The history and characteristics of isolates maintained at the Leprosy Research Center

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1 Jpn J Lepr 79, (2010) The history and characteristics of isolates maintained at the Leprosy Research Center Masanori MATSUOKA * Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan [Received / Accepted: 22 June, 2010] Key words : genotype, Leprosy Research Center, M. leprae strains, susceptibility to anti-leprosy drugs The origin, history, sensitivity to anti-leprosy drugs and genotypic characteristics for 27 strains maintained at the Leprosy Research Center in Tokyo are described. Strains are isolated and passaged in nude mouse footpads, and frozen bacillary suspensions with different generations are also maintained. The Leprosy Research Center provides bacillary materials as experimental resources at researchers request. Introduction Materials and Methods The Leprosy Research Center (LRC) has been providing Mycobacterium leprae grown in nude mice for research since the 1980 s. The Thai-53 strain is mainly used for supplying bacillary material and is used for many studies not only in Japan but also by researchers in other countries 1). Strains other than Thai-53, which have been succeeded from other laboratories or isolated by the LRC, are also maintained at the LRC. The origin, history, genotypic characteristics and sensitivity to antileprosy drugs of the isolates are described. *Corresponding author : Leprosy Research Center National Institute of Infectious Diseases 4-2-1, Aobacho, Higashimurayama-shi, Tokyo, , Japan. TEL : FAX : matsuoka@nih.go.jp Of 27 strains, 7 were isolated by two other researchers and provided as previously inoculated nude mouse footpads, and 20 strains were obtained from biopsied tissue sent to the LRC. Materials were kept in a freezer at -84 C until bacillary suspension preparation or else inoculated without freezing. Bacillary suspensions for footpad inoculation were prepared by Nakamura s method 2). Bacilli grown in nude mouse footpads were recovered when footpad swelling was recognized after about one year, passaged to the next generation and frozen as aliquots suspended in Hanks balanced salt solution. The origin of each strain, date obtained, form of material and provider are described in Table 1. All isolates have been succeeded to and are maintained by Dr. M. Kai at the LRC. 247

2 Susceptibility to anti-leprosy drugs was determined by the mouse footpad method as described elsewhere 3, 4, 5, 6). Susceptibility to dapsone, rifampicin, ofloxacin, sparfloxacin, clofazimine, clarithromycin, and minocycline was examined. Mutations in the drug resistance determining region (DRDR) of the folp1, rpob and gyra genes, which confer resistance to dapsone, rifampicin and quinolone, respectively, were determined by direct PCR sequencing 6). Genotypes of variable number tandem repeats (VNTRs) were determined according to the method described in our previous study using bacilli grown in nude mouse footpads 7). Single nucleotide polymorphisms (SNPs) were ascertained according to the method of Monot et al 8). Results and Discussion Origins of the strains. The disease type and nationality of the patient, date obtained, form of the material, generation and provider are described in Table 1. Of 27 isolates, 7 strains were provided as inoculated nude mouse footpads which showed swelling due to sufficient bacillary growth. The others were inoculated bacillary suspensions prepared at the LRC from tissue biopsied. The bacilli that first grew in the inoculated footpad were designated as 1 st generation. Three isolates are from Thais, one is from an Indonesian, another one is from a Korean and the other 22 strains are from Japanese. There are 15 samples from new cases, 11 from relapsed cases and one from an intractable case. Of the 27 cases, 23 are from lepromatous leprosy patients (LL type) and 4 are from borderline lepromatous patients (BL type) (Table 1). Susceptibility to anti-leprosy drugs. Susceptibility to 7 anti-leprosy drugs was examined for 21 strains by the mouse footpad method. Out of 21 strains, 10 are susceptible to all anti-leprosy drugs, 9 isolates are resistant to dapsone, 7 isolates are resistant to rifampicin and 3 isolates are resistant to quinolone (Table 2). Among these strains, 1 isolate is intermediate dapsone resistance 6), 2 isolates are resistant to three drugs such as dapsone, rifampicin and quinolone 3, 4) and 4 strains are resistant to dapsone and rifampicin. Two strains are resistant to only dapsone, while the others are resistant to rifampicin or quinolone. All isolates examined are susceptible to clofazimine, clarithromycin, and minocycline. Mutations in the DRDR of the folp1, rpob and gyra genes. All 9 dapsone-resistant strains harbor a mutation at amino acid 53 or 55 in the folp1 gene. Three kinds of mutations were detected: 53 Thr (ACC) Ile (ATC) 3), 55 Pro (CCC) Leu (CTC) 5), and 55 Pro (CCC) Ser (TCC) 4). Of 6 rifampicinresistant strains, 4 harbor mutation 425 Ser (TCG) Leu (TTG) 3, 4) in the DRDR of the rpob gene. Additionally, one strain harbors mutation 410 Asp (GAT) Try (TAT) 5) and another one has mutation 420 His (CAC) Try (TAC) in the DRDR of the rpob gene (Table 3). Airtaku-3 tested as resistant to rifampicin by the mouse footpad method, but no mutations were found in the DRDR of the rpob gene. Genotypes of VNTRs and SNPs Representative VNTRs and SNPs for the isolates are shown in Table 4. All isolates originating from Okinawa, and the isolates originating from Amami, Thailand and Indonesia have the 3-copy genotype of a 6bp tandem repeat (3 copy type); almost all of the isolates from the main island of Japan, Honshu, and Korea have the 4-copy genotype of a 6bp tandem repeat (4 copy type) 9). The genotypes 248

