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Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis.

ABSTRACT: Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDR_PZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p _-138) and the N terminus of ClpC1 (ClpC1_Val63Ala). Mutations have emerged in both these regions under POA selection in vitro, and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1_Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1_Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDR_PZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1_Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

SUBMITTER: Modlin SJ

PROVIDER: S-EPMC8315952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

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Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis.

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