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.
Highlights
To survive in the hostile host environment, bacteria may take two separate paths
Unlike the NCVs, all three corresponding bacterial small colony variants (SCVs) isolates were auxotrophic, unable to grow in glucose only M9 medium
Chemical rescue, as well as NCV cross-feeding, we found heme-production pathway lesions to be responsible for the SCV phenotype in two of the three isolates, while lipoic acid synthesis was responsible for the third
Summary
To survive in the hostile host environment, bacteria may take two separate paths. The first and most commonly discussed is an arms race of iron competition and acquisition of antimicrobial resistance genes and pathogenicity factors[1,2,3]. Characterized bacterial SCV species include Staphylococcus aureus, Escherichia coli, Neisseria gonorrhoeae, Stenotrophomonas maltophilia, Enterococcus, and Salmonella[8,9,10,11,12]. In addition to their decreased growth rate, bacterial SCV isolates are characterized by auxotrophism for components directly involved in the electron transfer chain, such as heme and menaquinone. Our lab has since implemented a systematic, culture-based approach that checks for colony variation in size, texture, color, or hemolysis, and inability to grow on the standard Mueller–Hinton (MH) medium for susceptibility testing Using this systematic approach, we identified 3 pairs of clinical NCV and SCV Enterobacteriaceae co-isolates from blood and urine cultures. Confirmatory chemical and genetic rescues were performed on the SCVs to determine which mutations were causal for the altered phenotype
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