The role of EcN’s microcins against cEc, AIEC and STm was tested by in vitro competitive growth assay in iron-rich and iron-limiting conditions. Strains were grown in Nutrient Broth supplemented with 0.2mM 2,2′-dipyridyl (Sigma) aerobically at 37 °C overnight. Approximately 5×103 CFU/mL from an overnight culture were inoculated into 0.1 mL of tissue culture medium (DMEM/F12 with 10% fetal bovine serum (FBS); Invitrogen), as previously described23 (link),24 (link). When indicated, media was prepared with 1 μM ferric iron citrate (Sigma). Wild-type EcN or EcN mutants were inoculated in competition with wild-type STm, STm mutants, cEc, or AIEC. CFUs of each strain were enumerated by plating serial dilutions at 0, 5, 8, 11 and (for some assays) 16 h post-inoculation.
Protocol detail
Microcin-mediated Competition Among Pathogens
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Assays
Culture medium
Dilutions
Ferric citrate
Fetal bovine serum
Inoculation
Iron
Iron conditions
Microcins s
Nutrient
Strain
Tissue
Corresponding Organization :
Other organizations : University of California, Irvine
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Variable analysis
independent variables
- Presence of 2,2′-dipyridyl (iron-limiting condition)
- Presence of 1 μM ferric iron citrate (iron-rich condition)
- Wild-type EcN or EcN mutants inoculated in competition with wild-type STm, STm mutants, cEc, or AIEC
- Colony Forming Units (CFUs) of each strain enumerated by plating serial dilutions at 0, 5, 8, 11 and (for some assays) 16 h post-inoculation
- Nutrient Broth as the growth medium
- Aerobic incubation at 37 °C overnight
- Inoculum size of approximately 5×10^3 CFU/mL from an overnight culture
- DMEM/F12 with 10% fetal bovine serum (FBS) as the tissue culture medium
- Positive control: Not mentioned
- Negative control: Not mentioned
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