Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para-Aminosalicylic Acid Resistance

Single nucleotide polymorphisms (SNPs) involved in the development of resistance represent powerful markers for the rapid detection of first- and second-line resistance in clinical Mycobacterium tuberculosis complex (MTBC) isolates. However, the association between particular mutations and phenotypic resistance is not always clear-cut, and phylogenetic SNPs have been misclassified as resistance markers in the past. In the present study, we investigated the utility of a specific polymorphism in thyA (Thr202Ala) as a marker for resistance to para-aminosalicyclic acid (PAS). Sixty-three PAS-susceptible MTBC strains comprising all major phylogenetic lineages, reference strain H37Rv, and 135 multidrug-resistant (MDR) strains from Germany (comprising 8 PAS-resistant isolates) were investigated for the presence of Thr202Ala. In both strain collections, the Thr202Ala SNP was found exclusively in strains of the Latin American Mediterranean (LAM) lineage irrespective of PAS resistance. Furthermore, PAS MICs (0.5 mg/liter) for selected LAM strains (all containing the SNP) and non-LAM strains (not containing the SNP), as well as the results of growth curve analyses performed in liquid 7H9 medium in the presence of increasing PAS concentrations (0 to 2.0 mg/liter), were identical. In conclusion, our data demonstrate that the Thr202Ala polymorphism in thyA is not a valid marker for PAS resistance but, instead, represents a phylogenetic marker for the LAM lineage of the M. tuberculosis complex. These findings challenge some of the previous understanding of PAS resistance and, as a consequence, warrant further in-depth investigations of the genetic variation in PAS-resistant clinical isolates and spontaneous mutants.para-Aminosalicylic acid (PAS) was one of the first drugs used in the treatment of tuberculosis (TB) but initially caused severe side effects, resulting in poor patient compliance. Thanks to a new formulation of the drug leading to improved gastrointestinal tolerance (5), PAS has since then become one of the components in second-line drug treatment to which even highly drug-resistant isolates are susceptible (3, 12, 16).Rengarajan et al. (15) first reported that PAS targets the folate pathway: selection for PAS resistance using transposon mutagenesis led to the isolation of strains with transposon-disrupted thyA (Rv2764c) which exhibited a reduced thymidylate synthase activity. Unlike the wild-type thyA from Mycobacterium bovis BCG (MtbBCG), the expression of the mutated thyA gene failed to rescue a thyA-negative phenotype in Escherichia coli. Similarly, only the expression of the wild-type MtbBCG thyA in the same E. coli background restored sensitivity to trimethoprim (TMP) (15).Complementing these results, two mutations in thyA (Thr202Ala in strains R1 and R3 and Arg222Gly in strain R2) were identified in clinical PAS-resistant Mycobacterium tuberculosis strains. Both alleles failed to fully restore sensitivity to TMP in the thy-negative E. coli background described above. Lastly, the effect of expressing the wild-type thyA gene as well as both clinical alleles in one of their original transposon-disrupted thyA MtbBCG mutants was investigated. Again, only the expression of the wild-type gene completely restored susceptibility to PAS (15).The involvement of thyA in PAS resistance has also been supported by a study of clinical strains from China. Of the 51 PAS-sensitive and 44 PAS-resistant strains investigated, only the resistant strains showed mutations in thyA. However, these accounted for only 36% of the phenotypic resistance, pointing to one or several unknown resistance mechanisms (20).In the attempt to identify further the mechanism leading to PAS resistance, six genes of the folate and thymidine biosynthetic pathways have been analyzed by Mathys et al. (11). Although some mutations were identified in these genes, they did not appear to be relevant for PAS resistance. Instead, the detection of 25 distinct mutations in thyA reaffirmed the role of this gene. Notably, the Thr202Ala mutation discussed earlier was the mutation most frequently identified in 23 clinical isolates and in 1 spontaneous mutant. It was predicted to have a structurally destabilizing effect (11).Our initial analysis of all previously sequenced M. tuberculosis complex (MTBC) strains revealed that this single nucleotide polymorphism (SNP) was present in the following strains: 98-R604, F11, GM 1503, KZN 506, KZN 605, KZN 1435, KZN 2475, and KZN 4207. Given that these eight genomes encompass different subfamilies of the Latin American Mediterranean (LAM) genotype, on the basis of their spoligotype patterns, which were found to accurately define this group (1), this indicated that thyA Thr202Ala might be a specific mutation or even a marker for this lineage.To investigate this striking finding and to define the actual role of thyA Thr202Ala in PAS resistance development, we carried out a more systematic investigation. Sequence analyses of a fragment of the thyA gene of MTBC strains of two different collections, one comprising 63 PAS-susceptible MTBC strains of all major phylogenetic lineages and reference strain H37Rv and the other comprising 135 multidrug-resistant (MDR) strains from Germany, 8 of which were PAS resistant, was carried out. In addition, PAS MIC determinations and growth curve analyses in 7H9 medium with increasing PAS concentrations were performed for selected strains with and without the mutation.