Despite the lack of correlation between bacteriolysis and intracellular replication, we hypothesized that cytosolic survival was essential for virulence. To assess virulence ex vivo, we tested these mutants for plaque formation within L2 fibroblasts (32) (Fig. 2B). menD::Tn and menF::Tn mutants did not form visible plaques even after 6 days of infection. lmo1602::Tn and yvcJ::Tn mutants had intermediate virulence phenotypes, while nrdD::Tn mutants made plaques similarly to wild-type L. monocytogenes. Strikingly, pdhC::Tn mutants were capable of forming intermediate-sized plaques despite being cleared in macrophages, consistent with the pdhC::Tn mutant’s minimal bacteriolysis phenotype in fibroblasts (Fig. S3B).