Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed the isogenic basis of SCV emergence, as there were only 4 single nucleotide variants in SCV for E. coli, 5 in C. freundii, and 8 in E. hormaechei (along with a 10.2kb chromosomal deletion), with respect to their NCV co-isolate. Intriguingly, each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved iron capture. Chemical rescue confirmed the causal role of heme biosynthesis in E. coli and C. freundii and lipoic acid in E. hormaechei SCV isolates. Genetic rescue restored normal growth under aerobic conditions for fes and hemL in C. freundii; hemL in E. coli; and lipA in E. hormaechei SCV isolates. Prototrophic growth in all 3 SCV was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo by abandoning the highly energetic lifestyle in an iron-limiting environment. We propose that the selective loss of functions in oxidative respiration and iron acquisition is indicative of bacterial virulence attenuation for niche specialization and persistence in vivo.

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of small-colony variant Enterobactericeae