pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis: spread of a monoresistant strain in Quebec, Canada

Abstract

Pyrazinamide (PZA) is an important first-line tuberculosis drug that is part of the currently used short-course tuberculosis chemotherapy. PZA is a prodrug that has to be converted to the active form pyrazinoic acid by pyrazinamidase (PZase) activity, encoded by the pncA gene of Mycobacterium tuberculosis, and loss of PZase activity is associated with PZA resistance. To further define the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we sequenced the pncA gene from a panel of 59 PZA-resistant clinical isolates from Canada, the United States, and Korea. Two strains that did not contain pncA mutations and had positive PZase turned out to be falsely resistant. Three PZase-negative strains (MIC, >900 microgram of PZA per ml) and one PZase-positive strain (strain 9739) (MIC, >300 microgram of PZA per ml) did not have pncA mutations. The remaining 53 of the 57 PZA-resistant isolates had pncA mutations, confirming that pncA mutation is the major mechanism of PZA resistance. Various new and diverse mutations were found in the pncA gene. Interestingly, 20 PZA-monoresistant strains and 1 multidrug-resistant isolate from Quebec, Canada, all had the same pncA mutation profile, consisting of an 8-nucleotide deletion and an amino acid substitution of Arg140-->Ser. Strain typing indicated that these strains are highly related and share almost identical IS6110 patterns. These data strongly suggest the spread of a PZA-monoresistant strain, which has not previously been described.

FIG. 1

IS6110 strain typing analysis of PZA-resistant M. tuberculosis strains. Lanes 1 and 2, strains T63168 and M52997, respectively, which share the same mutation of Gly97→Ser. Lanes 3 and 4, H2374 and H1033, respectively, which share the same mutation of nucleotide G insertion at position 420. Lanes 5 to 8, 10350, 10003, 9155, and 10257, which all share the same pncA mutation profile of an 8-bp deletion at nucleotide position 446 followed by Arg140→Ser.