CHARACTERIZATION OF PNCA MUTATIONS IN PYRAZINAMIDE-RESISTANT MYCOBACTERIUM-TUBERCULOSIS

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy, Resistance to PZA is usually accompanied by loss of pyrazinam idase (PZase) activity in Mycobacterium tuberculosis. PZase converts P ZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance, The gene (pncA) encoding the M. tuberc ulosis PZase has recently been sequenced, and mutations in pncA were p reviously found in a small number of PZA-resistant M. tuberculosis str ains, To further understand the genetic basis of PZA resistance and de termine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants mad e in vitro, Thirty-three of 38 PZA-resistant clinical isolates had pnc A mutations. Among the five strains that did not contain pncA mutation s, four were found to be falsely resistant and one was found to be bor derline resistant to PZA, The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleoti de substitutions, insertions, or deletions in the pncA gene, The ident ified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Th r142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation poly morphism (SSCP) analysis was shown to be useful for the rapid detectio n of pncA mutations in the PZA-resistant strains, We conclude that a m utation in the pncA gene is a major mechanism of PZA resistance and th at direct sequencing by PCR or SSCP analysis should help to rapidly id entify PZA-resistant M. tuberculosis strains.