Genetic variation in Mycobacterium tuberculosis isolates from a London outbreak associated with isoniazid resistance.

BACKGROUND: The largest outbreak of isoniazid-resistant (INH-R) Mycobacterium tuberculosis in Western Europe is centred in North London, with over 400 cases diagnosed since 1995. In the current study, we evaluated the genetic variation in a subset of clinical samples from the outbreak with the hypothesis that these isolates have unique biological characteristics that have served to prolong the outbreak. METHODS: Fitness assays, mutation rate estimation, and whole-genome sequencing were performed to test for selective advantage and compensatory mutations. RESULTS: This detailed analysis of the genetic variation of these INH-R samples suggests that this outbreak consists of successful, closely related, circulating strains with heterogeneous resistance profiles and little or no associated fitness cost or impact on their mutation rate. CONCLUSIONS: Specific deletions and SNPs could be a peculiar feature of these INH-R M. tuberculosis isolates, and could potentially explain their persistence over the years.

Clinical use of whole genome sequencing for Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB) remains a major challenge to global health and to healthcare in the UK. In 2014, a total of 6,520 cases of TB were recorded in England, of which 1.4 % were multidrug-resistant TB (MDR-TB). Extensively drug-resistant TB (XDR-TB) occurs at a much lower rate, but the impact on the patient and hospital is severe. Current diagnostic methods such as drug susceptibility testing and targeted molecular tests are slow to return or examine only a limited number of target regions, respectively. Faster, more comprehensive diagnostics will enable earlier use of the most appropriate drug regimen, thus improving patient outcomes and reducing overall healthcare costs. Whole genome sequencing (WGS) has been shown to provide a rapid and comprehensive view of the genotype of the organism, and thus enable reliable prediction of the drug susceptibility phenotype within a clinically relevant timeframe. In addition, it provides the highest resolution when investigating transmission events in possible outbreak scenarios. However, robust software and database tools need to be developed for the full potential to be realized in this specialized area of medicine.

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315.

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50-90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.