PZA has remarkable sterilizing activity on the so-called persistent bacterial population.37 Although the antimycobacterial activity of PZA has been known for 7 decades, its mechanism of action is not yet fully understood (Fig 238‐40). The main mechanism of PZA-R in mycobacteria relies on the inactivation of the nicotinamidase/pyrazinamidase enzyme (PncA, encoded by the pncA gene), physiologically involved in the metabolism of nicotinamide and responsible of the conversion of PZA to its active form (pyrazinoic acid).38,41 In general, the frequency of mutations at pncA in PZA-resistant isolates ranges between 72% and 98%.37 Mutations affecting the pncA gene are mainly missense, single nucleotide changes causing amino acid substitutions. However, nonsense mutations, insertions, and deletions are not rare. Few substitutions occur also in the promoter region of the gene. The variety of the mutations occurring in the pncA gene of PZA-resistant isolates is demonstrated by > 400 different single nucleotide polymorphisms (SNPs) described in 78 published studies. Further complexity is added by insertions and deletions, and by the fact that new studies, such as that by Napiórkowska and colleagues,42 are describing additional mutations. Although these mutations are scattered across entire gene (encoding a protein of 187 amino acids), some degree of clustering seems to occur in three regions of the PncA protein: positions 3-17, 61-85, and 132-142.37 Indeed, loss of pyrazinamidase activity is associated with mutations affecting the active site (eg, D8, W68), disrupting the protein core (eg, Q10, I6, V44, V139, M175, F94, H137) and directly or indirectly interfering with the coordination of the iron (II) ion (eg, D49, H51, H57, H71).43,44 The iron (II) ion is required for the correct binding and positioning of PZA at the active site of the PncA enzyme, and depletion of metal ions abrogates pyrazinamidase activity.43,44 It should be noted that some mycobacterial species, including M bovis and M canettii, are intrinsically resistant to PZA.45 Enzymatic activity seems unaffected by mutations at specific codons, including 102, 171, and 12.46‐48 Recently, mutations unrelated to drug resistance have been identified and proposed as phylogenetic markers49; thus, a better correlation between SNPs and drug-resistant phenotype is needed.