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

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 genetic and biochemical 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, with respect to their NCV co-isolate. In addition, a 10.2kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV 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 Enterobacteriaceae species 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.\n\nImportance\n\nSmall colony variant (SCV) bacteria are routinely isolated in the clinical microbiology laboratory and can be notoriously difficult to treat. Most studies of the genetic underpinnings of SCV clinical isolates have examined Staphylococcus aureus and few have looked at how SCV emerge in Gram-negative bacteria. Here, we undertook detailed characterization of three clinical isolates of SCV in Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei along with their NCV co-isolates. Genomic sequencing revealed that each SCV had at least one coding change in genes involved in both bacterial oxidative respiration and iron capture. Chemical and genetic rescue revealed that both pathways could be responsible for the small colony variant. Each of the SCV showed no growth defect compared to NCV when incubated under anaerobic conditions, indicating a potential mechanism for SCV survival in vivo. We hypothesize that by retreating to anaerobic environments and avoiding escalating iron competition with the host, SCV have adapted to live to see another day.