Shiomi Yoshida

Clinical and Microbiological Differences between Mycobacterium abscessus and Mycobacterium massiliense Lung Diseases

ABSTRACT In recent years, many novel nontuberculous mycobacterial species have been discovered through genetic analysis. Mycobacterium massiliense and M. bolletii have recently been identified as species separate from M. abscessus. However, little is known regarding their clinical and microbiological differences in Japan. We performed a molecular identification of stored M. abscessus clinical isolates for further identification. We compared clinical characteristics, radiological findings, microbiological findings, and treatment outcomes among patients with M. abscessus and M. massiliense lung diseases. An analysis of 102 previous isolates of M. abscessus identified 72 (71%) M. abscessus, 27 (26%) M. massiliense, and 3 (3%) M. bolletii isolates. Clinical and radiological findings were indistinguishable between the M. abscessus and M. massiliense groups. Forty-two (58%) patients with M. abscessus and 20 (74%) patients with M. massiliense infections received antimicrobial treatment. Both the M. abscessus and M. massiliense groups showed a high level of resistance to all antimicrobials, except for clarithromycin, kanamycin, and amikacin. However, resistance to clarithromycin was more frequently observed in the M. abscessus than in the M. massiliense group (16% and 4%, respectively; P = 0.145). Moreover, the level of resistance to imipenem was significantly lower in M. abscessus isolates than in M. massiliense isolates (19% and 48%, respectively; P = 0.007). The proportions of radiological improvement, sputum smear conversion to negativity, and negative culture conversion during the follow-up period were higher in patients with M. massiliense infections than in those with M. abscessus infections. Patients with M. massiliense infections responded more favorably to antimicrobial therapy than those with M. abscessus infections.

Population Structure Analysis of the Mycobacterium tuberculosis Beijing Family Indicates an Association between Certain Sublineages and Multidrug Resistance

ABSTRACT Our population-based study of the Mycobacterium tuberculosis Beijing family examined the frequency of occurrence of each sublineage of this family, classified by using 10 synonymous single-nucleotide polymorphisms. The results revealed the overabundance of two evolutionary sublineages in a population of multidrug-resistant and extensively drug-resistant tuberculosis bacteria.

Genetic diversity of Mycobacterium avium subsp. hominissuis strains isolated from humans, pigs, and human living environment.

Mycobacterium avium subsp. hominissuis (MAH) strains are genetically diverse and cause infections in pigs and humans. To elucidate the geographical and host-dependent variations in the genetic diversity of MAH, we performed variable numbers of tandem repeat (VNTR) analysis targeting 19 loci for MAH samples from humans (n=146), bathroom environments (n=37), and pigs (n=75) in Japan; these data were then compared with previously reported VNTR data from other countries. The minimum spanning tree (MST) and the multi-dimensional scaling (MDS) analyses based on the VNTR data indicated a high degree of genetic relatedness between isolates from humans and bathrooms in Japan, but a low degree of similarity with the isolates from France and Finland. Moreover, the comparison showed a higher similarity of isolates from Japanese pigs with those from French humans and pigs and Finnish humans and pigs than with other isolates from humans and bathrooms in Japan. The singularity of the Japanese MAH was characterized as the prevalence of hsp65 sequevar code 15 and ISMav6 for the human and bathroom isolates; however, none of the isolates obtained from the pigs belonged to the code 15 or possessed ISMav6. The genetic diversity of MAH and its regional variations imply a possible regional or local specific source of infection and route of transmission of MAH for humans.

Population Structure Dynamics of Mycobacterium tuberculosis Beijing Strains during Past Decades in Japan

ABSTRACT We used 909 strains to compare the population structures of the Mycobacterium tuberculosis Beijing family between different birth-year cohorts in Japan. The results revealed that the spread of a modern sublineage that has high transmissibility is currently increasing, while the spread of an ancient sublineage, STK, has significantly decreased in younger generations.

Comprehensive multicenter evaluation of a new line probe assay kit for identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis.

ABSTRACT We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n = 316), Mycobacterium avium (n = 71), Mycobacterium intracellulare (n = 51), Mycobacterium kansasii (n = 54), and other Mycobacterium species (n = 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.

Comparison of rifabutin susceptibility and rpoB mutations in multi-drug-resistant Mycobacterium tuberculosis strains by DNA sequencing and the line probe assay

We compared rifabutin susceptibility and rpoB mutations in 98 multi-drug-resistant strains of Mycobacterium tuberculosis (MDR-TB) by DNA sequencing and with a line probe assay using the commercially available INNO-LiPA Rif. TB kit (the LiPA). Our results indicated that rifabutin continues to remain active against MDR-TB strains harboring certain genetic alterations and also that the LiPA might be useful in identifying MDR-TB strains susceptible to rifabutin.

