Horizontal Spread of Rhodococcus equi Macrolide Resistance Plasmid pRErm46 across Environmental Actinobacteria

Abstract

Conjugation is one of the main mechanisms involved in the spread and maintenance of antibiotic resistance in bacterial populations. We recently showed that the emerging macrolide resistance in the soilborne equine and zoonotic pathogen Rhodococcus equi is conferred by the erm(46) gene carried on the 87-kb conjugative plasmid pRErm46. Here, we investigated the conjugal transferability of pRErm46 to 14 representative bacteria likely encountered by R. equi in the environmental habitat. In vitro mating experiments demonstrated conjugation to different members of the genus Rhodococcus as well as to Nocardia and Arthrobacter spp. at frequencies ranging from ∼10^-2 to 10^-6 pRErm46 transfer was also observed in mating experiments in soil and horse manure, albeit at a low frequency and after prolonged incubation at 22 to 30°C (environmental temperatures), not 37°C. All transconjugants were able to transfer pRErm46 back to R. equi Conjugation could not be detected with Mycobacterium or Corynebacterium spp. or several members of the more distant phylum Firmicutes such as Enterococcus, Streptococcus, or Staphylococcus Thus, the pRErm46 host range appears to span several actinobacterial orders with certain host restriction within the Corynebacteriales All bacterial species that acquired pRErm46 expressed increased macrolide resistance with no significant deleterious impact on fitness, except in the case of Rhodococcus rhodnii Our results indicate that actinobacterial members of the environmental microbiota can both acquire and transmit the R. equi pRErm46 plasmid and thus potentially contribute to the maintenance and spread of erm(46)-mediated macrolide resistance in equine farms.IMPORTANCE This study demonstrates the efficient horizontal transfer of the Rhodococcus equi conjugative plasmid pRErm46, recently identified as the cause of the emerging macrolide resistance among equine isolates of this pathogen, to and from different environmental Actinobacteria, including a variety of rhodococci as well as Nocardia and Arthrobacter spp. The reported data support the notion that environmental microbiotas may act as reservoirs for the endemic maintenance of antimicrobial resistance in an antibiotic pressurized farm habitat.

Keywords: R. equi; antimicrobial resistance; conjugation; erm(46); macrolides; pRErm46; soil.

FIG 1

Acquisition of pRErm46 has a different fitness cost depending on the recipient species. pRErm46 was conjugally transferred from R. equi to R. rhodnii, R. erythropolis, R. fascians, A. paraffineus, and N. globerula. Shown are growth curves with each isogenic set composed by a pRErm46+ (red) and a pRErm46- (turquoise) isolate in rich complex medium (BHI) and chemically defined medium (mREMM; see Materials and Methods).