3 Jpn J Lepr 79, (2010) of other VNTRs have been reported in studies on the usefulness of genotyping in transmission analysis 10, 11). The SNP genotype of all isolates from the main island is type III (Table 4). Two isolates from Okinawa and two isolates from Thailand have SNP type I. One isolate from Thailand and another Indonesian isolate have SNP type II. Acknowledgement The author greatly appreciates the provision of original bacillary materials by the doctors listed in Table 1. References 1) Matsuoka M: The history of Mycobacterium leprae Thai-53 strain. Lepr Rev 81: 137, ) Nakamura M: Elimination of contaminants in a homogenate of nude-mouse footpad experimentally infected with Mycobacterium leprae. Jpn J Lepr 64: 47-50, 1994 (in Japanese) 3) Matsuoka M, KashiwabaraY, Namisato Y: A Mycobacterium leprae isolate resistant to dapsone, rifampin, ofloxacin, and sparfloxacin. Int J Lepr Other Mycobact Dis 68: , ) Matsuoka M, Kashiwabara Y, Zhang L, Goto M, Kitajima S: A second case of multidrugresistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy. Int J Lepr Other Mycobact Dis 71: , ) Zhang L, Matsuoka M, Namisato M: A mutation at codon 516 in the rpob gene of Mycobacterium leprae confers resistance to rifampin. Int J Lepr Other Mycobact Dis 72: , ) Matsuoka M: Drug resistance in leprosy. Jpn J Infect Dis 63: 1-7, ) Zhang L, Budiawan T, Matsuoka M: Diversity of potential short tandem repeats in Mycobacterium leprae and application for Molecular typing. J Clin Microbio 43: , ) Monot M, Honore N, Garmier T, Araoz R, Copee JY, Lacroiz C, Sow S, Spencer JS, Truman RW, Williams DL, Gelber R, Virmond M, Flageul B, Cho SN, Ji B, Paniz-Mondolf A, Convit J, Young S, Fine PE, Rasolofo V, Brennan PJ, Cole ST: On the origin of leprosy. Science 308: , ) Matsuoka M., Maeda S, Kai M, Nakata N, Chae G, Gillis TP, Kobayashi K, Izumi S, Kashiwabara Y: Mycobacterium leprae typing by genomic diversity and global distribution of genotypes. Int J Lepr Other Mycobact Dis 65: , ) Groathouse NA, Rivoire B, Kim H, Lee H, Cho S-N, Brennan PJ, Vissa. VD: Multiple polymorphic loci for molecular typing of Mycobacterium leprae. J Clin Microbiol 42: , ) Zhang L, Budiawan T, Matsuoka M: Diversity of potential short tandem repeats in Mycobacterium leprae and application for Molecular typing. J Clin Microbiol 43: ,

4 250 Table 1. History of strains.

5 Jpn J Lepr 79, (2010) 251

6 252

7 Jpn J Lepr 79, (2010) Table 2. Susceptibility to anti-leprosy drugs examined by mouse footpad method. 253

8 Table 3. Mutations in drug resistance-determining regions of folp1, rpob and gyra genes. 254

9 Jpn J Lepr 79, (2010) Table 4. Genotypes of VNTRs and SNPs 255

10 ハンセン病研究センターにおいて分離継代されているらい菌株の由来と性状 * 松岡正典国立感染症研究所ハンセン病研究センター受付掲載決定 :2010 年 6 月 22 日キーワード : 遺伝子型ハンセン病研究センターハンセン病治療薬剤感受性らい菌株 nude マウス footpad において増殖しハンセン病研究センターにおいて継代されている計 27 株についてその由来薬剤感受性 drug resistance determining region の変異 single nucleotide polymorphisms (SNPs) および variable number tandem repeats (VNTRs) を報告した * Corresponding author: 国立感染症研究所ハンセン病研究センター東京都東村山市青葉町 TEL: FAX: matsuoka@nih.go.jp 256

National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi; 2

National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi; 2 Jpn. J. Infect. Dis., 65, 52-56, 2012 Original Article SNP Genotypes of Mycobacterium leprae IsolatesinThailandand Their Combination with rpot and TTC Genotyping for Analysis of Leprosy Distribution and