Treatment strategies impacting ceftiofur resistance among enteric bacteria in cattle

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyHarvey Morgan ScottA randomized controlled field trial was designed to evaluate the effects of two treatment strategies on ceftiofur and tetracycline resistances in feedlot cattle. The two strategies consisted of administering ceftiofur crystalline-free acid administration (CCFA) at either one or else all of the steers within a pen, and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. Both strategies were hypothesized to reduce ceftiofur resistance. The effects of treatment strategies were evaluated via metagenome-based and culture-based assays. In this 26-day study, 176 steers were allocated to 16 pens of 11 steers each. The two strategies were randomly assigned to the pens in a two-way full-factorial manner resulting in four treatment groups. The blaCMY-2, blaCTX-M, tet(A), tet(B), and 16S rRNA gene copies/g feces were quantified using qRT-PCR from fecal community DNA. Antimicrobial susceptibility profiles were determined using microbroth dilution technique from the non-type-specific (NTS) E. coli isolates (n=1,050). The NTS E. coli DNA was screened for the presence of blaCMY-2, tet(A), and tet(B) genes. Pens in which all the steers received CCFA treatment showed an increase in blaCMY-2 and blaCTX-M log10 gene copies/g feces and in the proportion of ceftiofur-resistant and blaCMY-2 positive NTS E. coli. This was in contrast to the pens where only one animal received CCFA treatment. There was a significant decrease in quantities of tetracycline genes in community DNA in pens where all animals received CCFA treatment. In contrast to metagenome-based assay results, culture-based assays indicated an increase in the proportion of tetracycline resistant NTS E. coli upon CCFA treatment. Thereafter, chlortetracycline administration led to rapid expansion both of ceftiofur (blaCMY-2, blaCTX-M) and tetracycline [tet(A) and tet(B)] log10 gene copies/g feces. Chlortetracycline treatment delayed the return of the ceftiofur resistance prevalence to baseline among NTS E. coli and thus did not lead to the hypothesized decrease in ceftiofur resistance. Our data suggest that chlortetracycline use is contraindicated when attempting to avoid expansion of resistance to critically important 3rd generation cephalosporins in feedlot cattle. Further studies are required to better establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together at varying ratios on the levels of antimicrobial resistance

Frequency distribution of <i>E. coli</i> by phenotypic multidrug resistance counts for the four treatment groups.

<p>(A) CCFA administered to all steers within pens without subsequent CTC administration at the pen level; (B) CCFA administered to one out of 11 steers within pens without subsequent CTC administration at the pen level; (C) CCFA administered to all steers within pens followed by CTC administered at the pen level; (D) CCFA administered to one out of 11 steers within pens followed by CTC administered at the pen level.</p

Prevalence of tetracycline-resistant <i>E. coli</i> isolates, modeled as marginal predicted probabilities, over days.

<p>Solid line represents the proportion of NTS <i>E. coli</i> isolates expressing phenotypic tetracycline resistance; dashed line represents the proportion of NTS <i>E. coli</i> isolates harboring the <i>tet</i>(A) gene; dotted line represents the proportion of NTS <i>E. coli</i> isolates harboring the <i>tet</i>(B) gene. The four treatment groups are: (A) CCFA administered to all steers within pens without subsequent CTC administration at pen level; (B) CCFA administered to 1 out of 11 steers within pens without subsequent CTC administration at pen level; (C) CCFA administered to all steers within pens followed by CTC administered at pen level; (D) CCFA administered to 1 out of 11 steers within pens followed by CTC administered at pen level.</p

PCR primers used for PCR reactions.

a<p>Primer set used is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080575#pone.0080575-Alali1" target="_blank">[31]</a>.</p>b<p>Primer set used is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080575#pone.0080575-Ng1" target="_blank">[54]</a>.</p>*<p>Sequence used for primer design.</p

Survival curve of <i>E. coli</i> isolates over each increasing ceftiofur concentrations tested, among treatment groups.

<p>Each survival curve represents the predicted probabilities of growth of NTS <i>E. coli</i> isolates on each of the increasing ceftiofur concentration tested on the NARMS plate as shown on a log scale in the figure. Dotted (large) line represent pens with CCFA administered to all steers within pens without subsequent CTC administration at pen level; dashed line represents CCFA administered to 1 out of 11 steers within pens without subsequent CTC administration at pen level; solid line represents CCFA administered to all steers within pens followed by CTC administered at pen level; dotted (small) line represents CCFA administered to 1 out of 11 steers within pens followed by CTC administered at pen level.</p

Probabilities of <i>E. coli</i> isolates to be pan-susceptible, penta-resistant, or deca-resistant among treatment groups.

<p>The three sub-graphs represent modeled marginal predicted probabilities for <i>E. coli</i> isolates to be (A) pan-susceptible, (B) penta-resistant, or (C) deca-resistant among the four treatment groups. The four treatment groups are represented by four lines in all three sub graphs. Dotted (large) line represent pens with CCFA administered to all steers within pens without subsequent CTC administration at pen level; Dashed line represents CCFA administered to 1 out of 11 steers within pens without subsequent CTC administration at pen level; solid line represents CCFA administered to all steers within pens followed by CTC administered at pen level; dotted (small) line represents CCFA administered to 1 out of 11 steers within pens followed by CTC administered at pen level.</p