| Escherichia coli has the characteristics of pathogenic, conditions pathogenic and non-pathogenic, as thus, E. coli is a symbiotic bacteria of humans and animals, and it is also an important mediator of propagation resistance gene, which has threatened the safety of livestock products even food. Currently, there are few reports on the relationship among drug-resistance, multi-drug resistance and the integron analysis in animal food production chain (farms-slaughterhouse-sales markets).To understand the multi-drug resistant and the integration of E. coli from pork production chain ((farms-slaughterhouse-market)) analyze the relationship between integration and the multi-drug-resistant strains, provide basic data and theoretical basis to control the spread of resistance on E. coli, this study was conducted.1. Isolation and contamination rate of E. coli in the pork production chain178samples of pig feces and pig meat were collected from pork production chain(farms-slaughterhouse-market), by Eosin methylene blue medium plate separation, gram staining, lactose fermentation test and indole test, methyl red experiment, Valerie plame, citric acid salt use test (IMViC),158E.coli were identified, the total contamination rate was89.67%(158/178), the contamination rates in farms, slaughterhouses and market were100.00%(60/60),94.00%(47/50) and75.00%(51/68). during the pork production chain, the contamination rate of E.coli from farm samples was the highest, followed by the slaughterhouses samples and market samples.2. Analysis of resistance of E. coliin the pork production chain158E. coli were tested against10antibiotics (combination) with broth micro-dilution method recommended by Clinical and Laboratory Standards Institute (CLSI). The results show that all the isolates were most resistance to trimethoprim/sulfamethoxazole (77.85%), and more resistance to tetracycline (68.33%), ciprofloxacin (36.08%), the resistance rate of ceftiofur was only3.80%.50kinds of drug resistance patterns were presented. The drug resistance patterns with most advantages were NAL-TET-SXT (39/158), SPE-TET-SXT (29/158) and CIP-AMP-AMC-SXT (26/159). The multi-resistance rate was62.66%. The E. coli from farms were resistant to3～10kinds of antibiotics, the strains form slaughterhouse and market were resistant to3～6kinds of antibiotics. Most strains demonstrated were multi-drug resistance to4kinds of antibiotics (25.32%) followed by3kinds of antibiotics(9.49%) and5kinds of antibiotics(9.49%), One strain was resistant to10antibiotics (combined). The resistance from the upstream to the downstream part showed a downward trend.3. Detection of integron of multi-drug-resistant E. coli in the pork production chainAccording to the Ⅰ, Ⅱ, Ⅲ class of integrase’s degenerate primers,90strains(the strains from farms, slaughterhouse and market were30,30,30, respectively)were tested by PCR to detect the integron, and it’s fragment disposed by restriction endonuclease Hinf I. The results showed that67of E. coli amplified integron, and the detection rate was74.44%(67/90) All the E. coli detected class I integron. The integron detection rates of E. coli from farm, slaughterhouse and market were96.67%(29/30),70%(21/30),56.67%(17/30), The sub-trend of integron detection rate in the pork production chain was that it is higher in the process of upstream than in the process of downstream.4. Detection integron gene cassette of multi-drug-resistant E. coli in the pork production chainClass I integron integrase gene primers were designed for PCR. PCR was used to the amplification of its variable region and the PCR products were identified by DNA sequencing, and through GenBank search, the selection and arrangement of the class I integron variable region gene cassette were determined. The results showed that42out of67integron-positive strains was amplified fragments of varying sizes. The detection rate of gene cassette carrying was42.69%(42/67). The product sizes were about300to3,000bp, and2000bp(23.81%),1000+1500bp(23.81%)and1000bp(19.05%) production sizes were predominant. The most important sub-resistant gene cassettes were aadB-aadA2(aminoglycoside resistance gene) and dhfrI the-aadA2-SULI (an oxygen trimethoprim sulfa-aminoglycoside resistance gene).Vast majority of strains carrying trimethoprim sulfa drug-resistance gene cassette were resistanct to trimethoprim/sulfamethoxazole. At the same time, those strains carrying aminoglycoside drug-resistance gene cassette were resistant to gentamicin and spectinomycin. During the pork production, the rate of carrying integron gene was higher in the process of upstream than in the process of downstream. There is a trend which is that the resistance gene may migrate from the upstream to the downstream.This study analyzed the resistance and the integration-cassette of E. coli in pork production chain (farms-slaughterhouse-market). It provided basic data for the monitoring of E. coli resistance. It also provided the theoretical basis for the spreading of resistance genes of multiple drug-resistant E.coli. Thesis research results have certain theoretical significance and practical value. |
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