Detection Of Novel Gene Mutations Associated With Pyrazinamide Resistance In Multidrug-Resistant Mycobacterium Tuberculosis Clinical Isolates In Southern China

Objective: Pyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis.Methods: Drug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA (Rv2044c-pncA-Rv2042c) and PZase assay were performed for 448 M tuberculosis clinical isolates.Results: Our data showed that among 448 M. tuberculosis clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drugresistance. A total of 60.04% (269/448)M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZA(R) isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZA(R). Compared with phenotypic data, 84.38% (227/269) PZA(R) strains with mutations in pncA(+) upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C-114del) in upstream of pncA(wt) nullified the effect of A-11G mutation and induced positive PZase activity, divergent from five PZase negative A-11G PZA(R) mutants. Twenty-six PZA(R) strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms.Conclusion: Our study revealed that PZA(R) rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA(+)UFR, rpsA, panD, Rv2783c, and Rv2044c provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.