This population-based study was used to investigate strong associations between phenotypes and genotypes.

Hypervariable loci should be included in standardized panels because they can provide consistent comparable results in multiple settings.

e Virulence factors (VFs) contribute to the emergence of new human Mycobacterium tuberculosis strains, are lineage dependent, and are relevant to the development of M. tuberculosis drugs/vaccines. VFs were sought within M. tuberculosis lineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity. Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX, caeA, and ponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozyme in vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across, M. tuberculosis lineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established. Human and animal tuberculosis (TB) is caused by a group of closely related bacteria (Mycobacterium tuberculosis, M. canettii, M. africanum, M. microti, M. bovis, M. caprae, M. pinnipedii, M. mungi, and M. orygis) collectively referred to as the Mycobacterium tuberculosis complex (MTBC) (1-6). M. tuberculosis is the main agent of TB in humans. Within M. tuberculosis itself, single nucleotide polymorphisms (SNPs) and large sequence polymorphisms (LSPs) are used to further classify M. tuberculosis into four (sensu stricto) major lineages, each of which is found to strongly associate with specific geographic human populations (7). While strong lineage associations with specific human populations or regions of the world are well acknowledged (8-12), the causes and relevance of this to bacterial virulence and clinical presentation are still only partly understood (13-16). It has become clear, however, that the previously unrecognized genetic diversity among M. tuberculosis isolates has an impact on the outcome of infection (17-19), and different approaches have been undertaken towards a better understanding of what makes a strain...