Investigation of the population structure of Mycobacterium abscessus complex strains using 17-locus variable number tandem repeat typing and the further distinction of Mycobacterium massiliense hsp65 genotypes

Mycobacterium abscessus complex is a significant pathogen in patients with non-cystic fibrosis (non-CF). Nevertheless, there is little description of the genetic diversity of this species. The aims of this study were to investigate the distribution of M. abscessus complex isolated from respiratory specimens by variable number tandem repeat (VNTR) typing. The results of 104 clinical isolates from 104 non-CF patients were compared using PFGE, hsp65 genotypes and clarithromycin susceptibility. The allelic diversity (Hunter-Gaston Discriminatory Index) of the 17 loci examined by VNTR typing was high (0.977). We determined that C28 sequevar erm(41) genotypes and clarithromycin-acquired resistance isolates were scattered in the minimum spanning tree. Intriguingly, VNTR typing and PFGE were highly congruent and revealed that there were clear examples of grouping of isolates from different individuals amongst both M. abscessus and M. massiliense, and showed five clusters of distinct identical isolates. Within these clusters, M. massiliense hsp65 type I formed three different clusters. Although the distribution of M. massiliense hsp65 type II-1 was low (9.3 %), M. massiliense hsp65 type II-1 isolates separated from clusters contained hsp65 type I isolates. Thus, M. massiliense hsp65 genotypes could be discriminated by analysing VNTRs with sufficient genetic distance for intra-species-level discrimination.

Population Structure and Local Adaptation of MAC Lung Disease Agent Mycobacterium avium subsp. hominissuis

Abstract Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.

Intra-subspecies sequence variability of the MACPPE12 gene in Mycobacterium avium subsp. hominissuis

The PE (Pro-Glu) and PPE (Pro-Pro-Glu) multigene families are unique to mycobacteria, and are highly expanded in the pathogenic members of this genus. We determined the intra-subspecies genetic variability of the MACPPE12 gene, which is a specific PPE gene in Mycobacterium avium subsp. hominissuis (MAH), using 334 MAH isolates obtained from different isolation sources (222 human isolates, 145 Japanese and 77 Korean; 37 bathroom isolates; and 75 pig isolates). In total, 31 single-nucleotide polymorphisms (SNPs), which consisted of 16 synonymous SNPs and 15 nonsynonymous SNPs, were determined through comparison with the MACPPE12 gene sequence of MAH strain 104 as a reference. As the result, the 334 MAH isolates were classified into 19 and 13 different sequevars at the nucleic acid level (NA types) and amino acid level (AA types), respectively. Among the 13 AA types, only one type, the AA02 type, presented various NA types (7 different types) with synonymous SNPs, whereas all other AA types had a one-to-one correspondence with the NA types. This finding suggests that AA02 is a longer discernible lineage than the other AA types. Therefore, AA02 was classified as an ancestral type of the MACPPE12 gene, whereas the other AA types were classified as modern types. The ubiquitous presence of AA02 in all of the isolation sources and all different sequevars classified by the hsp65 genotype further supports this classification. In contrast to the ancestral type, the modern types showed remarkable differences in distribution between human isolates and pig isolates, and between Japanese isolates and Korean isolates. Divergence of the MACPPE12 gene may thus be a good indicator to characterize MAH strains in certain areas and/or hosts.

Association between sequevar and antibiotic treatment outcome in patients with Mycobacterium abscessus complex infections in Japan

Purpose. Macrolide susceptibility differs between subspecies in the Mycobacterium abscessus complex, likely due to differences in erm(41) sequevars. Patients with M. abscessus complex infection generally show poor clinical outcomes in response to antibiotic treatment. Here, the association between genotype and treatment outcome was investigated. Methodology. We collected 69 isolates from 35 patients with non‐cystic fibrosis bronchiectasis: 24 had M. abscessus complex lung disease and non‐cystic fibrosis bronchiectasis, and 11 were colonized. Outcome analysis was performed in the 24 infected patients. Molecular analyses, including erm(41) and rrl sequencing, and variable‐number tandem‐repeat (VNTR) analysis of 69 isolates, from 24 infected and 11 colonized patients, were performed to elucidate the influence of genotype on antibiotic susceptibility. Results. Among the 24 patients, 18 (14 infected with M. abscessus subsp. abscessus and 4 with M. abscessus subsp. massiliense) showed unfavourable outcomes; six (three infected with M. abscessus subsp. abscessus and three with M. abscessus subsp. massiliense) exhibited favourable outcomes. Patients with unfavourable outcomes showed acquired clarithromycin resistance (33.3 vs 0 %), mixed sequevars (38.9 vs 16.7 %) and differing VNTR patterns between initial and serial isolates (33.3 vs 16.7 %). In contrast, in the 11 colonized patients, M. abscessus subsp. abscessus C28 (sequevar 02) and M. abscessus subsp. massiliense were the most prevalent subspecies. Conclusion. Patients infected with multiple sequevars and genotypes were more likely to exhibit treatment failure and/or recurrence. The precise identification of subspecies and analyses of mycobacterial characteristics may help to predict treatment outcomes in patients with M. abscessus complex lung disease.