Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets

Abstract

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.

Fig 1

Schematic representation of the study design. Linpro is a commercial feed supplement containing an extruded blend of flax seed and field peas with added vitamins and minerals.

Fig 2

Failure function graph showing cumulative susceptibilities of tcrB-positive and tcrB-negative isolates to increasing copper concentrations.

Fig 3

Southern blot hybridization of tcrB probe with S1 nuclease-digested genomic DNA of tcrB-positive enterococcal isolates. Lane 1, midrange PFG marker II; lanes 2, 3, and 4, S1 nuclease-digested genomic DNA of E. faecium isolate; lanes 5, 6, and 7, hybridization with tcrB probe.

Fig 4

eBURST-based population snapshot of E. faecium on 455 available MTs in the E. faecium MLVA database (http://www.umcutrecht.nl/subsite/MLVA). eBURST clustering of 20 two MTs of E. faecium isolates from feedlot cattle is indicated in boldface and enclosed in ovals. Each MT represents one node with a difference in one locus. The dotted circles indicate MTs that are closely related to the existing predominant clones in the database (solid circles).