Studies On Class 1 Integron-associated Antimicrobial Resistance In E. Coli From Beef Cattle Fed Antibiotics

Antimicrobial growth promoters (AGP) administration in agriculture promotes the emergence and the propagation of antimicrobial-resistant bacteria in food animals and also it can be transferred from animals to humans via the food chain. At present, the spead of antimicrobial resistance that is exerted by the use of AGP has been raised as a public health issues with global attention, and this also incurs a potential risk for the development of livestock husbandry and human health. To characterize class 1 integron-associated antimicrobial resistance in Escherichia coli from beef cattle fed subtherapeutic antibiotics, 204 tetracycline-resistant E. coli originating from beef cattle subtherapeutically administered chlortetracycline (A44, n=40), a mixture of chlortetracycline and sulfamethazine (AS700, n=53), or no antimicrobials (control, n=111) were studied. Susceptibilities to 11 antimicrobials were conducted on all isolates (n=204). The presence and characterization of class 1 integrons and inserted gene cassettes and phylogenetic analysis were performed by PCR, restriction enzyme analysis and sequencing.After that, the genetic relationship among integron-carrying isolates was determined by pulsed-field gel electrophoresis (PFGE). The prevalence of resistance genes associated with the major resistance phenotypes in bovine E. coli isolates was carried out by PCR. Moreover, the associations between resistance genes and class 1 integrons were performed using SAS-PC-System version 9.1 for Windows.Overall, susceptibility tesing results showed that the frequencies of resistance to various antimicrobial agents were higher in isolates from either A44 or AS700 steers, as compared to control steers. Among the isolates from different treatments, significantly higher (P<0.01) frequencies of resistance to ampicillin, chloramphenicol and streptomycin were detected in isolates from A44 and AS700 steers than the ones from control steers. Prevalence of genes coding for class 1 integrase was higher (P<0.001) in isolates from AS700 (33%) and A44 (28%) steers as compared to isolates from control animals (7%). Sequencing analysis revealed eight gene cassettes were prevalent and most integron gene cassettes belonged to the aad or dfr families. Eight gene cassettes that encoded resistance to aminoglycosides (aadA1, aadA2, aadA4, aadA5), trimethoprim (dfrA1, dfrA12, dfrA17) and chloramphenicol (catB3) were detected in the four different integrons. Of them, the gene cassette arrays aadA1 and dfrA12-orfF-aadA2 were most prevalent. To our knowledge, the gene cassette array dfrA1-catB3-aadA4 was firstly reported in bovine E. coli isolates.As revealed by analyzing the association between resistance phenotypes and class 1 integron, integron-carrying isolates were more likely to be multi-drug resistant. Isolates that contained a class 1 integron were highly resistant to streptomycin, sulfisoxazole, ampicillin and chloramphenicol, but not to quinolones and amoxicillin/clavulanic acid. Eighty-six percent of the integron-positive isolates belonged to phylogenetic group B1. As determined by PFGE, both closely and distantly related isolates harbored integrons with identical gene cassette arrays. Resistance genes aadA1, blaTEM1, sul1, and cat were more (P<0.001) frequently detected in isolates from A44 and AS700 steers. Positive correlations were found between the tet(A) gene and the genetic elements sul1 (r=0.44), aadA1 (r=0.61), cat (r=0.58) and intI1(r=0.37).In conclusion, the long-term use of AGP is strongly associated with the increases of the frequency of antimicrobial resistance in bovine E. coli isolates. Subtherapeutic administration of AGP to feedlot cattle may promote the presence of class 1 integron in bovine E. coli isolates. Dissemination of class 1 integron in beef cattle feedlots were probably result from both vertical and horizontal transfer. The association of antimicrobial resistance determinants with transferable elements such as integrons may promote the dissemination of antibiotic resistance among E. coli and possibly other enteric bacteria.