| The tetracycline antibiotics are important anti-infective agents in human medicine and veterinary clinic.The tetracycline antibiotics play an important role in ensuring the healthy development of human health and livestock.However,with the irrational use of tetracyclines,the resistance spectrum of bacteria to tetracyclines were extended and the resistance levels were increased.The antibiotic resistance in bacteria posed a serious threat to the health and safety of humans and animals.In reference to the standard of Clinical and Laboratory Standard Institute(CLSI),the sensitivity and multidrug resistance of 783 strains Escherichia coli to 6 kinds of tetracycline antibiotics,which were isolated from large-scale pig farms in Guizhou province was determined by broth microdilution method.The results showd that the drug resistance rates of the tested strains to oxytetracycline,tetracycline,aureomycin,doxycycline,mirocycline,and diacycline were 97.3%,97.6%,96.6%,89.3%,48.0%and 34.2%,respectivel.The resistance situation of Escherichia coli to the veterinary tetracyclines,such as oxytetracycline,tetracycline,aureomycin and doxycycline was serious,and their MIC50 were 256ug/mL,128ug/mL,64ug/mL,and 32 ug/mL,respectively.The level of tetracycline resistance is the highest,and its effective treatment concentration(MIC90)was above 512ug/mL,which is 32 times of the clinical recommended treatment amount,and its resistance rate in 8 districts in guizhou province exceeded 92%.The second high of resistance rate is oxytetracycline.Its MIC90 was 512ug/mL,16 times higher than the recommended therapeutic amount.It is belong to the level of moderate resistant.The resistance rate in Zunyi and Liupanshui city to oxytetracycline was the higher than orther districts.and the lowest resistance rate wasin Qiannan.The resistance levels of chlortetracycline anddoxycycline are relatively low and in moderately resistant level.The arear of the lowest resistance rate was Qianxinan and Tongren.The mirocycline and tigecycline had the lowest resistance levels and their MIC90 were 32ug/mL and 4ug/mL,respectively.However,their intermediation rate were quite high(21.5% and 18.5%respectively).It was shown the highest resistance rate to mirocycline in Qiannan,Zunyi and Bijie region and to tigecycline in Tongren and Qianxinan.The results of analysis for the MIC distribution and Cross resistant rate shown that the sensitive strains to the first generation of tetracyclines were also sensitive to second and third generation tetracyclins.But if bacteria was resistant to third tetracyclins,it was resistant to 1st~2rd generation tetracyclins.Multi-drug resistance of tested strains posed a serious situation,which the maximum Multi-drug resistance was five drug(45.98%)and the minimum was one drug(0.38%),but the 4-6 Multi-drug resistance were in the majority.The multidrug-resistant type consisted with oxytetracycline,chlortetracycline and doxycycline was dominated(92.1%).The tetracycline resistance genes of 783 Escherichia coli isolates was detected by PCR method.The results showed that the total positive rates of tetracycline resistance genes was 99.74%,and tetA,tetB,tetC,tetD,tetM,tetX were 92.60%,41.50%,58.75%,58.62%,70.10%,78.80%,respectively.Resistant strain mainly carry complex polygenic type.The bacteria carried 4 types resistant genes was in majority(34.2%),followed 5 type(26.4%)and 3 type(21.1%).The detection rate of bacteria with single resistant gene was low(2.7%),and the tetA was most gene(1.53%)in bacteria with single resistant gen.The result of statistical analysis on resistance phenotypes and resistance gene carryover shown that the drug resistance of E.coli to oxytetracycline,tetracycline,chlortetracycline,doxycycline,minocycline were extremely significantly correlated with the detection rate of carring tetB and tetM genes(P < 0.01).The detection rate of tetC was extremely significantly correlated with the drug resistance of minocycline(P<0.01),which was significantly related to the replacement of tigecycline(P<0.05).The detection rate of tetD was extremely significantly correlated with the drug resistance of minocycline andtetracycline(P<0.01).The detection rate of tetX was extremely significantly correlated with the tolerance of tetracycline(P<0.01).The relative expression of tetB and tetM genes in tested stain were detected by fluorescence quantitative PCR.Induced by different drug concentrations of oxytetracycline,chlortetracycline and doxycycline,respectively,the expression quantity of tetB and tetM gene in intial stage increased gradually,then decreased late following the increase of drug induced concentration.The highest expression quantity of the two genes was at 1/64 MIC concentration of oxytetracycline and chlortetracycline.Subsequently,due to the increase of drug inhibition,bacterial life activity was gradually inhibited,and drug resistance gene expression gradually decreased,too.The highest expression quantity of the two genes was at 1/16MIC(4ug/mL)concentration of doxycycline.TetB and tetM genes were not expressed in the environment of absence drugs.Induced by same drug concentrations(0.5ug/mL)of oxytetracycline and chlortetracycline,the expression quantity of tetM gene in1/128 MIC was outclass in 1/1024 MIC.The all results indicated that tetB and tetM genes were induced expression by tetracyclines,and carring those genes did not increase its survival fitness cost.The survivability of strains carring tetB and tetM genes and no-resistant genes strains was no difference in no-drug environment.But tetB and tetM were induced expression in drug environment,it made strains with the genes resist inhibition effect of drugs effectively,and became the advantage bacterium group gradually.So,it is difficult for bacteria to recover their sensitivities by stopping drug utilization once they are resistant to tetracycline antibiotics. |
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