Background 

Emergence and dissemination of bacteria that are resistant to a broad array of antimicrobial agents is of growing concern. Accumulation of multiple unrelated resistance determinants has arisen, at least in part, from antimicrobial selection pressure; however other non-antimicrobial-use factors also contribute. In our preliminary work, we documented that administration of long-acting ceftiofur favors expansion of E. coli that are phenotypically resistant to ceftiofur as well coresistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline (ACSSuT + Cef phenotype). This expansion of a broad ensemble of resistance determinants may have occurred through clonal expansion or dissemination of a mobile/mobilizable plasmid. It is important to distinguish between the clonal expansion and plasmid dissemination. If the latter occurred, we anticipate that an indistinguishable plasmid (and resistance phenotype) will present within a broad variety of E. coli genotypes. This signifies likely horizontal transfer of antimicrobial resistance. If so, it would guide future research toward targets and mechanisms to curtail the dissemination of multidrug-resistant pathogenic bacteria. 

In previous research (funded by America's Beef Producers), of 126 Salmonella Newport isolates, 67% (n=85) were resistant to ceftiofur and all carried the beta lactamase blaCMY-2 gene that confers resistance to extended-spectrum cephalosporins. Salmonella Newport isolates were also typed using PBRT. Ninety-seven percent (n=97) of the S. Newport isolates carried the Inc A/C incompatibility plasmid, presumably where the blaCMY-2 gene is located. These studies demonstrated that incompatibility type is important for plasmid transfer. 

Better characterization of the potential role that non-type-specific E. coli play in the horizontal dissemination of resistance determinants, particularly on plasmids, is greatly needed. Once the objectives of this study are met, we will have the necessary data to begin future studies aimed at targeting the mobile/mobilizable plasmids that confer resistance to a broad array of drugs. This may ultimately lead to mechanisms to cure bacteria of plasmids through innovative means. The long-term goal of our research team is to develop novel interventions to 1) reduce the burden of resistance in cattle presented for harvest; and 2) maintain agriculture’s access to efficacious antimicrobial drugs. The stated objectives for this work were: 

• Describe the genetic components conferring the phenotypically expressed resistance of our preliminary research.  PCR-based replicon typing (PBRT) of E. coli isolates was conducted to determine plasmid (replicon) types carried by the isolates. This also 1.     included PCR assays to determine the type of beta-lactamase gene(s) that may be carried by the isolates.
  
• Demonstrate that transmissibility of the resistant phenotype to recipient E. coli and Salmonella in vitro, evaluate the rate of plasmid transfer to recipient E. coli with and without the presence of ceftiofur, and that transmissibility occurs via a mobile/mobilizable plasmid.
  
• Molecularly characterize the isolates using pulsed-field electrophoresis (PFGE) to determine if resistance occurs in a single (or a limited number of) clones or in a broad array of genotypes.
  
• Molecularly characterize the resistance-encoding plasmid to identify plasmids shared among different strains.
  

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