Re-growth of Mycobacterium tuberculosis populations exposed to antibiotic combinations is due to the presence of isoniazid and not bacterial growth rate.

Modulation of growth rate in Mycobacterium tuberculosis is key to its survival in the host; particularly with regard to its adaptation during chronic infection when the growth rate is very slow. The resulting physiological changes will influence the way this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of frontline antimycobacterials, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), using continuous cultures. Time-course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (23.1. mean generation time (MGT)) or slow growth rate (69.3h MGT), were analysed with the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat, and profiled kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a higher kill rate associated with INH (compared to RIF or PZA) and the appearance of re-growth. In line with this observation, re-growth was not observed with RIF-exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate re-growth. We consider here that faster, early bactericidal activity is not what is required for successful sterilisation of M. tuberculosis, but instead slower elimination of bacilli followed by reduced recovery of the bacterial population.

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.