Study On The Mechanism Resistance To β-lactam Antibiotics Of Enteropathogenic E.coli From Deer

Escherichia coli is one of the common pathogenic bacterium in medical science and veterinarian clinical infection. The serological type of it is multiple, so there was no ideal vaccine to prevent (it is difficult to prevent). When it breaks out, we treated it with antibiotics. But with the generally, persistently and unreasonably use of the antibiotics, the quantity of persister of E.coli increased unceasingly. Since the mechanism resistance diversified frequently, sometimes we could not find out an effective antibiotic. In recent years, the occurrence of the colibacillosis of deer presented upgrade tendency, thus it caused large loss deer farming. Therefore, the study on the drug resistance of enteropathogenic E.coli from deer is significance to theory and practice.In this study, the enteropathogenic E.coli was isolated from sick deer in Jilin, Henan and other provinces or cities and those drug resistances were investigated by drug sensitivity experiment. Then, 10 strains were selected and examined by minimal inhibitory concentration (MIC) test to 6 β-lactam antibiotics. The drug resistance gene related to β-lactam antibiotics blaAmpC, pbpA, ompFA and ompCA were investigated in the experiment with polymerase chain reaction (PCR), cloning and sequencing to analyze the relationship between them with the drug resistance level. In the same time, we determined the transcriptional level of mRNA and the expression level of protein of the blaAmpC gene quantitatively which encoding extended spectrum β-lactamases (ESBLs) by real time fluorescent quantitation PCR and Western-blot.The result indicated that the MIC of these strains was from 2μg/mL to 256μg/mL. The MIC of these strains to Ampicillin and Amoxicillin which applied generally in clinic was≥128μg/mL, besides the MIC of 6, 8 strains to Ampicillin was 16μg/mL, 18μg/mL respectively. The result of sequencing of the masculine clone plasmid showed, to contrast with the reference sequence in GeneBank: the pbpA gene encoding penicillin-binding protein (PBP) had 5 mutation basic group and 1 changed amino acids, the ompFA gene encoding outer membrane protein (OMP) had 7 mutation basic group and corresponding 7 changed amino acids, and the ompCA gene had 11 mutation basic group and 7 changed amino acids; the blaAmpC had 2 mutation basic group and no amino acids changed. The result of enzyme linked immunosorbent assay (ELISA) indicated that we obtained the antibody against blaAmpC and the antibody titer reach to 10,000. The result of real-time PCR and Western-blot showed that the strain with high drug resistance, the expression level of mRNA and ESBLs was also in high level.The results also indicated that the drug-resistance of E.coli which isolated in clinic from deer was...