Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

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.