Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia.

BACKGROUND: The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region. METHODS: Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates. RESULTS: Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94-32 and 100-32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region. CONCLUSION: In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

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

OBJECTIVES: The 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. METHODS: Spontaneous 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. RESULTS: Results confirm that the in vitro mutation rate for isoniazid resistance (3.2 x 10(-7) mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 x 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. CONCLUSIONS: These 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.

Specific mutations in the Mycobacterium tuberculosis rpoB gene are associated with increased dnaE2 expression.

In Mycobacterium tuberculosis (MTB), rifampicin resistance is almost invariably due to mutations in the rpoB gene, whose function is critical for cell viability. Most of these mutations, at least initially, impair the fitness of the bacteria but confer a selective advantage when antibiotic pressure is exerted. Subsequent adaptation may be critical to restore fitness. The possibility was considered that MTB with mutations in the rpoB gene elicits a constitutive stress response, increasing the probability of subsequent adaptation. In order to test this hypothesis, the expression of recA and dnaE2, an inducible putative error-prone DNA polymerase, was determined in six different isogenic laboratory-generated rpoB-mutants of MTB. Expression levels were determined with real-time PCR and the data obtained were compared with those of the wild-type parent. In four of the six rpoB mutants, a two- to fivefold induction of dnaE2 was detected (P<0.05). Thus, the presence of specific mutations in rpoB is not only associated with impaired fitness but also results in a detectable, moderate yet persistent increase in the expression of dnaE2 but not recA.

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency.

BACKGROUND: Mutations 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. METHODS: Mycobacterium 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 microg/ml but remained sensitive to rifabutin (MIC<0.8 microg/ml). This mutant was subjected to a second round of selection with rifabutin. RESULTS: All 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. CONCLUSION: First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.