Studies On Epidemiology Of Class â…  Integron Among Bovine E. Coli And Mechanism Of Its Resistance To Florfenicol

The development of antimicrobial resistance from pathogen of animal origin has become a major public health problem attracting the global attention and posing potential danger to animal production and human health. In order to perform hazard analysis and assessment of bacterial resistance of animal origin, in this study, the pathogenic bovine Escherichia coli isolates which were collected from beijing surrounding region were characterized for serogroups and antimicrobial susceptibility pattern. The investigation and analysis on the molecular epidemiology of integrons/gene cassettes in these E. coli isolates by PCR technique and direct sequencing were carried out.Subsequently, three floR genes were cloned from calf pathogenic E. coli strains. Based on analysis of the possible upstream regulatory region of the floR gene, the florfenicol-resistant strain JM109-R was constructed. In order to investigate whether the phenotype of E. coli resistance to florfenicol was due to expression of the putative efflux of the floR protein, a modified high performce liquid chromatography (HPLC) method was developed to determine florfenicol accumulation in the susceptible and resistant E. coli.The investigation on the integrons/gene cassettes in twenty-third E. coli isolates associated with calf diarrhea indicated that 57% of them were positive for intll gene. Sequencing analysis revealed six gene cassettes prevailed, which encoded resistance to trimethoprim (dfrAl, dfrAlT), aminoglycosides (aadB, aadAl and aadAS) and chloramphenicol (cmlA), respectively. Of them, the gene cassette arrays dfrAl-orf (45%) and aadB-orf-cmlA (32%) were found most prevalent among these isolates. The gene cassette arrays aadB- aadAl-cmlA was firstly reported to our knowledge. These data revealed the high prevalence of class I integrons among calf pathogenic Escherichia coli isolates in Beijing surrounding region in China, which may provide some important useful surveillance information reflecting the antibiotic selective pressure in this specific region.The investigation of the antimicrobial spectrum of twenty-three Escherichia coli isolates and analysis of the class I integron/cassette in twenty-three Escherichia coli isolates found that wether the isolates carried the integron/cassttte was closely related to the antimicrobial spectrum of bacterial isolates.The isolates whose resistanct rates were not high nearly didn't carry the class I integron. The isolates carrying the class I integron were all resistanct to the sulfonamides and most of them were to resistant to trimethoprim, streptomycin and/or spectinomycin and chloramphenicol. While, the resistant phentype of the bacterial isolates to the fluoroquinolones, tetracyclines, and phenicillins wasn't strongly correlated whether the bacterial isolates carried the class I integron.Three floR genes were cloned from calf pathogenic florfenicol-resistance E. coli strains, The floR genes resulted in a 1356-bp fragment covering the ORF in region 66-1280 coding for 404 amino acids. Nucleotide sequence alignment with other published floR gene sequences from GenBank isolated from Escherichia coli strains revealed that several nucleotide residue substitutions were observed (for instance, at sites 26,105,175,439,441,671,678,798,943 and 1070).In order to elucidate the detailed molecular mechanism of resistance of E. coli to florfenicol, apositive clone that comprises the entire floR ORF and possible regulatory site was obtained and named plasmid pDux/floR, then the plasmid pDux/floR was transformed into the JM109 successfully. A significant resistance to florfenicol and chloramphenicol was found in this strain (64ug/ml for florfenicol). Subsequently, a modified high performce liquid chromatography (HPLC) method for determination of florfenicol accumulation in the bacterial sample was developed. The detection limit determining florfenicol in the bacterial sample was 0.0125ug/ml. In the bacterial sample fortified at 0.125ug/ml, lug/ml and 8ug/ml, the average recoveries ranged from 93.2% to 101.4%, with the coefficients of...