The Establishment Of Multiplex PCR For Porcine Enterotoxigenic Escherichia Coli And Analysis Of Integron Which Mediated It Multiresistance

Enterotoxigenic E. coli (ETEC) is a pathogenic E.coli, which can usually cause acutewatery diarrhea and rapid dehydration death in piglets, mortality and morbidity rates reachvery high, and gives the aquaculture industry brought huge economic losses. In recentyears, ETEC have showed more severe multi-drug resistance, the resistance has become animportant factor affecting the human health and the development of aquaculture. Accuratediagnosis the type of ETEC virulence factors and monitoring the drug resistance of ETECis a prerequisite for effective prevention and treatment of diarrhea in piglets which inducedby ETEC.In this experiment, to establishment a multiplex PCR method which cansimultaneously detect porcine ETEC heat-labile enterotoxin、heat-stable enterotoxin andadhesin gene (F4). Bulid an optimize conditions for the senstivity、optimum annealingtemperature and specific experimental of multiplex PCR. Then collected swine diarrhea E.Coli which isolation in Jiangxi surrounding area, and had detected79positive ETECstrains that the rate of ETEC detection reach to65.8%（79/120）. The most popularvirulence genotypes of ETEC is STa and K88.In addition, to measured the clinical strains and20kinds of commonly used antibioticsof drug sensitivity by using the KB method, which randomly selected40positive ETECstrains. The results showed that the highest resistance rate is cotrimoxazole (SMZ),tetracycline(TCY), rifampicin (RIF) and chloramphenicol (CHL), reached100%. Thelowest resistance resistance rate was ampicillin/sulbactam (SAM), which accounted for25%(10/40).27kinds of resistance spectrum type were be formed in ETEC, resistant ninekinds or more antibiotics of strains were accounted for97.5%(39/40). The most commonare resistant19and18antibiotics, which had accounted for22.5%(9/40) and12.5%(5/40).Tetracycline with other antibiotic are still have cross-resistance. These results suggest thatenterotoxigenic Escherichia coli is based on multi-drug resistant, the resistancephenomenon is quite serious.Based on the important role of integron in the multi-drug resistance mechanisms forbacteria, this study was research on integron-cassette system detection for ETEC, and tomakesure where is the location of class I gene in bacteria. The results showed that thepositive rate of class I integron is73.5%(29/40), class II integron accounted for32.5%(13/40), and both two types of integron were be detected in13strains.All of the class Iintegron were located on positive strains plasmids.To study the correlation of the integron gene-cassette system with the multi-drugresistance phenotype in ETEC. Amplified and sequenced the variable region of29ETECwhich class I integron positive. At last, five different gene cassette fragment were amplified which the size were650bp,1009bp,1197bp,1586bp,1913bp, and found sixdifferent combinations.Among them, coding of sat gene cassette accounting for58.6%(17/29). Followed by the encoding trimethoprim dihydrofolate reductase andaminoglycoside adenyltransferase which is dfrA1-aadA1gene cassette, accounting for44.8%(13/29). The most common combination is sat+dfrA1-aadA1, accounting for43.5%(9/29). It is consistent with the resistant phenotype of the isolation bacteria. Studies haveshown that, class I integron mediated multi-drug resistance of enterotoxigenic Escherichiacoli and play an important role in large-scale pig farms.