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 shortcourse 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 g of PZA per ml) and one PZase-positive strain (strain 9739) (MIC, >300 g 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 Arg1403Ser. 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.

Characterization ofpncAMutations of Pyrazinamide-ResistantMycobacterium tuberculosisin Korea

“…These M. tuberculosis isolates have been observed previously (17,21). In addition, PZA-resistant isolates retaining PZase activity, which are expected to carry a normal pncA gene, have also been reported (21,26,27).…”

“…More than 200 PZA-susceptible M. tuberculosis strains failed to show any mutations of the gene including silent substitutions (18)(19)(20)23). In contrast, diverse mutations at various sites of the pncA gene were identified in PZA-resistant strains and the frequencies of mutation varied among studies (75-100%) (17)(18)(19)(20)(21)(22)(23). In this study of Korean isolates, the frequency of pncA mutation appeared to be very high (97%) in PZA-resistant M. tuberculosis.…”

“…Mutation of the pncA gene was suggested as the major mechanism of PZA resistance. Several subsequent studies including 20-40 PZAresistant M. tuberculosis isolates from different countries confirmed the close correlation between pncA mutation and PZA resistance (72-97%) (17)(18)(19)(20)(21)(22)(23). These observations raised the possibility of utilizing DNA-based methods as an alternative PZA susceptibility test.…”

“…In addition, recent reports have suggested that mutations in the pncA gene could be specific to subtypes of M. tuberculosis (23) and/or to the geographic source of the isolate (17). In this study, the pncA gene sequence was characterized in 95 multidrug resistant, PZase negative M. tuberculosis strains isolated from Korean patients to define any possible geographic preferences for mutations in the gene, and to evaluate the utility of a DNAbased detection test for PZA-resistant M. tuberculosis isolates in Korea.…”

A New Approach for Pyrazinamide Susceptibility Testing inMycobacterium tuberculosis

“…This cycle is repeated, resulting in a lethal combination of POA accumulation and intracellular acidification, causing disruption of membrane permeability and cellular damage. 3,17,19 The major mechanism of PZA resistance in M. tuberculosis arises from mutations in the PZAse, resulting in a loss of enzymatic activity, preventing conversion of PZA to POA. 17 The same mechanism is responsible for the natural resistance to PZA of Mycobacterium bovis and Mycobacterium kansasii.…”