At this codon, the substitution from AGC to ACC leading to the amino acid change serine to threonine (S to T), seen in 166 (74.1%) isolates. In addition, a single nucleotide polymorphism (SNP) from AGC (S) to AAC (N) was seen in 9 isolates; and from AGC (S) to ACG (L) was noted for 3 isolates. In other regions of the katG gene, substitution SNPs were identified at codons 258, 299 and 300 (Table 1). We also screened for check details mutations in oxyR-ahpC and inhA (ORF and regulatory) gene loci previously reported to be associated with INH resistance. Mutations were also identified including in oxyR-ahpC (8.9%, n = 20 isolates), inhA regulatory gene region (9.8%, n

= 22 isolates), and inhA ORF gene region (1.3%, n = 3 isolates) (see Table 1). Figure 1 depicts correlation of MIC level with frequencies of individual mutations and cumulative mutations. As shown, 99.8% of isolates with

MIC Nutlin3a ≤ 8 μg/mL present at least one mutation. The data suggest that with increasing MIC levels, the assessed mutations could account for or is associated with an increasingly greater proportion of isolates having the quantified resistance MIC level. Table 1 Mutations identified in 224 INH resistant M. tuberculosis isolates from South America   Specific mutation in each loci (number of isolates with mutation)   katG only OxyR-ahpC only inhA (reg) only inhA (ORF) only KatG and inhA (reg) KatG and ahpC No mutation* Brazil (176) S315T (121) S315N (5) S315I (3) G258D*** (1) STK38 C(-15)T (1) I20I (1)**/*** C(-39)T (3) C(-30)T (1) G(-6)A (2) G(-32)A (1) C(-15)T PF-02341066 molecular weight (7) G(82)R*** (1) W300R***/C(-15)T (1) S315T/C(-15)T (8) S315N/I20I**/***

(1) G299S/G(-9)A (1) S315T/G(-48)A (1) 17 Peru (34) S315T (19) S315N (2) C(-10)T (1) C(-15)T (3) S(94) R*** (1) S315T/C(-15)T (1) S315N/C(-10)A*** (1) S315T/C(-10)A*** (3) S315T/C(-15)T (1) 2 Argentina (14) S315T (9) C(-15)T (1) C(-10)T (1) — S(93)A*** (1) S315T/C(-15)T (1) — 1 Total 224 N = 160 N = 12 N = 10 N = 3 N = 11 N = 8 N = 20 *No mutation in studied loci. **Silent mutation in the codon 20 of the ahpC gene. ***Not reported in the literature. Figure 1 Correlation or MIC levels and percentage of strains bearing the studied mutations in Kat G, ahp C and inh A gene loci. Cumulative percent at each MIC level is derived by the number of isolates with any of the assessed mutations divided by all isolates × 100. Country specific mutation frequency The proportion of M. tuberculosis isolates with any katG mutation in the different countries was; Brazil (81.3%, n = 143), Peru (82.4%, n = 28), and Argentina (71.4%, n = 10) (p > 0.05); and the S315T katG mutation was: Brazil (74.4%, n = 131), Peru (73.5%, n = 25), and Argentina (71.4%, n = 10). Spoligopatterns The INH resistant M. tuberculosis isolates (n = 224) were spoligotyped and segregated in strain families in which 86 different spoligotype patterns were identified.