Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

Abstract

The distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.Rifampin (RIF) is a key drug in the four-drug treatment regimen of tuberculosis. It interacts with the ␤-subunit of the RNA polymerase and inhibits the early steps of transcription. Resistance to RIF is almost entirely coupled (Ͼ97%) to mutations within an 81-bp region of the rpoB gene, called the RIF resistance-determining region (RRDR) (5, 23). Mutations in the rpoB gene of Mycobacterium tuberculosis clinical isolates were analyzed by Telenti et al. (23), and many studies have since followed (1,4,7,13,21,22). A few studies of in vitrogenerated RIF resistance mutations in M. tuberculosis have also been conducted; however, these were limited to the H37Rv reference strain and a single clinical isolate (3,14,16).Strains of the M. tuberculosis Beijing genotype have been spreading increasingly and are often associated with multidrugresistant (resistance to at least RIF and isoniazid) tuberculosis (6, 15, 17-19, 24, 25). Isolates of this genotype have been shown not to have a higher in vitro mutation rate for the development of RIF resistance (27). A few clinical studies have analyzed RIF-resistant Beijing isolates, for which no clear differences in occurring RRDR mutations compared to those of other genotypes have been seen (8,18,19,24,25). There are, however, no data on in vitro-selected rpoB mutations in Beijing strains.In the present study, we wanted to investigate the frequency and distribution of rpoB mutations occurring in vitro among several independent M. tuberculosis parental strains and subsequently compare these to available data on clinical isolates. Additionally, we determined whether Beijing genotype mutants display differences in the types and/or the frequencies of rpoB mutations compared to those of mutants of non-Beijing genotypes. Finally, we wanted to measure the levels of resistance associated with the various RRDR mutations.By use of the Luria and Delbrück fluctuation assay (12), 189 RIF-resistant mutants were previously selected (27). By setting a series of parallel cultures from a single parent strain, this method selects spontaneously occurring, independent mutational events in nonselective broth. Then, by inoculation of the cultures on selective media (2 mg/liter RIF), the mutants are obtained. In all, nine fully drug-susceptible parent strains with unique DNA fingerprint patterns (26) were used to select RIFresistant mutants. Eight were clinical isolates, of which one was a Dutch Harlingen isolate (11) and one was the H37Rv (ATCC 25618) reference strain. Based on their spoligotype ...

Spread of a Low-Fitness Drug-Resistant Mycobacterium tuberculosis Strain in a Setting of High Human Immunodeficiency Virus Prevalence

“…These isolates clustered with the atypical Beijing strains from the WC which had a nsSNP at codon 516 (GAC3GTC) and the Ϫ17 inhA promoter mutation, suggesting interprovincial spread. This finding was contrary to previous reports, which suggested that atypical Beijing strains are attenuated in their ability to transmit (9,14), while the mutation at codon 516 (GAC3GTC) would have been expected to further compromise the ability of these strains to transmit unless compensatory mutations were present or the 4 529 CGA3CCA 1 CGA3CAA 1 CGA3GGA 1 531 TCG3TGG 17 8 2 3 3 2 2 TCG3TTG 132 169 110 54 5 58 42 5 11 TCG3CAG 3 3 TCG3TTC 1 1 (4,7,10). c Under-represented in the in vitro generated rifampin-resistant mutants (z-test ͓P Ͻ 0.001͔).…”

“…d Over-represented in the in vitro-generated rifampin-resistant mutants (z-test ͓P Ͻ 0.001͔). e Eleven different deletion events (7,10 epidemiological context allowed transmission to occur. Analysis of the host population in the EC region showed human immunodeficiency virus (HIV) coinfection to be a risk factor for the spread of the atypical Beijing strains (z-test for the hypothesis that a proportion of HIV-positive cases ϭ 0.42, P ϭ 0.029).…”

Mutations in rpoB and katG genes in Mycobacterium isolates from the Southeast of Mexico

“…A similar mutation at this codon (S→L) was recently reported to correlate with a low level of resistance in clinical isolates and to be associated with a bacterial fitness deficit (Mariam et al 2004, Huitric et al 2006. Thus, this isolate could help in understanding the real value of this codon change in the biology of Mycobacterium and the sensitivity of the BACTEC system for the diagnosis of DR-TB.…”

Insights into the processes that drive the evolution of drug resistance in Mycobacterium tuberculosis

“…For instance, some mutations in rpo B gene, such as rpo B S531L, H526Y, H526D and H526R, are often associated with high levels of rifampicin resistance, while mutations including rpo B L511P, H526L, H526N, L533P and I572F are generally linked to low levels of rifampicin resistance (Huitric et al., 2006; Van Deun et al., 2009). Similar to that, kat G mutations are often associated with high levels of isoniazid resistance, whereas inh A mutations with low levels (Fenner et al., 2012; van Soolingen et al., 2000; Vilcheze & Jacobs, 2014).…”

“…For instance, the predominant mutations associated with high level of drug resistance and a low or no biological cost, such as kat G S315T, rpo B S531L, rps L K43R and gyr A D94G (conferring resistance to isoniazid, rifampicin, streptomycin and fluoroquinolones respectively), are more frequently found in clinical drug‐resistant isolates (Billington et al., 1999; Bottger et al., 1998; Campbell et al., 2011; Casali et al., 2014; Gagneux, Burgos, et al., 2006; Gagneux, Long, et al., 2006; Mariam et al., 2004; Pym et al., 2002). Indeed, some of these resistance mutations do not reduce bacterial fitness in the absence of treatment (Bergval, Schuitema, Klatser, & Anthony, 2009; Huitric et al., 2006; Mariam et al., 2004). It is worth noting that MDR and XDR strains associated with outbreaks often carried these mutations explaining the successful spread of these highly drug‐resistant strains in the community (Casali et al., 2014; Cohen et al., 2015; Niehaus, Mlisana, Gandhi, Mathema, & Brust, 2015; de Vos et al., 2013).…”