Gene Detection And Resistance Control On Esbls In The Pathoginic E.coli

Ceftiofur is a high activity, good pharmacokinetic, little side effects, and low residual, animal-specific medicine, which is used to treat bacterial diseases in swine, chickens and ruminants. But in recent years, bacteria, especially Gram - negative bacteria has generated resistance for its pressure of survival, with the third - generation cephalosporin widely used and abused, and the resistant strains increased year by year. Results showed that: the main resistant mechanism of gram-negative bacteria to the third generation cephalosporins and the new class ofβ-lactam antibiotics is producing extended spectrumβ‐lactamase (ESBLs) and Ampc enzyme, bacteria producing ESBLs may carry other resistant plasmid to quinolones, aminoglycosides, and sulfa drugs so on, showed cross-resistance and multiple-resistance, lead to serious threat to clinical treatment, and it attracted the focus of clinical attention. In order to guide farmers to use drugs scientific and legitimately in clinics, to reduce the harm leaded from bacteria producing ESBLs, some experiment had been done in this paper.1. Detection of ESBLs in pathogenic E. coli and research on plasmid dissemination(1)To the 177 clinical E.coli strains, the minimal inhibitory concentrations (MICs) of ceftiofur, ceftriaxone, amoxicillin and cefotaxime were determined with the two-fold dilution method. Results showed that MICs of ceftiofur, amoxicillin, ceftriaxone, and cefotaxime to the clinical E. coli in chicken and swine were higher than their MICs to the standard strains.(2)Detection of ESBLs were done by double disc synergy test(DDST), three primers were designed: SHV, TEM and CTX, ESBLs gene type was amplified,two strains from dofferent citys were selected, their ESBLs were sequenced and analyzed by PCR. For all E. coli strains, ESBL-positive rate with the double disk synergy test was 20.34%, ESBL-positive rate with PCR was 19.77%. There was not significant difference between the two results. And two strains were detected to have produced TEM type ESBLs and SHV type ESBLs; one strain was detected to have produced TEM type ESBLs and CTX type ESBLs.(3)Results of analysis about TEM type ESBLs gene showed that: E. coli in swine and in chicken from Xinzheng and E. coli in swine from Zhongmou produced the same ESBLs; there were close relationship between ESBLs produced by E. coli in swine and in chicken from Xingyang. (4)Conjugation and transformation about the plasmid of ESBLs were tested, as ampicillin used as an alternative antibiotic, E. coli DH5αwas used as the recipient bacteria in the test. Results showed that E. coli in swine and chicken can transfer the resistant ESBLs plasmid to the recipient strain.2. Study on the inhibited and protective effect of tazobactam(1)MICs of ceftiofur, CEF-T and CEF-T-EN to C83907, C83845 and E.coli Xingyang-ESBLs were determined. Results showed that compound of ceftiofur with tazobactam showed higher activity to the E.coli Xingyang-ESBLs, the MIC reduced for 12.5 times than ceftiofur, compound of ceftiofur - tazobactam -enrofloxacin showed the highest activity, the MIC was 1.2μg/mL.(2)The killing curve map of CEF-T to E.colixingyang-ESBLs was similar with CEF to C83845, but it was worse than CEF-T-EN slightly.(3)PAE of ceftiofur to the standard E.coli were similar to CEF-T, but compand of CEF-T-EN was longer than them at same MICs. PAE of ceftiofur, CEF-T and CEF-T-EN to E.colixingyang-ESBLs were similar. The PLIE map of Tazobactam showed that it have轻微的PLIE.(4)The 7 day old chicks were infected to make into artifical E.coli infection model successfully by E.coli Xingyang-ESBLs (0.4mL, IP). The chickens infected but not treated were used as control, other infected chickens were treated by ceftiofur powder, ceftiofur suspension, CEF-T and CEF-T-EN. The results of drug treatment were analyzed by SPSS11.5 software and X2 test. The mortality rates of every treatment group were lower than the control group significantly. The cure effect of ceftiofur sodium powder was higher than ceftiofur suspension, the cure effect of CEF-T was higher than CEF, and the cure effect of CEF-T-EN was the highest of all those drugs.3. Research on reversal of ESBLs- resistant plasmid(1)After the standard E.coli were induced to produce ESBLs by ceftiofur with sub-MIC method, with SDS as control, the elimination rate of chlorpromazine, promethazine, diazepam, enrofloxacin ciprofloxacin to the ESBLs plasmid in vitro were determined. After the mice were got rid of pollution by tetracycline (TC), erythromycin (EM), rifampicin (RIF), elimination rate of chlorpromazine, promethazine, diazepam, norfloxacin, ciprofloxacin to the induced strains in vivo were studied. Results showed that the elimination rate of every plasmid-cure agent in vitro were almost the same as it's in vivo. And the the tricyclic psychotropic drugs showed better elimination effect to ESBLs, they were suitable for to prevent bacterial resistance from spreading and developing.(2)Bacter eliminated in vivo and in vitro were collected, the MICs of them and original bacter to ceftiofur, ceftriaxone, amoxicillin, ampicillin were determined. Results of MICs showed bacteria eliminated have restored to be sensitive to ceftiofur etc. Ampicillin was used as inducer; resistance stability of bacteria eliminated was study. They were resistant to amoxicillin and ampicillin after they were induced for 50 generations, but they were sensitive to ceftiofur and ceftriaxone after they were induced for 100 generations.