Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance

Abstract

ObjectivesThe high prevalence of isoniazid-resistant Mycobacterium tuberculosis is often explained by a high mutation rate for this trait, although detailed information to support this theory is absent. We studied the development of isoniazid resistance in vitro, making use of a laboratory strain of M. tuberculosis.MethodsSpontaneous isoniazid-resistant mutants were characterized by molecular methods allowing identification of the most commonly encountered resistance-conferring mutations. Additionally, we determined the in vitro mutation rates for isoniazid and rifampicin resistance, and characterized the genome of a triple-resistant strain.ResultsResults confirm that the in vitro mutation rate for isoniazid resistance (3.2 × 10−7 mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 × 10−9 mutations/cell division). However, in the majority of the in vitro mutants katG was partially or completely deleted and neither of the two most common in vivo mutations, katG-S315T or inhA-C(-)15T, were found in 120 isogenic mutants. This implies that clinically prevalent resistance mutations were present in <0.8% of isoniazid-resistant strains selected in vitro (95% CI 0%–2.5%). The triple-resistant strain had acquired isoniazid resistance via a 49 kbp deletion, which included katG. Apart from previously identified resistance-conferring mutations, three additional point mutations were acquired during sequential selection steps.ConclusionsThese outcomes demonstrate that the in vivo mechanism of isoniazid resistance is not reflected by in vitro experiments. We therefore conclude that the high in vitro mutation rate for isoniazid resistance is not a satisfactory explanation for the fact that isoniazid monoresistance is significantly more widespread than monoresistance to rifampicin.

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

“…We observed a biphasic pattern of killing in our in vitro persistence experiment. However, we observed less bactericidal activity of Inh compared to Rif, which might be associated with tolerance of metabolically less active persisters against Inh (67)(68)(69). We also observed that polyP deficiency is associated with increased susceptibility of M. tuberculosis to both Inh and Levo, therefore, suggesting that accumulation of polyP in M. tuberculosis induces a metabolic state that enables the bacteria to persist better in the presence of drugs.…”

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

“…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).…”

“…As demonstrated by Bergval et al. (Bergval et al., 2009), drug resistance mutation patterns of in vitro selected‐resistant mutants do not always reflect mutation profiles obtained in clinical isolates. Indeed, mutations associated with high biological cost of resistance detected in in vitro drug‐resistant mutants are rarely found in clinical drug‐resistant isolates (Bergval et al., 2009; Gagneux, Long, et al., 2006; Huitric et al., 2006).…”

“…(Bergval et al., 2009), drug resistance mutation patterns of in vitro selected‐resistant mutants do not always reflect mutation profiles obtained in clinical isolates. Indeed, mutations associated with high biological cost of resistance detected in in vitro drug‐resistant mutants are rarely found in clinical drug‐resistant isolates (Bergval et al., 2009; Gagneux, Long, et al., 2006; Huitric et al., 2006). For example, mutations in the kat G gene that lead to complete loss of the catalase‐peroxidase enzyme function (conferring resistance to isoniazid), such as frame‐shift nucleotide deletions or insertions, are found less frequently in clinical than in in vitro mutants (Bergval et al., 2009).…”

Therapeutic Potential of Gnidia Capitata L. F.: Investigations on Its Antityrosinase, Antibacterial, Antioxidant and Anticancer Activities

“…Perhaps, different solvents used for extraction could be one of the possible explanations for the differences in anti-TB activities recorded in the present study. The H37Rv isolate was earlier found to be resistant against some reference drugs including rifampicin and isoniazid, in addition to some other first line and second line drugs (Bergval et al, 2009). Growth inhibition of H37Rv strains by some of the G. capitata extracts reported is an indication that this species could be used as a good anti-TB agent against drug resistant strains of M. tuberculosis.…”