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Mycobacterium tuberculosis (Mtb) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original Mtb isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent Mtb are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of Mtb Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.

Background Each of the currently available (1→3)-β-D-glucan (BDG) measurement kits follows a different measurement method and cut-off value. Comparisons of diagnostic performance for invasive fungal infections (IFIs) are desirable. Additionally, ecological considerations are becoming increasingly important in the development of new measurement kits. Methods The plasma BDG levels in clinical samples were measured using the following currently available kits: the Fungitec G test MKII, the Fungitec G test ES, Fungitell, the β-Glucan test Wako, and the newly developed Wako kit (Wako-Eu). Wako-Eu uses a pre-treatment solution that conforms to European regulations for the registration, evaluation, authorisation, and restriction of chemicals. The values obtained for the samples using each kit were studied and compared. Results Of the 165 patients evaluated, 12 had IFIs, including pneumocystis pneumonia, aspergillosis, and candidiasis. BDG values obtained using the kits were moderately correlated with each other. Clinical diagnoses of the evaluated cases indicated that 21 false positives were diagnosed by at least one kit. The sensitivity of the Fungitell kit was relatively low, but those of the other four were over 90%. The specificity was above 90% for all kits. For positive predictive value, the Wako and the Wako-Eu methods were superior to the others owing to fewer false positive results. Conclusions The newly developed Wako-Eu method, which considers ecological concerns, shows diagnostic performance equivalent to that of its predecessor. To improve the diagnostic accuracy of IFIs, it is necessary to interpret the results carefully, giving due consideration to the characteristics of each measurement kit.

DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria’s eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria.