title: [Genetic research about Mycobacterium avium complex].
abstract: A variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, Clin Microbiol.) Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections. Mycobacterium avium (n=81) from patients with pulmonary infections who were HIV-negative and isolates (n=33) from HIV-positive patients were subjected to genetic analysis by PCR detection of three M.avium-specific insertion sequences (IS901, IS1245 and IS1311) and nucleotide sequencing of the heat-shock protein 65 (hsp65) gene. All clinical isolates were identified as 'M. avium subspecies hominissuis' by sequence analysis of hsp65. Compared with clinical isolates of M. avium reported elsewhere, IS1245 was found less frequently in Japanese isolates (96/114 isolates, 84%) and IS901 was detected more frequently (76/114 isolates, 67%). One isolate was found to lack IS1311, which has not been reported previously for 'M. avium subsp. hominissuis.' Nucleotide sequence analysis of the PCR products for IS901 revealed that all clinical isolates had the same new insertion sequence, designated ISMav6, which had 60 point mutation compared with the nucleotide sequence of the original IS901. These results suggest that 'M. avium subsp. hominissuis' with ISMav6 is prevalent in Japan. ISMav6 may have implications for the virulence of M. avium and contribute to an increase of M. avium infections in this country. Clarithromycin (CAM) is the key drug in the various treatment regimens of Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility has been shown to correlate with a clinical response in these diseases. A point mutation at position 2058 or 2059 of the 23S rRNA gene has been reported to occur in high-level CAM-resistant isolates. This study examined a correlation between the results from a drug susceptibility test and the mutation of genes involved in drug resistance in MAC. Furthermore, we adapted a rapid detection method using amplification refractory mutation system (ARMS)-PCR to identify a mutation in 23S rRNA gene in MAC isolates. Using MICs of CAM, drug susceptibility was tested for 253 clinically-isolated MAC strains. Of these, 28 CAM-sensitive strains and 26 CAM-resistant strains were analysed by sequence analysis and ARMS-PCR. The drug susceptibility test showed that a bimodal distribution was associated with 227 CAM-sensitive strains and 26 CAM-resistant strains. Sequence analysis revealed that all 28 CAM-sensitive strains tested were wild type, whereas 24 of the 26 CAM-resistant strains were mutant type. The sensitivity of the sequence and ARMS-PCR analyses were 92.3% and 84.6%, respectively, and specificity of both was 100%. We found a correlation between drug susceptibility testing and the mutation of drug-resistant genes in this study. ARMS-PCR allowed rapid determination of CAM resistance, even when samples consisted of the mixed type of CAM-sensitive and CAM-resistant strains. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 49 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter-Gaston discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay highly discriminating and stable over time.
authors.affiliation: Department of Clinical Research, National Hospital Organization, Higashinagoya National Hospital, 5-101, Umemorizaka, Meito-ku, Nagoyashi, Aichi 465-8620, Japan. ogawak@toumei.hosp.go.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Ogawa
mesh_terms: D017291:Clarithromycin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D018598:Minisatellite Repeats; D015269:Mycobacterium avium Complex / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D017291:Clarithromycin