Construction Of Staphylococcus Aureus Transposon Library And Study Of Staphylococcal Enterotoxin A Gene

Staphylococcus aureus(S.aureus)is an important food-borne pathogens,and the main cause of food poisoning is staphylococcal enterotoxins(SEs).Staphylococcal food poisoning(SFP)is an intoxication that results from the ingestion of foods containing S.aureus and SEs.Symptoms of SFP have a short onset,and include nausea,vomiting,abdominal cramps or diarrhea.S.aureus is widely distributed in the air,dust and the surface of food processing equipment,and makes food easily produce cross-contamination.Although the food processing is strictly controlled,the food can also be polluted by S.aureus.Food-borne animals and the hand or respiratory secretions of the human are regarded as the main source of food contamination.Generally,staphylococcal enterotoxin A(SEA)is most strongly associated with food poisoning,followed by SED,SEC,SEB and SEE.Transposon is a unique DNA sequence with a specific translocation characteristic,which can be transferred horizontally between chromosomes,plasmids or phages of bacteria.The aim of this study is to investigate the effects of plasmid and transposon(Tn917)on SEA from food-borne S.aureus.In this study,a total of 48 S.aureus isolates carrying sea from different food sources were detected 18 kinds of enterotoxin genes,29 kinds of resistance genes and 12 kinds of antibiotics sensitivity testing after plasmid elimination.Then,Tn917 was inserted into two strains for establishing the transposon library.Finally,the mutant strains should be detected SEA by ELISA,biofilm formation ability and haemolysin,and compared with the original strains.It can clarify the molecular mechanism of SEA,and provide a theoretical basis for the prevention and control of food poisoning caused by S.aureus.The main findings are as follows:(1)A total of 48 S.aureus isolates were detected 18 kinds of enterotoxin genes,29 kinds of resistance genes and 12 kinds of antibiotics sensitivity testing after plasmid elimination.The results showed that plasmid elimination had an obvious influence on genes see,tetO and tetK,as well as antibiotics AMP and ERY.It indicated that tetO,tetK and the related genes of AMP and ERY might be on bacterial plasmids.The gene loci of enterotoxin genes and resistance genes are very complex.And it has irregularity because gene loci of the same gene are varying in different strains.In addition,there are some differences between the resistant genotype and phenotype.(2)The plasmid containing Tn917 was transferred into two strains.Then,they should be induced under the conditions of ERY and 42?.In the transposon library,we randomly chose 200 single colonies for resistance screening.Finally we got two replicon fusion strains,and the rest of them were transposon mutant strains.(3)The mutant strains were detected SEA by ELISA,biofilm formation ability and haemolysin,and compared with the original strains.Two strains of them were screened out because they didn't produce SEA and haemolysin.Besides,one mutant strain with weak biofilm formation ability was selected.