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Journal of Clinical Microbiology, June 2003, p. 2337-2340, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2337-2340.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Emergence of Rifampin-Resistant Rhodococcus equi with Several Types of Mutations in the rpoB Gene among AIDS Patients in Northern Thailand

Norichika Asoh,1* Hiroshi Watanabe,1 Marguerite Fines-Guyon,2 Kiwao Watanabe,1 Kazunori Oishi,1 Weerayut Kositsakulchai,3 Tippaya Sanchai,3 Khemrassamee Kunsuikmengrai,3 Sumpun Kahintapong,3 Banyong Khantawa,4 Prasit Tharavichitkul,4 Thira Sirisanthana,5 and Tsuyoshi Nagatake1

Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan,1 Service de Microbiologie, CHU Côte de Nacre, 14033 Caen Cedex, France,2 Nakornping Hospital,3 Departments of Microbiology,4 Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand5

Received 6 December 2002/ Returned for modification 12 February 2003/ Accepted 11 March 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The antimicrobial susceptibilities of 30 Rhodococcus equi isolates obtained from 30 patients between 1993 and 2001 in northern Thailand were investigated. The MICs showed a tendency toward resistance to various antibiotics but sensitivity to imipenem, minocycline, vancomycin, and teicoplanin (MICs, <=0.5 µg/ml) and relative sensitivity to meropenem, clarithromycin, and ciprofloxacin (MICs, <=2 µg/ml). Of the 30 isolates, 26 were susceptible (MICs, <=1 µg/ml), 1 showed low-level resistance (MIC, 8 µg/ml), and 3 showed high-level resistance (MICs, >=64 µg/ml) to rifampin. PCR amplification and DNA sequencing of the rpoB gene and molecular typing by pulsed-field gel electrophoresis (PFGE) were performed for eight R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001, including one low- and three high-level rifampin-resistant isolates. As a result, two high-level rifampin-resistant strains with PFGE pattern A had a Ser531Trp (Escherichia coli numbering) mutation, and one high-level rifampin-resistant strain with PFGE pattern B had a His526Tyr mutation, whereas one low-level rifampin-resistant strain with PFGE pattern C had a Ser509Pro mutation. Four rifampin-susceptible strains with PFGE patterns D and E showed an absence of mutation in the rpoB region. Our results indicate the presence of several types of rifampin-resistant R. equi strains among AIDS patients in northern Thailand.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rhodococcus equi, an aerobic, intracellular, gram-positive, acid-fast coccobacillus initially isolated from foals in 1923 as Corynebacterium equi (8), is a well-known pathogen in domestic animals, especially horses, which causes suppurative bronchopneumonia with a high mortality rate in young foals (15). It has been well known that this organism is an important pathogen in immunosuppressed human hosts since the first case of a human infection was reported in 1967 (6). A marked increase in the incidence of infection caused by R. equi has been reported since the human immunodeficiency virus (HIV) disease epidemic began in 1981 (4, 16, 21).

A combination of erythromycin and rifampin is considered to be effective for treating R. equi infections (9, 13, 19). However, the emergence of rifampin resistance in R. equi has been reported (10, 17), although it is still rare. Recently, Fines et al. reported that several mutations in the rpoB gene are associated with rifampin resistance in R. equi isolated from foals, as well as in Mycobacterium tuberculosis, Escherichia coli, and Staphylococcus aureus (5).

However, the issue of whether rifampin-resistant R. equi isolated from humans has such mutations is unclear. To address this issue, we conducted the study described here.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. Thirty R. equi strains were isolated from 22 patients, whose clinical histories, including HIV, were not known in detail, at Chiang Mai University Hospital in northern Thailand between 1993 and 1996 and from 8 AIDS patients who had been admitted to Nakornping Hospital in northern Thailand due to pneumonia or lung abscess caused by R. equi from 1998 to 2001. The strains were detected in sputum (n = 22), blood (n = 5), pleural effusions (n = 2), and gastric juice (n = 1). Cultures were performed using TSA II medium (Becton Dickinson Microbiology Systems, Cockeysville, Md.) supplemented with 7% rabbit blood agar for 24 h at 37°C. The morphology of the colonies was similar to that of Corynebacterium species. Subsequently, R. equi was distinguished from Corynebacterium spp. by its biochemical characteristics, including the fact that it is nitrate positive, phosphatase alkaline positive, and alpha-glucosidase positive.

