This is the first molecular characterization of resistance to macrolides, lincosamides and streptogramins B in R. equi. Resistance was due to the presence of a novel erm(46) gene mobilizable likely by conjugation, which has spread among equine isolates of R. equi in the USA.

bRhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. The genus Rhodococcus comprises a large number of metabolically diverse species that have attracted considerable biotechnological interest because of their ability to metabolize a wide variety of substrates, which finds applications in bioremediation and in the synthesis of precursors of pharmaceutical compounds (1). The genus contains only two pathogenic species: the plant pathogen Rhodococcus fascians and the animal pathogen Rhodococcus equi (2). Although the latter species was initially isolated from young foals, it has subsequently been isolated from a wide range of animals and humans (3). Disease in foals and immunosuppressed humans usually presents as pyogranulomatous pneumonia, although other manifestations, including osteomyelitis, may also occur. In pigs and cattle, R. equi is usually associated with submandibular lymphadenitis (3). In addition to having a pathogenic lifestyle, R. equi grows readily as a saprophyte in soils, as well as in the equine intestinal tract (3).R. equi is a parasite of macrophages, which prevents killing by the host cell through inhibition of phagosomal maturation, resulting in the formation of R. equi-containing vacuoles devoid of lysosomal markers, including cathepsin D and the proton-pumping vacuolar ATPase (vATPase) complex (4-6). The growth of...

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ϳ81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

the practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. the practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multidrug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (GaM; n = 19) and 19 untreated controls was performed. Treatment with MaR, but not GaM, significantly decreased fecal microbiota abundance and diversity, and expanded the abundance and diversity of antimicrobial resistance genes in feces. Soil plots experimentally infected with Rhodococcus equi (R. equi) and treated with MaR selected for MaR-resistant R. equi, whereas MaR-susceptible R. equi out-competed resistant isolates in GaM-treated or untreated plots. our results indicate that MaR use promotes multi-drug resistance in R. equi and commensals that are shed into their environment where they can persist and potentially infect or colonize horses and other animals. Despite their beneficial properties for treating infections, injudicious antimicrobial use can promote resistance in bacteria in both human and animal populations 1,2. The United States leads the list of high-income countries for antibiotic consumption, with ~80% of its annual antimicrobial consumption used for treating disease or promoting growth in animals 3,4. Furthermore, several studies suggest that the use of antibiotics in animals contributes to the crisis of antibiotic-resistant infections in humans 5-7. Although antimicrobial use in food animal production has received considerable attention 8-14 , antimicrobial resistance in equine medicine has received relatively limited attention. Pneumonia caused by Rhodococcus equi (R. equi) in foals is an important problem for the equine breeding industry worldwide 15-17. Foals are exposed to R. equi from birth 18,19 , and the disease generally progresses insidiously with onset of signs typically between ages 1 and 5 months 19,20. The combination of a macrolide with rifampin has been the standard of care for foals infected with R. equi in North America for over 30 years 21. Because of its insidious onset, many farms in North America have implemented serial thoracic ultrasonographic screening of foals to identify foals with pneumonia prior to the onset of clinical signs (i.e., subclinical pneumonia) and treatment of subclinical pneumonia 21-23. Evidence exists that most foals with subclinical pneumonia attributed to R. equi will not develop clinical signs of pneumonia 21,24,25. Consequently, thoracic ultrasonographic screening combined with antimicrobial treatment of foals with subclinical pneumonia results in overuse of antimicrobials. Resistance to MaR in R. equi has been increasing in prevalence over recent years in cent...

Pneumonia caused by remains an important cause of disease and death in foals. The combination of a macrolide (erythromycin, azithromycin, or clarithromycin) with rifampin has been the recommended treatment for foals with clinical signs of infection caused by since the early 1980s with, until recently, only rare reports of resistance. Resistance to macrolides and rifampin in isolates of cultured from horses is increasing, with isolates resistant to all macrolides and rifampin now being cultured from up to 40% of infected foals at some farms. This text reviews the available data regarding antimicrobial resistance in, with emphasis on the molecular mechanisms of the recent emergence of resistance to macrolides and rifampin in equine isolates of .