Clonal multidrug resistance recently emerged in
Rhodococcus equi
, complicating the therapeutic management of this difficult-to-treat animal and human pathogenic actinomycete. The currently spreading multidrug-resistant (MDR) “2287” clone arose in equine farms upon acquisition, and co-selection by mass macrolide-rifampin therapy, of the pRErm46 plasmid carrying the
erm
(46) macrolides-lincosamides-streptogramins resistance determinant, and an
rpoB
S531F
mutation. Here, we screened a collection of susceptible and macrolide-rifampin-resistant
R. equi
from equine clinical cases using a panel of 15 antimicrobials against rapidly growing mycobacteria (RGM), nocardiae and other aerobic actinomycetes (NAA).
R. equi
–including MDR isolates– was generally susceptible to linezolid, minocycline, tigecycline, amikacin and tobramycin according to
Staphylococcus aureus
interpretive criteria, plus imipenem, cefoxitin and ceftriaxone based on Clinical & Laboratory Standards Institute (CLSI) guidelines for RGM/NAA. Ciprofloxacin and moxifloxacin were in the borderline category according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Molecular analyses linked pRErm46 to significantly increased MICs for trimethoprim-sulfamethoxazole and doxycycline in addition to clarithromycin within the RGM/NAA panel, and to streptomycin, spectinomycin and tetracycline resistance. pRErm46 variants with spontaneous deletions in the class 1 integron (C1I) region, observed in ≈30% of
erm
(46)-positive isolates, indicated that the newly identified resistances were attributable to C1I’s sulfonamide (
sul1
) and aminoglycoside (
aaA9
) resistance cassettes and adjacent
tetRA
(33) determinant. Most MDR isolates carried the
rpoB
S531F
mutation of the 2287 clone, while different
rpoB
mutations (S531L, S531Y) detected in two cases suggest the emergence of novel MDR
R. equi
strains.