Antimicrobial susceptibility test. The MIC was determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (12). The susceptibilities of 30 R. equi isolates to the following 20 antibiotics were tested: amikacin (Meiji Seika Kaisha, Tokyo, Japan), ampicillin (Meiji Seika Kaisha), cefazolin (Fujisawa Pharmaceutical Co., Osaka, Japan), cefotiam (Takeda Chemical Industries, Osaka, Japan), ceftriaxone (Nippon Roche Co., Tokyo, Japan), chloramphenicol (Sankyo Co., Tokyo, Japan), ciprofloxacin (Bayer Yakuhin, Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), clindamycin (Pharmacia K.K., Tokyo, Japan), erythromycin (Dainippon Pharmaceutical Co., Osaka, Japan), fosfomycin (Meiji Seika Kaisha), gentamicin (Schering-Plough K.K., Osaka, Japan), imipenem (Banyu Pharmaceutical Co., Tokyo, Japan), meropenem (Sumitomo Chemical Co., Tokyo, Japan), minocycline [Lederle (Japan), Tokyo, Japan], penicillin G (Meiji Seika Kaisha), rifampin (Daiichi Pharmaceutical Co., Tokyo, Japan), teicoplanin (Aventis Pharma, Tokyo, Japan), tetracycline (Lederle), and vancomycin (Shionogi Co., Osaka, Japan).

PCR and DNA sequencing. PCR and DNA sequencing in the rpoB gene were performed for eight R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001 as described previously (5). A set of primers, MF (5'-CGACCACTTCGGCAACCG-3') and MR (5'-TCGATCGGGCACATCCGG-3'), was chosen to amplify a portion of the rpoB region of R. equi that includes the rifampin resistance-determining region.

PFGE. Pulsed-field gel electrophoresis (PFGE) was also performed for eight R. equi isolates as mentioned above in order to determine genetic relatedness, as described previously (11). The DNA was digested with PshBI (Takara Bio Co., Shiga, Japan) at 37°C overnight. A CHEF Mapper pulsed-field electrophoresis system (Bio-Rad Life Science Group, Hercules, Calif.) was used for electrophoresis, with a potential of 6 V/cm, switch times of 0.47 and 63 s, and a run time of 20 h and 18 min. After the gels were stained with ethidium bromide, the interpretation of PFGE patterns was based on criteria described by Tenover et al. (18).


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antimicrobial susceptibility test. The MICs of 20 antibiotics for 30 R. equi isolates showed that the isolates had a tendency to be resistant to various antibiotics but that they were sensitive to imipenem, minocycline, vancomycin, and teicoplanin (MICs, <=0.5 µg/ml) and relatively sensitive to meropenem and ciprofloxacin (MICs, <=2 µg/ml). Of the 30 isolates, 28 showed sensitivity, but 2 isolates detected from 1993 to 1996 showed low-level resistance (MICs, 8 and 2 µg/ml, respectively) to erythromycin and clarithromycin. Twenty-six isolates were susceptible (MICs, <=1 µg/ml), one showed low-level resistance (MIC, 8 µg/ml), and three showed high-level resistance (MICs, >=64 µg/ml) to rifampin (Table 1). The one low- and three high-level rifampin-resistant isolates were detected in four AIDS patients between 1998 and 2001.


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  		TABLE 1. Distribution of MICs of various antibiotics against 30 strains of R. equi in northern Thailand

 
AIDS patients. Eight AIDS patients (seven males and one female; mean age, 32.6 years), who presented with fever, a productive cough, and sputum, were admitted to Nakornping Hospital in northern Thailand due to pneumonia or lung abscess caused by R. equi from 1998 to 2001. The mean CD4 lymphocyte count was 9.4/mm3 (CD4/CD8 ratio, 0.04). Five patients were treated with a combination of erythromycin and rifampin; two patients were treated with antituberculosis drugs, including rifampin; and one patient was treated with a combination of penicillin G and gentamicin. Four patients improved after treatment, three patients died of respiratory failure, and the prognosis of one patient was unknown because of transfer. Two of the three patients who died were infected by high-level rifampin-resistant strains (Table 2).


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  		TABLE 2. Clinical characteristics of R. equi isolates detected in AIDS patients, rifampin MICs, RpoB amino acid substitutions, and PFGE patterns

 
Amino acid sequence analysis and susceptibility to rifampin. Two high-level rifampin-resistant strains had a Ser531Trp mutation and one high-level rifampin-resistant strain had a His526Tyr mutation in the rpoB gene, whereas one low-level rifampin-resistant strain had a Ser509Pro mutation. Four rifampin-susceptible strains showed an absence of mutation in the rpoB region (Table 2).

