Resistance Of Escherichia Coli From Animal Origin And Study On The Strategies Of Drug Combination Using

Escherichia coli was an important flora in intestines of human and animal,which was also a conditional pathogenic bacteria.With the use of a large number of antibiotics in the medical and animal husbandry industries,more and more drug-resistant pathogenic E.coli strains emerged and they carried some drug resistance gene elements which could be spread horizontally.It was prominent to make the characteristics of zoonosis to the significance of public health.On the basis of study of drug resistance of animal source E.coli in Shandong Province,multiple high resistant E.coli strains were analyzed for drug resistance genes,virulence genes and MLST typing to explore the intrinsic association of these characteristics.The potential of drug combinations were explored to control the multiple high resistant strains to maximize the use of existing antibiotics in the best way and to find the most effective way to cope with the emergence and spread of drug resistance.1.Investigation on drug resistance of animal source E.coli in Weifang and Liaocheng-Samples of liver,lung and anal swab 89 cases from sick chicken or pigs were collected in various areas Wei and Liaocheng of shandong province.110 strains of E.coli were isolated,including 21 cases from pigs and 89 cases from chickens.The susceptibility tests to ampicillin,cefotaxime,ceftriaxone,cefotaxime,levofloxacin,ciprofloxacin,enrofloxacin,doxycycline,florfenicol and gentamicin were carried out by agar dilution method.According to the EUCAST criteria,the results showed that E.coli strains were resistant to many kinds of antibiotics,and the drug resistance rates to florfenicol and ampicillin were over 90%,and the resistance rate to ciprofloxacin was 68.18%.At the same time,almost all E.coli strains showed multiple resistant.There were 103 strains of E.coli resistant to 6 or above 6 kinds of antibiotics,which accounted for 93.64%.The proportion of E.coli resistant to 10 kinds of antibiotics was 37.27%,the proportion of E.coli resistant to 7 kinds of antibiotics was21.83%.Not only that,the strains tested were highly resistant,but the minimum inhibitory concentration of more than 50%strains to some drugs was found.The results showed that drug resistance of animal source E.coli was very serious,and it was a huge threat to public health.2.Molecular characteristics of multiple high resistant E.coli strainsMLST genotyping of multi resistant and highly pathogenic E.coli was carried out.It was found that 32 strains had 20 spectrum types and were very dispersed.No prevalent epidemic strains were found.Type ST69 was dominant,but only 5 cases.The molecular pattern characteristics of E.coli greatly increase the difficulty of its prevention and control.The detection of resistance genes of all kinds of antibiotics used in multi and highly resistant E.coli was detected.It was found that the resistance genes with high carrying rate were found for the common antibiotics.It was found that bla_CTX,bla_TEMEM and bla_SHVHV genes related to broad-spectrum?-slactamase existed widely in the strains.The contents of bla_CTXTX and bla_TEMEM genes were both above 90%,and that of bla_SHVHV gene was less than 10%.By resistance genes of aminoglycoside antibiotics detected,it was found that some resistance genes,like oqxA,oqxB,qnrA and qnrS,and their contents were respectively 84.38%,96.88%,6.25%and 37.5%.Tetracycline resistance genes of texA and texB and florfenicol resistance gene floR were detected.The texA was not found,and the tex B was lower than other drug-resistant genes,but the proportion of floR gene was high to 81.25%.62.63%of the tested isolates contained more than 10 kinds of resistant genes,and the maximum of 13 resistance genes and the minimum of 6 resistance genes.For multiple high resistant strains in this study,the main virulence genes were detected to include fimC,hlyF,sitA,iucD,iss,cva/cvi,eaeA,papC,stx2 and vat,but stx1,astA,tsh,iucD and irp 2 genes were not found.The virulence gene fimC was the most common existence,while the virulence gene vat less.Different virulence genes were found in different number of strains,and different strains contained different virulence genes.The virulence genes in multiple high resistant strains were analyzed to find 9.38%multiple resistant strains contained up to 6 virulence genes,and there were no virulence gene only in 3 multiple resistant strains and other strains had got 1-5 virulence genes,of which about 1/3 strains with 3 virulence genes.The results showed that the MLST types of multiple high resistant strains were dispersed and there were no obvious rules.Multiple resistance genes and virulence genes coexisted generally in clinical E.coli strains and there was no obvious association between kinds and quantity.Even when the apparent drug resistance level of the same antibiotics was similar,the drug resistance genes were different.There was no significant correlation between the drug resistance genes and the virulence genes.The results indicated that there was no significant correlation between drug resistance level of E.coli from different animals,MLST type,drug resistance genes and virulence genes.3.Strategies to multiple high resistant pathogenic strainsMultiple high resistant strains were tested by chessboard dilution method to analyze the antibacterial effect of synergistic drugs in vitro.The following six combinations with potential application were found.Results:with the combination of enrofloxacin and ceftiofur to resistant strains,9.38%showed a synergistic effect and 21.88%additive effect;with enrofloxacin and cefotaxime in combination,18.88%showed a synergistic effect and 34.38%additive effect;with enrofloxacin and ceftriaxone in combination,50%showed a synergistic effect and 37.5%the additive effect;with combined use of enrofloxacin and ampicillin,18.75%showed a synergistic effect.With florfenicol and doxycycline in combination,12.5%showed synergism effect and 62.5%additive effect.With gentamicin and ceftriaxone in combination,96.88%showed synergistic antibacterial activity to resistant E.coli strains in vitro.The combination of gentamicin and ceftriaxone could reduce gentamicin the MIC to 1/4-1/256 MIC of some multidrug resistant E.coli,when ceftriaxone add 2?g/mL ceftriaxone,which for specific strains or even higher.The results suggested that the accurate use of antibiotics against pathogenic bacteria was valuable.The results of this study provided a foundation to control the multiple high resistant pathogenic E.coli and delayed the production of drug resistance.