澤井 宏太郎

Pulmonary disease caused by non-tuberculous mycobacteria (NTM-PD) is an emerging global health concern. Among NTM, Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of NTM-PD. Similar to Mycobacterium tuberculosis , MAH exhibits lineage-specific geographical distributions and host adaptations. Here, we characterized three MAH strains from the residential bathrooms of Mycobacterium avium complex pulmonary disease patients in Japan. A genetic population clustering analysis revealed that the three strains belong to the East Asia (EA) lineages that are predominant in Japan and Korea. Pan-genome analysis using the publicly available complete genome sequences of MAH and the newly sequenced MAH strains identified 3,313 core genes that are conserved among distinct MAH lineages. Identification of essential genes in the three strains was conducted using transposon insertion sequencing, and their gene essentiality profiles were compared to those of a previously studied sequence cluster 3 (SC3) lineage strain, MAC109. Despite their genetic diversity, nearly all essential genes were derived from the core gene set. In addition, we identified a set of common essential genes for the EA and SC3 lineages, as well as lineage-specific essential genes. Our results highlight the evolutionary and clinical importance of lineage-specific adaptations in MAH.

Abstract Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus . We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr , helR, and MAB_2807 . By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis , we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific β-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies. Importance Rifamycins are cornerstone antibiotics for the treatment of mycobacterial infections, yet they are ineffective against Mycobacterium abscessus . Although several key resistance mechanisms have been described, the full genetic basis of intrinsic rifamycin resistance remains to be elucidated. Here, we used transposon insertion sequencing to systematically identify genes that contribute to intrinsic rifamycin resistance in M. abscessus . Because recent studies have shown that chemical modification of rifamycins can overcome specific intrinsic resistance mechanisms, a comprehensive understanding of these determinants could facilitate the development of next-generation rifamycins with improved activity against M. abscessus . In addition, we unexpectedly found that rifamycin exposure alters cell envelope structure, the intracellular levels of a key metabolic intermediate in peptidoglycan biosynthesis, and susceptibility to certain β-lactams in M. abscessus . These findings may help guide the rational development of combination therapeutic strategies with next-generation rifamycins.

OBJECTIVE: Mycobacterium lentiflavum is a rare, non-tuberculous mycobacterium (NTM) which is implicated in some cases of active, pulmonary non-tuberculous mycobacterial disease. The outbreak of NTM in nosocomial settings occasionally occurs and outbreak investigation with implementation of concurrent countermeasure is essential. DESIGN: Outbreak investigation. SETTING: A tertiary care medical center. PATIENTS AND PARTICIPANTS: Hospitalized patients during the outbreak period. RESULTS: In April 2024, a cluster of patients with Mycobacterium lentiflavum-positive sputum cultures, presumed to be due to nosocomial transmission, was identified at the study center. A retrospective review of cases dating back to February 2023 revealed 27 patients with M. lentiflavum infection whose isolates had initially not been speciated. According to the American Thoracic Society (ATS) criteria for diagnosing pulmonary non-tuberculous mycobacterial (NTM) disease, two of these patients met the criteria for active disease. Multi-locus sequence typing of 12 isolates demonstrated 100% clonality, indicating a common source. A concurrent outbreak investigation identified contaminated faucet aerators in hospital wards as the likely source of transmission. All faucet aerators and caps were manually cleaned and disinfected using liquid sodium hypochlorite, after which no further cases were detected. CONCLUSION: The present study described a nosocomial cluster of M. lentiflavum colonization and infections at a tertiary care center, with contaminated faucet aerators identified as the likely source. Prompt identification of such NTM clusters in healthcare settings is essential to initiate timely treatment and prevent further transmission.

In Japan, outbreaks of H5N8 highly pathogenic avian influenza (HPAI) were reported between November 2020 and March 2021 in 52 poultry farms. Understanding HPAI epidemiology would help poultry industries improve their awareness of the disease and enhance the immediate implementation of biosecurity measures. This study was a simulation-based matched case-control study to elucidate the risk factors associated with HPAI outbreaks in chicken farms in Japan. Data were collected from 42 HPAI-affected farms and 463 control farms that were within a 5-km radius of each case farm but remained uninfected. When infected farms were detected as clusters, one farm was randomly selected from each cluster, considering the possibility that the cluster was formed by farm-to-farm transmission within an epidemic area. For each case farm, up to three control farms were selected within a 5-km radius. Overall, 26 case farms (16 layer and 10 broiler farms) and 75 control farms (45 layer and 30 broiler farms) were resampled 1000 times for the conditional logistic regression model with explanatory variables comprising geographical factors and farm flock size. A larger flock size and shorter distance to water bodies from the farm were found to increase infection risk in layer farms. Similarly, in broiler farms, a shorter distance to water bodies increased infection risk. On larger farms, frequent access of farm staff and instrument carriages to premises could lead to increased infection risk. Waterfowl visiting water bodies around farms may also be associated with infection risk.