Interpretation of PFGE. The PFGE patterns in two high-level rifampin-resistant strains with the Ser531Trp mutation were closely related to patterns A1 and A2, but one other high-level rifampin-resistant strain with a His526Tyr mutation showed a different pattern, B, while one low-level rifampin-resistant strain with a Ser509Pro mutation showed pattern C. The PFGE patterns among the four rifampin-susceptible strains with no mutation were different from those of rifampin-resistant strains, but two of four strains revealed identical patterns (D), and the two remaining cases were possibly related to patterns E1 and E2 (Table 2 and Fig. 1).



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  		FIG. 1. PFGE patterns of PshBI-digested DNAs from R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001. The PFGE patterns in two high-level rifampin-resistant strains were the closely related patterns A1 and A2 (lanes 1 and 3), but another high-level rifampin-resistant strain showed a different pattern, B (lane 2). One low-level rifampin-resistant strain showed pattern C (lane 4). The PFGE patterns of two rifampin-susceptible strains were identical (pattern D; lanes 5 and 6), and another two rifampin-susceptible strains showed the possibly related patterns E1 and E2 (lanes 7 and 8).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
R. equi is now recognized as an important pathogen in animals and immunocompromised humans, especially AIDS patients (3, 4, 15). In humans, mortality rates from 25 to 60% in R. equi pneumonia have been reported (3, 19, 20). The introduction of a combination of erythromycin and rifampin for the treatment of R. equi pneumonia in foals has improved survival rates considerably (7) and is also considered to be effective in R. equi infections in humans due to the low MICs, intracellular penetration, and synergistic action (9, 13, 19). The emergence of erythromycin (3, 10) and rifampin (10, 17) resistance in R. equi has been reported, although it is still rare. In our study, antimicrobial susceptibilities (R. equi was found to be resistant to various antibiotics but sensitive to imipenem, minocycline, vancomycin, and teicoplanin) were similar to those in previous studies, except that the susceptibility to aminoglycosides was lower (9, 13). Of 30 R. equi isolates, 2 isolates detected from 1993 to 1996 showed low-level resistance to erythromycin (MIC, 8 µg/ml), while among those detected from 1998 to 2001, one showed low-level resistance (MIC, 8 µg/ml) and three showed high-level resistance (MICs, >=64 µg/ml) to rifampin. In fact, two of three AIDS patients with pneumonia caused by high-level rifampin-resistant R. equi had fatal outcomes despite treatment that included rifampin. R. equi is largely a soil organism but is widespread in the feces of herbivores. Most human patients who have developed R. equi infections are known to have been in contact with herbivores, their manure, or soil (15). It is likely that the eight AIDS patients in our study had such contact, because they lived close to a farm in northern Thailand. On the other hand, AIDS is still prevalent in Thailand, and tuberculosis is the most commonly reported opportunistic infection (2). Therefore, AIDS patients in Thailand have many opportunities for treatment with antituberculosis drugs, including rifampin, and this may explain the selection for rifampin resistance in R. equi as well. In our study, two types of high-level rifampin-resistant strains and one low-level rifampin-resistant strain with mutations in the rpoB gene were confirmed despite their small numbers, and no rifampin-suscepible strains had such mutations. Moreover, molecular analysis by PFGE was consistent with this type of mutation. In a previous study of R. equi isolated from foals, the His526Asp mutation was found to be predominantly associated with high-level rifampin resistance (5). One high-level rifampin-resistant strain in our study had a mutation at position 526, but the amino acid substitution was not the same. However, two high-level rifampin-resistant strains had a Ser531Trp mutation. Although it has been reported that one low-level rifampin-resistant R. equi strain had a mutation at position 531 with a different amino acid substitution (5), a mutation at this position was associated with high-level rifampin resistance in M. tuberculosis (1, 14). Meanwhile, one low-level rifampin-resistant strain in our study showed a mutation at position 509 which had never been reported in R. equi.

In conclusion, our results demonstrated the presence of several types of rifampin-resistant R. equi among AIDS patients in northern Thailand. Therefore, treatment should be considered based on antimicrobial susceptibility and intracellular penetration.


   ACKNOWLEDGMENTS
 
We thank all the staff of the Department of Microbiology, Faculty of Medicine, Chiang Mai University, and of Nakornping Hospital for their help in the completion of this study. We also thank Akihiro Wada (Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University), Chieko Shimauchi (Miyazaki Prefectural Nursing University), and Matsuhisa Inoue (Kitasato University School of Medicine) for their help in the completion of the PFGE studies.

This study was supported by a Monbukagakusho Grant-in-Aid for Scientific Research (09045083) from the Japanese government.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Phone: 81 (95) 849-7842. Fax: 81 (95) 849-7843. E-mail: nori-a{at}net.nagasaki-u.ac.jp. Back


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Journal of Clinical Microbiology, June 2003, p. 2337-2340, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2337-2340.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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