INTRODUCTION

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First isolated by Magnusson from the lesions of an infected foal in 1923, Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus which causes suppurative pneumonia and ulcerative enteritis in foals aged from 1 to 3 months (17). R. equi has a worldwide distribution and is responsible for sporadic disease in general but can be devastating on some farms. Morbidity rates reach 5 to 17%, and mortality rates of up to 80% have been reported (14). As a result of the AIDS epidemic, R. equi has also been recognized as an important opportunistic pathogen in immunocompromised patients (8).

By virtue of good tissue and macrophage penetration combined with low MICs and a synergistic action, the combination of erythromycin and rifampin is often used for the treatment of R. equi infections in humans and foals and has dramatically reduced mortality rates since its introduction as therapy (4). However, although rifampin is used in combination with erythromycin to reduce the likelihood of selection of resistant mutants, several cases of emergence of rifampin resistance have been reported during treatment of humans and foals (6, 9, 15, 18). Reports of resistance to rifampin are still rare, and the mechanisms of rifampin resistance in R. equi have not yet been elucidated at the genetic level.

In several bacterial genera such as Mycobacterium tuberculosis (2, 10, 19), Escherichia coli (5), Staphylococcus aureus (1), and Neisseria meningitidis (3), resistance to rifampin results from the substitution of a limited number of highly conserved amino acids of the RNA polymerase  subunit encoded by the rpoB gene. These amino acids are clustered in three regions: clusters I, II, and III. Most of the substitutions occur in cluster I. We have thus amplified and sequenced portions of rpoB including the cluster I region from four rifampin-susceptible R. equi strains and from resistant strains isolated from foals. Mutants obtained in vitro were also studied.

	MATERIALS AND METHODS

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Bacterial strains. Twelve R. equi-resistant strains were isolated from three foals. Foal 1 died from R. equi pneumonia in Ontario, Canada, in 1992. Strain 143 was isolated from the foal's lung and was kindly given to us by J. Prescott. Foal 2 received a 2-month course of the combination of erythromycin and rifampin for R. equi infection before a rifampin-resistant strain was isolated, and foal 3 was autopsied at the age of 7 months after a 2-month course of treatment with rifampin alone (25 mg/kg of body weight three times a day). The characteristics of the strains are summarized in Table 1.

Antibiotic susceptibility testing. Determination of the rifampin MICs was done by an agar dilution method adapted from the recommendations of NCCLS (11). An inoculum of 10⁴ CFU per spot was used, and the MICs were read after 48 h of incubation at 30°C.

Selection of mutants resistant to rifampin. Approximately 10⁹ CFU of R. equi ATCC 33701 was plated onto brain heart infusion agar containing 1, 10, or 100 µg of rifampin per ml. After 48 h of incubation at 30°C, the number of colonies on the agar plates was counted. The resistance of the growing colonies to rifampin was checked by the disk agar diffusion method and was confirmed by determination of MICs. The mutation frequency was determined relative to the total count of viable organisms plated.

PCR amplification and DNA sequencing. Total DNA of R. equi strains was extracted and purified from a single colony by using the InstaGene DNA purification matrix as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, Calif.). A set of primers described by Bum-Joon Kim (7), primers MF (5'-CGACCACTTCGGCAACCG-3') and MR (5'-TCGATCGGGCACATCCGG-3'), was chosen to amplify a portion of the rpoB region of R. equi encompassing the rifampin resistance (Rif^r)-determining region. Mutations in this region are associated with rifampin resistance in M. tuberculosis as well as in E. coli and S. aureus. Ten microliters of the DNA extracts was added to 40 µl of a PCR mixture containing 0.2 U of Taq polymerase (Eurobio, Les Ullis, France), 1 mM MgCl₂, 20 pmol of each primer (GIBCO BRL, Life Technologies, Paisley, Scotland), 5 µl of 10× Taq buffer (Eurobio), and each deoxynucleoside triphosphate at a concentration of 200 µM. The reaction mixture was subjected to 30 cycles of amplification (with each cycle consisting of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s), followed by a 10-min extension at 72°C in a Gene Amp PCR System 2400 instrument (Perkin-Elmer Corp., Norwalk, Conn.). The PCR products were then electrophoresed in a 2% agarose gel in 1× TAE (Tris-acetate-EDTA) and purified on Microcon 100 columns (Millipore Corp., Bedford, Mass.) before sequencing in an automated ABI PRISM 310 system (Perkin-Elmer Corp.). Both strands were sequenced as a cross-check by using either primer MF or primer MR.

	RESULTS AND DISCUSSION

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Mutation frequency. The rifampin-associated mutation frequency of R. equi-sensitive strain ATCC 33701 (MIC 0.125 µg/ml) was nearly 10⁸ in each of three independent selection experiments on agar plates containing either 1, 10, or 100 µg of rifampin per ml. This frequency was similar to the 10⁷ in vivo rifampin-associated mutation frequency in R. equi (21) or to the 10⁷ to 10⁸ in vitro rifampin-associated mutation frequencies observed in S. aureus (1) or other pyogenic bacteria such as Streptococcus pneumoniae, N. meningitidis, and Streptococcus pyogenes. The six mutants obtained were all highly resistant to rifampin (MICs, >256 µg/ml), regardless of the rifampin concentration on which the mutant was grown. These results suggest that resistance to high levels of this antibiotic arises in a single-step event and substantiate the use of rifampin in combination with other antimicrobials for the treatment of R. equi infections. However, the use of this combination might not be sufficient to prevent the emergence of rifampin resistance, as illustrated by the history of foal 2 and previous reports (6).

