Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

Abstract

Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi. Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set’s bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at −20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except −20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing. IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.

A Common Practice of Widespread Antimicrobial Use in Horse Production Promotes Multi-Drug Resistance

“…We found that treatment with macrolides induce a selective pressure that selected for macrolide-resistant R. equi in the gut and in the environment, and that removing this pressure reduces the proportion of resistant isolates over time. This is likely the result of the resistance genes having a fitness burden for R. equi, consistent with other in vitro findings from our laboratory 64 . This suggests that removing the selective pressure induced by antibiotics could lead to resistant isolates being out-competed by susceptible isolates.…”

Effect of Macrolide and Rifampin Resistance on Fitness of Rhodococcus equi during Intramacrophage Replication and In Vivo

“…While there was no significant effect of macrolide or rifampin resistance alone, the dual-resistant strain-resistant to both macrolides and rifampin-led to bacterial killing, as indicated by a decrease in the numbers of CFU per monolayer over the course of 48 h. This is in contrast to what our laboratory previously observed regarding the effect of macrolide or rifampin resistance in R. equi in in vitro growth assays. When in vitro growth was analyzed in minimal medium or minimal medium without iron, an impairment of bacterial growth correlated with the rpoB mutation conferring rifampin resistance was observed (13). The data from the current study and those previously observed (13) demonstrate that there are differences between R. equi in vitro growth and the survival and replication of R. equi in the host macrophage.…”

“…Recently, our laboratory demonstrated that resistance to macrolides, rifampin, or macrolides and rifampin negatively impacted the in vitro growth of R. equi in various types of medium and in soil (13). Specifically, the possession of macrolide and rifampin resistance reduced the in vitro growth of R. equi in minimal medium lacking iron.…”

“…The objective of this study, therefore, was to elucidate the effects of carrying macrolide, rifampin, or dual macrolide and rifampin resistance mechanisms on intramacrophage replication in equine alveolar macrophages and in vivo virulence of R. equi in mice. Based on the decrease in R. equi in vitro fitness previously demonstrated in resistant strains (13), we hypothesized that macrolide and/or rifampin resistance would decrease intracellular R. equi replication in host macrophages. Additionally, we hypothesized that the presence of macrolide or rifampin resistance would impair the virulence of resistant R. equi in vivo in the absence of antimicrobial selection pressure but would provide a survival advantage to the resistant strains in the presence of macrolides or rifampin.…”

The novel and transferable erm(51) gene confers macrolides, lincosamides and streptogramins B (MLSB) resistance to clonal Rhodococcus equi in the environment

“…Recent studies have demonstrated that the presence of erm(46) in its plasmid does not impact fitness when compared with susceptible R. equi strains (Alvarez-Narvaez et al, 2019;Willingham-Lane et al, 2019a; however, rpoB mutations conferring rifampicin resistance decreased growth rate of R. equi in minimal media (Willingham-Lane et al, 2019a. In the study herein, we found two types of rpoB mutations, one not previously described in R. equi; the fitness impacts of these mutations are not known.…”