Abstract
Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10−8 to 2.49×10−7) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10−7, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10−8). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.
Highlights
The emergence, spread and persistence of drug resistance inhibits the successful treatment and control of tuberculosis (TB).In contrast to many other bacterial pathogens, the etiological agent of tuberculosis, Mycobacterium tuberculosis, does not acquire antimicrobial resistance via horizontally transferred plasmids or other mobile genetic elements, but almost exclusively via the acquisition of point mutations or, occasionally, through genomic deletions [1,2]
Acquired isoniazid resistance caused constrained genetic pathways in vitro Six isoniazid (INH)-resistant strains, carrying different drugresistance mutations, and their two parent strains were used for these experiments; laboratory-generated mutant strains H15, H26, H48, H71, H103 and their parent strain MTB72, and clinical isolate 2001–2184 and its isogenic INH-resistant clinical isolate 2001–2185 (Table 1)
Most of the RIF-resistant mutants carried the rpoB-S531L mutation when method 2 was used; for all strains, except 2001–2185, which had acquired an equal number of H526Y mutants (7/17 (41%)), this was the category with the highest number of representatives (Table 3)
Summary
The emergence, spread and persistence of drug resistance inhibits the successful treatment and control of tuberculosis (TB).In contrast to many other bacterial pathogens, the etiological agent of tuberculosis, Mycobacterium tuberculosis, does not acquire antimicrobial resistance via horizontally transferred plasmids or other mobile genetic elements, but almost exclusively via the acquisition of point mutations or, occasionally, through genomic deletions [1,2]. Genetic characterisation of drug-resistant clinical isolates indicates that only a small range of mutations is responsible for the majority of resistance in clinical isolates; screening for only 3 to 5 mutations in the rpoB gene detects more than 80% of all clinical M. tuberculosis isolates with resistance to rifampicin (RIF), a critical component of any successful anti-TB treatment and a marker for multidrug resistance [4,5,6,7] The majority of these mutations are located within an 81-bp region of rpoB, the gene that encodes the b-chain of RNA polymerase [8]. RIF resistance is often used as a proxy to study antibiotic resistance in the laboratory
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