Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae

Abstract

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 SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.

Figure 1

Case histories. Relevant past clinical microbiological and antibiotic selective pressures are indicated in the line histories for the isolation of NCV and SCV in Escherichia coli (a), Citrobacter freundii (b), and Enterobacter hormaechei (c).

Figure 2

Heme biosynthesis lesion as cause of small colony phenotype in Escherichia coli isolated from urinary tract infection. Genomic sequencing of the paired NCV and SCV isolates revealed genomic lesions in fepC, hemF, hemL, and mprA. (a) Only genetic rescue with hemL rescued normal growth from the Escherichia coli SCV. (b) Chemical rescue with heme partially restored normal growth in Escherichia coli SCV. (c) Cross-feeding from Escherichia coli NCV partially restores growth of SCV, consistent with a diffusible factor required for growth. All plates in this figure were incubated under aerobic conditions.

Figure 3

Heme biosynthesis along with iron availability lesion as cause of small colony phenotype in Citrobacter freundii isolated from urinary tract infection. Genomic sequencing of the paired NCV and SCV isolates revealed genomic lesions in araC, fdnG, fes, hemL, pta, and in the intergenic region upstream of the hemF gene. (a) Genetic rescue with fes and hemL rescued normal growth from the Citrobacter freundii SCV. (b) Chemical rescue with heme restored normal growth in Citrobacter freundii SCV. (c) Cross-feeding from Citrobacter freundii NCV partially restores growth of SCV, consistent with a diffusible factor required for growth. All plates in this figure were incubated under aerobic conditions.

Figure 4

Lipoic acid biosynthesis as cause of small colony phenotype in Enterobacter hormaechei isolated from a bloodstream infection in a patient with end-stage renal disease. Genomic sequencing of the paired NCV and SCV isolates revealed multiple genomic lesions including single nucleotide substitutions in entD, hemN, and pqqB along with large-scale rearrangements leading to disruption of the lipA gene. (a) Genetic rescue with lipA restored normal growth from the Enterobacter hormaechei SCV. (b) Chemical rescue with lipoic acid restored normal growth in Enterobacter hormaechei SCV. (c) Cross-feeding from Enterobacter hormaechei NCV restores growth of SCV, consistent with a diffusible factor required for growth. All plates in this figure were incubated under aerobic conditions.

Figure 5

Growth curve analysis in aerobic conditions demonstrates hemL fully restores the impaired growth kinetics of (a) Escherichia coli SCV and (b) Citrobacter freundii SCV isolates. The Citrobacter freundii SCV isolate was not rescued by fes. (c) Growth kinetics of the Enterobacter hormaechei SCV isolate are partially restored by overexpression of lipA.

Figure 6

SCV E. coli (a), C. freundii (b), and E. hormaechei (c) isolates grow on minimal media without additional chemical or genetic supplementation in anaerobic conditions. NCV and SCV E. coli (d), C. freundii (e), and E hormaechei (f) co-isolates display similar growth kinetics in anaerobic conditions.