Single nucleotide polymorphisms and other mutations have been associated with drug resistance in M. tuberculosis. Although some mutations are well-known and explain the many drug resistance cases, they do not explain all cases.
Pyrazinamide
Pyrazinamide (PZA) is an important first-line drug used to treat tuberculosis (TB) and has helped shorten the duration of chemthorapeutic regimens for TB. Unfortunately, reported cases of PZA-resistance in M.tuberculosis are on the rise, especially among MDR/XDR cases. PZA Drug Susceptibility Testing (DST) suffers from a relatively high false-resistance rate, motivating investigation into alternative susceptibility tests. This study provides a comprehensive assessment of a potential molecular diagnostic platform for detection of PZA resistance in M/XDR TB cases.
A set of 224 resistant and 72 susceptible, mostly MDR/XDR, clinical M. tuberculosis isolates from four high TB-burden countries were subjected to DST, confirmatory Wayne’s PZase assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, panD, and their promoters were investigated for their potential role as molecular markers of PZA resistance.
We report 90% sensitivity, but only 65% specificity for a pncA-based molecular platform. Adding rpsA and panD only provides potential markers for an additional 2% of resistant cases. Heterogeneity in pncA had a strong association with resistance and should be evaluated as a diagnostic tool. This was not the case in rpsA and panD. Further analysis indicates lineage-specific mutation patterns in pncA and that Euro-American isolates are most likely to escape molecular detection.
Overall, this study contributes to the body of knowledge regarding the mechanism of PZA resistance, finds pncA not sufficiently sensitive or specific in MDR/XDR cases as a sole basis for molecular diagnostics, and highlights the need for identification of alternative mechanism(s) of resistance beyond rpsA and panD.
Rifampicin
Rifampicin (RIF) is a first-line drug used to treat Mycobacterium tuberculosis (M. tuberculosis). Resistance to RIF is most often associated with chromosomal mutations in the rpoB gene, especially in the 81-bp “hot spot” region called the rifampicin-resistance-determining region (RRDR). This study considered 347 isolates (303 resistant, 44 susceptible) from India, Moldova, Philippines, and South Africa; there were 7 isoniazid-monoresistant isolates, 18 multi-drug resistant (MDR), 41 pre-extensively drug resistant (pre-XDR), 242 XDR, 29 pan-susceptible, and 9 with other variable drug profiles . In this study, the rpo complex (rpoA, rpoB, and rpoC) and the promoter regions of three efflux pump genes (emrB, pstB, and drrA) were investigated for the molecular basis of RIF resistance and associated compensatory mutations. We found a molecular basis for RIF resistance in 295 (97.4%) RIF resistant (RIFR) isolates while 36 (81.8%) RIF susceptible (RIFS) isolates harbored no resistance conferring mutations in the six genes. One novel nonsynonymous SNP (Ser450Phe) was identified in RRDR of one RIFR isolate, while eight (2.6%) RIFR isolates lacked a molecular explanation for their resistance in the six genes. Eight (18.2%) RIFS isolates harbored mutations reported to confer RIF resistance. In the efflux pump genes, one particular mutation in the promoter region of drrA, G3243630A, was found in 41 (13.5%) RIFR isolates and six (13.6%) RIFS isolates. Of these, 38 RIFR and one RIFS also had a mutation in the RRDR known to confer resistance. This mutation was also harbored by one (of the eight) RIFR isolates otherwise lacking a resistance-conferring mutation. Occurrence of the mutation among 6 susceptible isolates (two of which tested as resistant with site DST) may then be a result of the resistance level nearing the DST threshold.
Fluoroquinolone
Fluoroquinolones (FQs) are important second-line bactericides used in the treatment of Mycobacterium tuberculosis (M. tuberculosis). Due to the increasing prevelence of multi-drug resistance in new (3.3%) and previously treated (20%) Tuberculosis, use of fluoroquinolones to treat M. tuberculosis is increasing. In this study, we have performed susceptibility testing for ofloxacin (OFX) and moxifloxacin (MOX) and whole genome sequencing (WGS) on 331347 clinical M. tuberculosis isolates from India, Moldova, Philippines, and South Africa. Minimum inhibitory concentration (MICs) was performed for isolates that lacked canonical mutations. We have also collected previously published MIC levels associated with gyrA and gyrB mutations and compared them to MIC levels of our isolates without canonical mutations. A total of 82 different variants (34 SNPs, 4 insertions and 44 deletions) were observed in gyrA among all isolates. In gyrB, 55 variants (26 SNPs, 5 insertions and 24 deletions) were observed amongst all isolates in gyrB. We have determined the distribution of known-resistance conferring mutations in gyrase subunits gyrA and gyrB and searched for novel genomic correlates of FQ-resistance. We have identified novel gyrA and efflux pump mutations associated with MOX and OFX resistance. Susceptibility results were highly concordant between the two drugs despite recent recommendations to treat OFX resistant isolates with MOX. Isolates harboring mutations in QRDR consistently demonstrated high MOX MIC levels relative to OFX MIC levels, suggesting differential efficacy between the two drugs. Twenty-nine isolates had no known resistance conferring mutations but were phenotypically resistant. In these isolates, two novel variants within the gyrA QRDR and two outside the gyrB QRDR were discovered. Lineage analysis revealed that Asp94Gly was overrepresented in the East Asian lineage and Ala90Val in the Euro-American lineage. This study provides possible alternative mechanisms of resistance in isolates with unexplained FQ resistance and contributes to the growing information pool correlating MIC levels to mutations in gyrA and gyrB.
Injectables
We are investigating the association of specific mutations in rrs, eis promoter, and tlyA to amikacin, kanamycin, and capreomycin susceptibility testing. These are known mechanisms of resistance and contribute to the rapid spread of XDR-TB.
Phylogenetics
Whole-genome sequencing offers a high-resolution view of phylogenetics. Constructing phylogenetic trees with such data helps us study patterns of evolution of drug resistance in M tuberculosis.
Phylogeny
Lineage-specific differences in the Mycobacterium tuberculosis complex (MTBC) play an important role in virulence and emergence of drug-resistance. Recent research indicates that genome-wide single nucleotide polymorphisms (SNPs) allow for highly sensitive lineage and sublineage differentiation. We analyzed isolates collected from India, Moldova, South Africa, the Philippines, and Sweden and utilized Pacific Biosciences’ (PacBio) single molecule sequencing technology for our Whole Genome Sequencing, making this one of the first studies to use long read sequencing in order to include Mtb’s repetitive genomic regions in a phylogenetic analysis.