Kenji Ogawa

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for Typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245 Restriction Fragment Length Polymorphism Typing

ABSTRACT Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, 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 (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). 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.

Characterization of Mycobacterium avium clinical isolates in Japan using subspecies-specific insertion sequences, and identification of a new insertion sequence, ISMav6.

Clinical isolates of 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 mutations 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.

Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium complex isolates

OBJECTIVESnClarithromycin is the key drug in the various treatment regimens of Mycobacterium avium complex (MAC) diseases, and is the only drug for which drug susceptibility has been shown to correlate with clinical response in these diseases. A point mutation at either positions 2058 or 2059 of the 23S rRNA gene has been reported to occur in high-level clarithromycin-resistant isolates. In this study, we examined the correlation between the results from a drug susceptibility test and the mutation of the 23S rRNA gene involved in drug resistance in MAC. Furthermore, we adapted a rapid detection method using amplification refractory mutation system (ARMS)-PCR to identify mutations in the 23S rRNA gene in MAC isolates.nnnMETHODSnUsing a microdilution method based on the NCCLS/CLSI recommendation, the MIC of clarithromycin was determined for 245 clinical MAC isolates. Of these, 219 clarithromycin-susceptible and 26 clarithromycin-resistant strains were analysed by sequencing of the 23S rRNA gene and ARMS-PCR.nnnRESULTSnThe drug susceptibility test revealed a bimodal distribution of MICs for both the susceptible and resistant strains. Sequence analysis of the 23S rRNA gene revealed that all of the clarithromycin-susceptible strains were wild-type whereas 24 of the clarithromycin-resistant strains were mutant type. The sensitivity of the sequence and ARMS-PCR analyses was 92.3% and 84.6%, respectively, and the specificity of both was 100%.nnnCONCLUSIONSnWe found a correlation between MICs of clarithromycin and 23S rRNA gene mutations. ARMS-PCR for 23S rRNA mutations of MAC isolates is useful for rapid detection of clarithromycin resistance.

Molecular typing of Mycobacterium intracellulare using multilocus variable-number of tandem-repeat analysis: identification of loci and analysis of clinical isolates

In addition to its known status as a disseminated disease in HIV-positive patients, Mycobacterium avium complex (MAC) is increasingly recognized as a causative pathogen of respiratory disease in HIV-negative patients. 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, of which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 50 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter Gastons 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 are highly discriminating and stable over time.

INHIBITION OF CYCLIC GMP PHOSPHODIESTERASE BY XANTHINE DERIVATIVES RELAXES GUINEA‐PIG TRACHEALIS SMOOTH MUSCLE

1. For the purpose of clarifying the mechanism of the airways smooth muscle relaxant action of xanthines, cyclic guanosine monophosphate (GMP) phosphodiesterase (PDE) from guinea‐pig trachealis muscle was purified with diethylaminoethyl ether (DEAE) cellulose column chromatography.

High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae.

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials.