from 2003-2009 in our laboratory. Results showed that seven housekeeping genes chosen have good discrimination index for subtyping. Seventy-one strains were classified into 62 sequence types (STs). With all the 201 STs (included those from our laboratory) in the web database,4 Clonal Complexes(CCs),18 Groups and 141 Singletons were seen. Out of 141 singletons,54 were from our isolates. Other 8 STs scattered in 2CCs and 6 Groups. Phylogenetic results were generally in agreement with eBURST analysis. Genetic diversity ofâ…¤. parahaemolyticus was proved by both methods. eBURST software is a powerful tool to predict the founder of Clonal Complex. Strains in CC3 were considered as the founder strains responsible for worldwide outbreak of Vibriosis due to pandemic V. parahaemolyticus isolates.There were also CC3 strains in China from 2006-2009.The minimum evolution tree divided strains in 111 STs into four major branches. Vast majority of our isolates and those from abroad were into the two major branches, while 5 STs (representing 5 strains) were categoried in the other two minor branches. For source-tracking ofâ…¤. parahaemolyticus isolates isolates in epidemiological studies, we believe that virulence related genes could be complementary to the house-keeping genes.According to information, major antibiotics resistance genes of V. parahaemolyticus isolates are sulfonamides, aminoglycosides, tetracyclines and cephalosporins.To establish a rapid method to detect four major resistance genes, firstly positive strains and primers were identified by single PCR.Secondly, PCR conditions were optimized.Finally two duplex PCR systems were eatablished for detection of major antibiotic resistance genes.The detection was generally specific, and the sensitivity was moderate above 105CFU/mL.Eighty-eight strains isolated from NingBo were examined with these two methods. Results showed that the ratio of cephalosporins resistance gene (bla-TEM) was 91.7%, and tetracycline resistance gene (tetB) was not detected. Sulfonamides resistance gene (sul2) and aminoglycosides resistance gene (strB) were 16.7% and 43.3% respectively. These results showed that the methods could be used to detect the drug resistance genes ofâ…¤. parahaemolyticus in seafood.