Characterization of Mutations in the rpoB Gene Associated with ...

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2001, p. 2784–2787 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.8.2784–2787.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 8

Characterization of Mutations in the rpoB Gene Associated with Rifampin Resistance in Rhodococcus equi Isolated from Foals MARGUERITE FINES,1* STEPHANE PRONOST, KARINE MAILLARD, SAID TAOUJI,3 AND ROLAND LECLERCQ1 Service de Microbiologie, CHU Co ˆte de Nacre, 14033 Caen Cedex,1 Laboratoire De´partemental ´ tudes Frank Duncombe, 14053 Caen Cedex,2 and AFSSA, Laboratoire d’E ´ quine, Goustranville, et de Recherche en Pathologie E 14430 Dozule´,3 France Received 8 November 2000/Returned for modification 11 March 2001/Accepted 13 May 2001

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rifr)determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 ␮g/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 ␮g/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution. 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.

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

MATERIALS AND METHODS 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. Four rifampin-susceptible R. equi strains were used as control strains for rpoB sequencing. Strains E04, E07, and E09 were isolated from soil (strain E04) or horse dung (strains E07 and E09) in Normandy, France. Strain ATCC 33701 was used as a susceptible control strain and was kindly given to us by S. Takai; it was also used for the in vitro selection of rifampin-resistant mutants. 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 ␮g 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.

* Corresponding author. Mailing address: Service de Microbiologie, CHU Co ˆte de Nacre, Av. Co ˆte de Nacre, 14033, Caen cedex, France. Phone: 33 2 31 06 45 72. Fax: 33 2 31 06 45 73. E-mail: fines-m @chu-caen.fr. 2784

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TABLE 1. Origins, rifampin MICs, and RpoB amino acid substitutions for the R. equi strains tested Origin

Strain

Rifampin MIC (␮g/ml)

Amino acid substitution (E. coli numbering)

Foal (Japan)

ATCC 33701

0.125

None

Environment (France), soil

E04

0.06

None

Environment (France), horse dung

E07

⬍0.03

None

Environment (France), horse dung

E09

0.25

None

Foal 1 (Canada), lung

143

Foal 2 (France), right hip pus

428098

Foal 3 (France) Lung Lung abcesses Mesenteric nodes Intestinal abscesses Intestinal abscesses Proximal carpal joint Transtracheal aspirate Spleen Uterus Tracheobronchial nodes

RE1 RE2 RE5 RE6 RE7 RE9 RE10 RE3 RE8 RE4

Rifampin-resistant mutants ATCC 33701 Rifampin-resistant mutants ATCC 33701

128

His526Asp

8

Ser531Leu

128 128 128 128 128 128 128 8 8 2

His526Asp His526Asp His526Asp His526Asp His526Asp His526Asp His526Asp His526Asn His526Asn Asp516Val

M1, M2, M3

ⱖ256

His526Tyr

M4, M5, M6

ⱖ256

His526Arg

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⬘-TCGATCGGGCACATCC GG-3⬘), was chosen to amplify a portion of the rpoB region of R. equi encompassing the rifampin resistance (Rifr)-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 ␮l 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 ␮l of 10⫻ Taq buffer (Eurobio), and each deoxynucleoside triphosphate at a concentration of 200 ␮M. 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-acetateEDTA) and purified on Microcon 100 columns (Millipore Corp., Bedford, Mass.) before sequencing in an automated ABI PRISM 310 system (PerkinElmer Corp.). Both strands were sequenced as a cross-check by using either primer MF or primer MR.

RESULTS AND DISCUSSION Mutation frequency. The rifampin-associated mutation frequency of R. equi-sensitive strain ATCC 33701 (MIC 0.125 ␮g/ml) was nearly 10⫺8 in each of three independent selection experiments on agar plates containing either 1, 10, or 100 ␮g of rifampin per ml. This frequency was similar to the 10⫺7 in vivo rifampin-associated mutation frequency in R. equi (21) or to the 10⫺7 to 10⫺8 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 ␮g/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 ␮g/ml, respectively. Whereas Canadian strain 143 showed high-level rifampin resistance (MIC, 128 ␮g/ml), French strain 428098 appeared to be resistant to a lower level (MIC, 8 ␮g/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 ␮g/ml), and three strains were resistant at lower levels (MICs, 2 or 8 ␮g/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 ␮g/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 Rifr-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

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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 Rifr-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 ␮g/ml). Replacement of His526 by an aspartic acid led to low-level resistance (MIC, 8 ␮g/ml), as did replacement of Asp516 by a valine (MIC, 2 ␮g/ml) and Ser531 by a leucine (MIC, 8 ␮g/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 highlevel 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

