Research On The Testing And Transfer Of Tetracycline-Resistant Genes Of Swine Escherichia Coli In Guizhou Province

The irrational use of antibiotics has led to increasingly serious problems with bacterial drug resistance, which makes it significantly more difficult to manage bacterial diseases with antibiotics.In order to approach the resistant level of swine Escherichia coli to tetracycline antibiotics and the molecular epidemiology of the tetracycline-resistant genes in large-scale farms in Guizhou province, and to explore the modes of acquiring and transmitting bacterial drug resistance, a horizontal transfer model of tetracycline-resistant genes of Escherichia coli was established to investigate the transfer frequency and modes of tetracycline-resistant genes across bacteria; the solutions to drug resistance of Escherichia coli were also investigated, including reducing bacterial drug resistance by eliminating resistance plasmids with traditional Chinese medicine, in an effort to provide evidence for the management of swine colibacillosis and the development of new antibiotics.In reference to the standard of the Clinical and Laboratory Standards Institute(CLSI), in this study the resistance status of the Escherichia coli isolates to 12 drugs including tetracycline antibiotics was detected using the broth microdilution method, and the 7 tetracycline-resistant genes carried by these isolates were detected by PCR amplification.The results showed that the resistance rate of160 strains of Escherichia coli to tetracycline, aureomycin, oxytetracycline, minocycline, demeclocycline, doxitard, kanamycin, gentamicin,ciprofloxacin,ofloxacin,sulfadiazine and Cefamezin were 94.36%, 92.96%, 100%, 18.31%, 70.42%, 59.15%, 66.29%, 48.76%, 29.82%, 21.62%, 91.67%, 21.62%. All the E. coli showed multi-drug resistance, at least to 3 antibiotics, the most to 11 antibiotics.The positive rate of tetracycline resistance genes of tetA, tetB, tetC, tetD, tetE, tetM and tetO were 60.63%、74.38%、51.88%、11.88%、0、64.38% and 0. Tetracycline-resistance was positively correlated with the positive rate of tetracycline resistance genes.In vitro induction of tetracycline was used to identify the effect of drug-resistant genes on the drug-resistant level of Escherichia coli. The results showed that under the drug stress, the Escherichia coli strain containing tetB gene could be rapidly induced from sensitive bacteria into high drug-resistant bacteria. Therefore, tetB gene is one of the important factors influencing the high drug resistance of Escherichia coli to tetracycline. The transformation from drug-resistant plasmid containing tetB gene to Escherichia coli DH5α could successfully obtain transformants, indicating that the tetracycline-resistant plasmid in Escherichia coli can transfer tetB gene to Escherichia coli DH5α in the transformation way and stably encode efflux pump protein to show tetracycline resistance. In the conjugal transfer experiment of tetB, the conjugal transfer frequency of Escherichia coli EC1 and EC2 to Salmonella bacteria S1 was 1.68x10-3 and 2.24x10-3. And no transformant was obtained in the conjugal transfer experiment of two strains of Escherichia coli and Staphylococcus aureus, indicating Escherichia coli may not be able to transfer tetracycline resistance to Staphylococcus aureus in the conjugation way.The resistance of the plasmid mediated was reduced by eliminating resistance plasmid of traditional Chinese medicine, the results showed that the elimination rates ofEscherichia coli strains with drug resistance treated with 1/2 minimum bacteriostasis concentration Coptis Chinensis, Coneysuckle, Houttuynia Cordata and Galla Chinensis after 24 h to Gentamicin resistance were 5.34%、2.67%、10.01%、12.53%, After 48 h the drug-resistance elimination rates were 7.27%、3.47%、10.93%、13.68%.Research onthe loss of plasmid of the eliminated resistance colonies were detected and the restoreing drug-sensitivty, The MIC values of the eliminated resistance colonies to antibacterial agents were redued, and theirMIC valuesto demeclocycline,gentamicin, kanamycin and cefazolin were declined into “sensitivity” from “resistance” or “high-resistance” except the individual strain changed to “intermediate”.