Abstract
BackgroundMutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.MethodsMycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.ResultsAll 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.ConclusionFirst round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.
Highlights
It has been estimated that one third of the World's population is infected with Mycobacterium tuberculosis (MTB) resulting in 2 million deaths annually
Mutations in the 81-bp locus of the MTB rpoB gene were detected in cluster I in all 105 first round resistant mutants that were sequenced (Table 1); no mutations were detected in the parent strain and two blinded wild type isolates included in this panel as sequencing controls
No mutations were detected in the two parent strains (Mtb72 and R190) sequenced as controls
Summary
It has been estimated that one third of the World's population is infected with Mycobacterium tuberculosis (MTB) resulting in 2 million deaths annually. Annals of Clinical Microbiology and Antimicrobials 2005, 4:9 http://www.ann-clinmicrob.com/content/4/1/9 spontaneous mutation, as no horizontal transfer of genetic elements carrying a resistance genotype has been described. For this reason MTB strains or sub-populations with an unusual spectrum or rate of mutations are of considerable interest [1,2,3] as potentially they are more prone to develop resistance to antimicrobial drugs. Mutations within an 81-bp locus of MTB rpoB have been seen in almost all (> = 95%) rifamycin resistant isolates, whether they be clinical [4] or laboratory generated mutants [5]. Second round selection resulted in a changed frequency of specific mutations
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