J. CLIN. MICROBIOL.

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). In conclusion, similar to other bacterial genera, a single base change that led to a missense mutation in the rpoB cluster I region was associated with rifampin resistance in R. equi. As described for M. tuberculosis, E. coli, and S. aureus, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution. Moreover, the observation that similar changes were found at the same codon in the various bacterial genera confirms the universal site of action of rifampin among prokaryotes. ACKNOWLEDGMENTS This work was supported in part by a grant from the Conseil Ge´ne´ral du Calvados and the Fondation pour la Recherche Me´dicale. We thank J. Prescott and S. Takai for the gifts of strains. REFERENCES 1. Aubry-Damon, H., C. J. Soussy, and P. Courvalin. 1998. Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 42:2590–2594. 2. Billington, O. J., T. D. McHugh, and S. H. Gillespie. 1999. Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 43:1866–1869. 3. Carter, P. E., F. J. Abadi, D. E. Yakubu, and T. H. Pennington. 1994. Molecular characterization of rifampin-resistant Neisseria meningitidis. Antimicrob. Agents Chemother. 38:1256–1261. 4. Hillidge, C. J. 1987. Use of erythromycin-rifampin combination in treatment of Rhodococcus equi pneumonia.Vet. Microbiol. 14:337–342. 5. Jin, D. J., and C. A. Gross. 1988. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J. Mol. Biol. 202:45–58. 6. Kenney, D. G., S. C. Robbins, J. F. Prescott, A. Kaushik, and J. D. Baird. 1994. Development of reactive arthritis and resistance to erythromycin and rifampin in a foal during treatment for Rhodococcus equi pneumonia. Equine Vet. J. 26:246–248. 7. Kim, B. J., S. H. Lee, M. A. Lyu, S. J. Kim, G. H. Bai, G. T. Chae, E. C. Kim, C. Y. Cha, and Y. H. Kook. 1999. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J. Clin. Microbiol. 37:1714–1720. 8. Mascellino, M. T., E. Iona, R. Ponzo, C. M. Mastroiani, and S. Delia. 1994. Infections due to Rhodococcus equi in three HIV-infected patients: microbiological findings and antibiotic susceptibility. Int. J. Clin. Pharmacol. Res. 14:157–163. 9. McNeil, M. M., and J. M. Brown. 1992. Distribution and antimicrobial susceptibility of Rhodococcus equi from clinical specimens. Eur. J. Epidemiol. 8:437–443. 10. Musser, J. M. 1995. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin. Microbiol. Rev. 8:496–514. 11. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7–A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. 12. Nordmann, P., J. J. Kerestedjian, and E. Ronco. 1992. Therapy of Rhodococcus equi disseminated infections in nude mice. Antimicrob. Agents Chemother. 36:1244–1248. 13. Ohno, H., H. Koga, S. Kohno, T. Tashiro, and K. Hara. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob. Agents Chemother. 40:1053– 1056.

VOL. 39, 2001 14. Pozzi, G., M. Meloni, E. Iona, G. Orru, O. F. Thoresen, M. L. Ricci, M. R. Oggioni, L. Fattorini, and G. Orefici. 1999. rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy. J. Clin. Microbiol. 37:1197–1199. 15. Prescott, J. F. 1991. Rhodococcus equi: an animal and human pathogen. Clin. Microbiol. Rev. 4:20–34. 16. Pronost, S., G. Fortier, M. F. Legendre, K. Ogawa, Y. Sasaki, C. Collobert, S. Taouji, and S. Takai. 1998. Rhodococcose equine: nouvelles approches et bilan de deux anne´es d’e´tudes au laboratoire. Prat. Vet. Equine 30:268– 273. 17. Severinov, K., A. Mustaev, E. Severinova, M. Kozlov, S. A. Darst, and A. Goldfarb. 1995. The beta subunit Rif-cluster I is only angstroms away from the active

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center of Escherichia coli RNA polymerase. J. Biol. Chem. 270:29428–29432. 18. Takai, S. 1997. Epidemiology of Rhodococcus equi infections: a review. Vet. Microbiol. 56:167–176. 19. Takai, S., K. Takeda, Y. Nakano, T. Karasawa, J. Furugoori, Y. Sasaki, S. Tsubaki, T. Higuchi, T. Anzai, R. Wada, and M. Kamada. 1997. Emergence of rifampin-resistant Rhodococcus equi in an infected foal. J. Clin. Microbiol. 35:1904–1908. 20. Taniguchi, H., H. Aramaki, Y. Nikaido, Y. Mizuguchi, M. Nakamura, T. Koga, and S. Yoshida. 1996. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol. Lett. 144:103–108. 21. Wehrli, W. 1983. Rifampin: mechanisms of action and resistance. Rev. Infect. Dis. 5(Suppl. 3):S407–S411.