The accumulation of resistance mutations in Mycobacterium tuberculosis in vitro

Abstract number: R2097

Bergval I.L., Schuitema A.R.J., Klatser P., Anthony R.M.

Objectives: In Mycobacterium tuberculosis (MTB), antibiotic resistance is almost exclusively established by point mutations. Multidrug-resistance develops by sequential acquisition of mutations at different loci. Although the acquisition of resistance mutations is random, the genetic routes leading to the development of successful and virulent (multi)drug-resistant bacteria may be quite constrained (Weinreich DM et al. Science 2006) and probably influenced by the genetic background of the strain in question.

We have studied the acquisition of drug resistance mutations in different strains and isogenic mutants to better understand the development of MDR strains.

Methods: Cultures of well-characterised laboratory derived isogenic mutants and clinical isolates were used. Drug-resistant mutants were selected in vitro from either a single large liquid culture or a series of smaller liquid cultures by plating on solid antibiotic-containing medium and mutant colonies characterised using a multiplex ligation probe assay (MLPA) and DNA sequencing.

Results: Different clades have a preference for different distributions of specific rpoB mutations, this effect was most strongly evident when individual colonies were selected from a single large broth (frequency). Less clade specific variation was seen with multiple cultures(rate). Most notably we detected a marked increased in the proportion of rpoB C526T mutations (typically greater than 50%) from strain Mtb72(Group 3 genotype) when selected from a single large broth.

Initial results indicate a possible reduction in the diversity of rpoB resistance genotypes after selection of strains with pre-existing INH resistance.

Conclusion: Strain specific variation in favoured mutations was more evident when multiple colonies are characterised from a single large culture. We believe this indicates that the relative fitness of the different mutations varies based on the strain's genotype. Our results for the variation in distribution of spontaneous rpoB mutations between different clades shows similarity to variations in the distribution of mutations previously reported from clinical isolates within genotypic groups (Lipin MY et al. Clin Microbiol 2007). These results suggest the sequence of mutations leading to a successful MDR strain may indeed be quite constrained with certain mutations in certain genotypes probably being significantly more likely to become multiply resistant.