However, the PZase assay was still useful for screening PZA-resistant M. tuberculosis isolates and could be used as an alternative method, particularly for low-income countries where the assay was highly sensitive. The major mechanism of PZA resistance was associated with mutations of the gene coding for pyrazinamidase, pncA, in which mutations were scattered along the coding and promoter regions with high diversity [7]. In this study, mutations were found in 49 isolates, of which 39 were PZA-resistant and 10 were PZA-susceptible. However, 17 Selleck AZD3965 isolates (7 PZA-resistant and 10 PZA-susceptible isolates) showed either Ile31Ser or

Ile31Thr mutations. Of these, 15 isolates (except 2 PZA-resistant isolates) had PZase activity. Previous studies have demonstrated the catalytic residues of M. tuberculosis PZase that comprise the active (Asp-8, Trp-68, Lys-96, Ser-104, Ala-134, Thr-135 and

Cys-138) and metal-binding sites (Asp-49, His-51 and His-71) [30–32]. Taken together with our results, the click here mutation at Ile-31 did not appear to be associated with PZA resistance. Notably, two PZA-resistant isolates harboured the Ile31Ser mutant but possessed no PZase activity. One possible scenario is that these 2 isolates might have PZase activity that is below the limit of detection for the PZase assay. Twenty-two of 24 mutation types were detected in this study and showed a correlation selleck with PZA resistance (Table 2). Of these, 14 nucleotide substitutions Ponatinib order [13, 14, 29, 33–36] and 2 putative

promoter region [9, 33] mutations were previously reported. There were 6 novel mutation types, consisting of 3 nucleotide substitutions (Leu27Pro, Gly122Ser, and Thr174Ile), 2 nucleotide insertions (G insertion between nucleotide 411 and 412 and GG insertion between nucleotide 520 and 521), and 1 nonsense mutation at Glu127. In agreement with earlier studies, the mutations were diverse and scattered throughout the gene sequence, with the most frequently occurring mutation being His71Asp (8/49 = 16%). This is not surprising, as His71 is located in one of the three preferably mutated regions (positions 3 to 17, 61 to 76, and 132 to 142) [37] and in the metal-binding site. In addition, there were 13 PZA-resistant isolates (25%) with observed PZase activity and no mutations in pncA, implying that other unknown mechanisms are involved in PZA resistance. Conclusions This study showed the prevalence of PZA resistance in pan-susceptible and MDR-TB M. tuberculosis clinical isolates from Siriraj Hospital, Thailand. MDR-TB isolates had a much higher percentage of PZA resistance (49%) than susceptible isolates (6%). In this study, the sensitivities of the PZase assay and pncA sequencing were 65% and 75%, respectively. The results revealed that 25% of PZA-resistant isolates had wild-type pncA, indicating that phenotypic susceptibility testing was still necessary.