Susceptibility to rifampin of environmental and animal R. equi strains. The rifampin MICs for the rifampin-susceptible strains isolated from soil (strain E04) or horse dung (strains E07 and E09) were 0.06, <0.03, and 0.25 µg/ml, respectively. Whereas Canadian strain 143 showed high-level rifampin resistance (MIC, 128 µg/ml), French strain 428098 appeared to be resistant to a lower level (MIC, 8 µg/ml). Various levels of rifampin resistance could also be observed among the 10 R. equi strains isolated from various infected sites in the third foal, with most strains resistant to high levels of rifampin (MIC, 128 µg/ml), and three strains were resistant at lower levels (MICs, 2 or 8 µg/ml) (Table 1). The same observation was reported by Takai (18), who described 119 R. equi isolates obtained from several lesions of one infected foal euthanatized after several unsuccessful treatments and 1 month of monotherapy. Several levels of rifampin resistance were observed (MIC range, 12.5 to >100 µg/ml). Moreover, analysis of the strains revealed at least eight plasmid profiles or ribotypes, suggesting that the foal was infected with several R. equi strains.

Amino acid sequence analysis and susceptibility to rifampin. The amino acid sequence of the rpoB Rif^r-determining gene region from amino acids 454 to 554 (E. coli numbering) was deduced from the nucleotide sequences determined for susceptible strains ATCC 33701, E04, E07, and E09, for 12 rifampin-resistant strains isolated from three foals, and for in vitro mutants. The nucleotide sequences of R. equi ATCC 33701, E04, E07, and E09 showed 100% identity to the sequence of R. equi ATCC 10146 (GenBank accession number AF057494), a susceptible R. equi strain studied by Kim et al. (7). These sequences appeared to be very closely related to the M. tuberculosis H37Rv rpoB Rif^r-determining region, differing only by a leucine in place of a glycine at position 468. This amino acid substitution also occurs in nontuberculous mycobacteria as well as in E. coli and S. aureus without conferring rifampin resistance (7). Compared to the DNA sequences of rifampin-susceptible R. equi strains ATCC 33701 and ATCC 10146, the DNA sequences of the clinical resistant strains and the in vitro mutants showed a single base pair mutation that introduced an amino acid substitution. Six mutational changes were found at three positions. For 16 of 18 strains, the missense mutations occurred at position 526 (E. coli numbering), in which the initial histidine was replaced by either an aspartic acid (8 strains), an asparagine (2 strains), a tyrosine (3 in vitro mutants), or an arginine (3 in vitro mutants). In the two remaining strains, an aspartic acid at position 516 and a serine at position 531 were replaced by a valine and a leucine, respectively (Table 1). In our study, all the missense mutations involved in R. equi rifampin resistance fell within the so-called cluster I region, which encompasses 27 amino acids from amino acids 507 to 533. In particular, residues 516 to 540 in E. coli are known to be part of the rifampin target (20) and participate along with amino acids 1065 and 1237 in the formation of the initiation site when the  subunit is assembled in the RNA polymerase complex (16). In fact, 96% of missense mutations involved in M. tuberculosis rifampin resistance are found in the cluster I region (10), and similar data have been obtained for E. coli (5) and S. aureus (1). Moreover, the residues associated with M. tuberculosis rifampin resistance and with the highest mutation frequencies are His526 (36%), which is usually replaced by a tyrosine; Ser531 (43%), which is replaced by a leucine; and Asp516 (8%), which is replaced by a valine (10). The same substitutions except for those of His526 were found in R. equi, in which His526 was replaced by an aspartic acid more frequently than it is in M. tuberculosis. This suggests that these amino acids are critical sites for rifampin resistance. Sequence analysis of clusters II and III involved in rifampin resistance in E. coli (5) and M. tuberculosis (19) was not carried out in our study, and it could not be excluded that substitutions in these regions are responsible for rifampin resistance in Rhodococcus. Comparative analysis of the level of rifampin resistance in R. equi and of the mutation sites indicated that high-level resistance correlated with replacement of His526 by an aspartic acid, an arginine, or a tyrosine (MIC, 128 µg/ml). Replacement of His526 by an aspartic acid led to low-level resistance (MIC, 8 µg/ml), as did replacement of Asp516 by a valine (MIC, 2 µg/ml) and Ser531 by a leucine (MIC, 8 µg/ml) (Table 1). Low-level resistance conferred in R. equi by replacement of Ser531 by a leucine is surprising since this high-frequency change is always associated with high-level resistance in M. tuberculosis, S. aureus, and E. coli (12, 13). In our study, only one strain harbored this mutation, and this result should be confirmed. In M. tuberculosis, the substitution of His526 is most frequently associated with high-level resistance, but rare substitutions (His526Leu or Val) can lead to low-level resistance, depending on the new amino acid incorporated (12). The replacement of His526 by an asparagine observed in an R. equi strain with low-level rifampin resistance is scarcely observed in M. tuberculosis and could not be associated with a particular resistance level in the latter species since this mutation was combined with a mutation associated with high-level resistance in one study (12), and no correlation was found in an other study (10). The last alteration observed in R. equi strains with low-level rifampin resistance, in which an aspartic acid was replaced by a valine at position 516, is frequently described in other bacterial genera and is associated with low-level or a moderate level of resistance in M. tuberculosis (12, 13).