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Antimicrobial Agents and Chemotherapy, September 2000, p. 2291-2295, Vol. 44, No. 9
0066-4804/00/$04.00+0

Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis

Glenn P. Morlock,1,* Jack T. Crawford,1 W. Ray Butler,1 Suzanne E. Brim,1 David Sikes,1 Gerald H. Mazurek,2 Charles L. Woodley,1 and Robert C. Cooksey1

Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases,1 and Division of Tuberculosis Elimination, National Center for HIV, STD, and TB Prevention,2 Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 11 June 1999/Returned for modification 27 August 1999/Accepted 5 June 2000

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were >= 100 µg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 µg/ml, 3 had the same mutation (Thr47right-arrowAla) and 18 had the wild-type sequence. For the three Thr47right-arrowAla mutants PZA MICs were 12.5 µg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 µg of PZA per ml and the third was resistant to 800 µg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to >= 100 µg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47right-arrowAla mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to >= 100 µg of PZA per ml.

* Corresponding author. Mailing address: 1600 Clifton Rd., Mail Stop F08, Atlanta, GA 30333. Phone: (404) 639-1280; Fax: (404) 639-1287. E-mail: gpm0{at}cdc.gov.

Antimicrobial Agents and Chemotherapy, September 2000, p. 2291-2295, Vol. 44, No. 9
0066-4804/00/$04.00+0


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