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Cited by 8Bacterial Genetics and BiotechnologyEscherichia coli research studiesAntibiotic Resistance in Bacteria

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

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Greninger, Alexander L., Addetia, Amin, Tao, Yue, Adler, Amanda, Qin, Xuan·2021·Scientific Reports
DOI 10.1038/s41598-021-86764-4PMC PMC8018945

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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.

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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 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).CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Greninger, A. L., Addetia, A., Tao, Y., Adler, A., & Qin, X.
Affiliations
  • Seattle Children's Hospital (US)
  • Shanghai Jiao Tong University (CN)
Corresponding author
Qin, Xuan
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2021-04-02
Volume
11
Issue
1
Pages
7457-7457
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-021-86764-4
PMC
PMC8018945
OpenAlex
W3145054816
Cited by
8
References
6
Citations by year
2026: 1 · 2025: 2 · 2023: 1 · 2022: 2 · 2021: 2
FWCI
0.71
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
  • Health Sciences › Medicine › Microbiology (medical) › Antibiotic Resistance in Bacteria
Keywords
small colony variants, Enterobacteriaceae, Escherichia coli, Citrobacter freundii, Enterobacter hormaechei, oxidative respiration, iron acquisition, heme biosynthesis, lipoic acid biosynthesis, single nucleotide variants, chromosomal deletion, genetic rescue, anaerobic growth, Gram-negative bacteria
MeSH terms
Whole Genome Sequencing, Aerobiosis, Anaerobiosis, Child, Enterobacteriaceae, Female, Genes, Bacterial, Heme, Humans, Infant, Iron, Kinetics, Thioctic Acid, Male, Microbial Sensitivity Tests, Phenotype, Genetic Variation, Colony Count, Microbial, Gene Silencing, Drug Resistance, Bacterial, Biosynthetic Pathways

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