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

Abstract

25Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and 26 can fundamentally change the outcome of the associated infections. Bacterial SCVs often 27 emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The genetic 28 and biochemical basis of SCV emergence in vivo is not well understood in Gram-negative 29 bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of 30 NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter 31 hormaechei. We confirmed the isogenic basis of SCV emergence, as there were only 4 single 32 nucleotide variants in SCV for E. coli, 5 in C. freundii, and 8 in E. hormaechei, with respect to 33 their NCV co-isolate. In addition, a 10.2kb chromosomal segment containing 11 genes was 34 deleted in the E. hormaechei SCV isolate. Intriguingly, each SCV had at least one coding 35 change in a gene associated with bacterial oxidative respiration and another involved iron 36 capture. Chemical rescue confirmed the causal role of heme biosynthesis in E. coli and C. 37 freundii and lipoic acid in E. hormaechei SCV isolates. Genetic rescue restored normal growth 38 under aerobic conditions for fes and hemL in C. freundii; hemL in E. coli; and lipA in E. 39 hormaechei SCV isolates. Prototrophic growth in all 3 SCV Enterobacteriaceae species was 40 unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo 41 by abandoning the highly energetic lifestyle in an iron-limiting environment. We propose that the 42 selective loss of functions in oxidative respiration and iron acquisition is indicative of bacterial 43 virulence attenuation for niche specialization and persistence in vivo. 44 45 Importance 46 Small colony variant (SCV) bacteria are routinely isolated in the clinical microbiology laboratory 47 and can be notoriously difficult to treat. Most studies of the genetic underpinnings of SCV 48 clinical isolates have examined Staphylococcus aureus and few have looked at how SCV 49 emerge in Gram-negative bacteria. Here, we undertook detailed characterization of three 50 clinical isolates of SCV in Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei 51 along with their NCV co-isolates. Genomic sequencing revealed that each SCV had at least 52 one coding change in genes involved in both bacterial oxidative respiration and iron capture. 53Chemical and genetic rescue revealed that both pathways could be responsible for the small 54 colony variant. Each of the SCV showed no growth defect compared to NCV when incubated 55 under anaerobic conditions, indicating a potential mechanism for SCV survival in vivo. We 56 hypothesize that by retreating to anaerobic environments and avoiding escalating iron 57 competition with the host, SCV have adapted to live to see another day. 58 59 65 SCVs were first described in Salmonella typhi over a hundred years ago, prior to the antibiotic 66 era (4). Isolation of SCVs is especially common...