Extended Data Fig. 4 |. VAN-P assays predict PDIM levels across different Mtb strains and lineages and enable re-isolation of single PDIM(+) clones.

a, VAN10-P assays for a range of virulent Mtb strains belonging to different lineages. Bacterial growth was measured after 7, 10, and 14 days of incubation as indicated. Data are from the same experiment in Fig. 2a and show additional time points plus an independent experimental repeat measured on day 10 (hatched bars with unfilled symbols). b, VAN10-P assays for strains with an H37Rv background including mc²7902 and mc²8398. H37Rv-SC is a single PDIM(+) clone isolated from H37Rv-B by VAN10-P colony screening. This clone was used as our PDIM(+) H37Rv wildtype strain throughout this work and was used to construct H37Rv ΔppsD and ΔppsD::comp isogenic mutants (Supplementary Table 2). Data in (a,b) show mean ± SD for n = 9 pairwise comparisons between triplicate wells, except for the day 10 repeat in (a) where n = 4 pairwise comparisons between duplicate wells. c, VAN-P MICs of H37Rv stocks and H37Rv-SC. d, VAN-P MICs of non-H37Rv strains from (a). e–g, VAN-P MICs of single PDIM(+) clones isolated from Rag^−/− mice using VAN10-P colony screening for (e) Erdman, (f) HN878, and (g) CDC1551 (see also Extended Data Table 1). Data are plotted together with MIC data from (d) for comparison. MIC data in (c–g) show mean ± SD for n = 4 biological replicates from two independent experiments. h–j, Determination that our Mtb mc²6230 stock is a mixed population and re-isolation of a single PDIM(+) clone by VAN10-P screening. (h) VAN10-P assay of single colonies isolated from our mc²6230 stock (n = 40) and (i) following a single passage in 10 μg/ml vancomycin (n = 20). Vancomycin significantly enriched for PDIM(+) bacilli (P < 0.0001 two-tailed Mann-Whitney test), facilitating re-isolation of low-frequency PDIM(+) clones. Each colony was assayed in triplicate and data points represent mean VAN10-P growth%. Lines indicate the median. j, VAN-P MICs of PDIM(+) (AE1601) and PDIM(−) (AE1611) mc²6230 clones identified by VAN10-P colony screening (see also Extended Data Table 1). Mean ± SD for n = 6 biological replicates from two independent experiments.