Iron citrate

Plate count was established as the methodology to determine the bacterial growth of each of the strains in multispecies biofilms after incubation periods of 24, 48, and 72 h. To do so, the surfaces were washed twice with 3 mL of sterile distilled water to remove the unattached cells and then placed in a sterile flask containing 3.5 g of glass beads and 10 mL of peptone water. The samples were then vortexed for 90 s at 40 Hz to dislodge the attached cells from the surface for quantification [36 (link)].
The resulting suspension was decimally diluted in peptone water and transferred to a plate for its quantification. Since the biofilms consisted of two species, a culture medium was designed that enabled them to be differentiated. The media consisted of esculin, since L. gasicomitatum was observed to ferment the sugar, while P. fragi and L. reuteri did not. This enabled a medium composed of TSA, esculin (Sigma-Aldrich, Madrid, Spain), and iron citrate (Sigma-Aldrich, Madrid, Spain) to be developed, which turned the colonies of L. gasicomitatum black, making it easily distinguishable from the other two strains used. Differences were observed based on colony morphology. The plates were incubated at 30 °C for 48 h and then counted.

EcN microcins were tested in vitro against EHEC O157: H7, SE, and ST under iron-limited and iron-rich conditions. Strains were first cultured overnight at 37 °C in the resistant LB added with 0.2 mM 2,2′-dipyridyl (Sangon Biotech, Shanghai, China) (Supplementary Figure S2a) aerobically. EcN and pathogenic enterobacteria were inoculated in a 1: 1 ratio. Approximately 1 × 10⁶ CFU mL⁻¹ of overnight culture (calculated according to the formula of the growth curve, Supplementary Figure S1) was inoculated into 10 mL of iron-limited conditions (DMEM with 10% fetal bovine serum (FBS); Procell, Wuhan, China) and iron-rich medium (LB supplemented with 4 mM iron citrate; Sigma, Darmstadt, Germany) (Supplementary Figure S2b), previously described [25 (link)]. Four EcN strains (EcN wt, EcN ΔmcmA, EcN ΔmchB, and EcN ΔmcmA ΔmchB) were respectively inoculated in competition with pathogenic enterobacteria (EHEC O157: H7, SE, and ST). At 0, 4, 8, 12, and 24 h after inoculation, serial dilutions of each strain were plated to determine the strain counts. In addition, four EcN strains and pathogens were screened with chloramphenicol and ampicillin-resistant plates, respectively.

All strains used in this study are isogenic, except EGY48 from Clontech, 3B9 (BY4741) and 21A5 (double mutant fet3Δ fet4Δ) (Table 1). Gene disruptions were made by standard PCR-based methods. Yeast strains were grown in YPD medium (1% yeast extract, 2% bacto peptone, 2% glucose). For YPD plates, 2% agar was added. For URA3-containing plasmid selection, yeasts were grown on Synthetic Complete (SC) medium lacking uracil (SC–URA) (0.67% yeast nitrogen base, adenine, histidine, lysine, leucine, tyrosine, methionine, tryptophan, 40 mg/liter for each, 2% glucose, 3% agar). HU, iron citrate and bathophenantroline (BPS) were purchased from Sigma. 30 mM stock solution of BPS was prepared in ethanol. For cell cycle experiments, 20 and 40 mM HU were used in exponential yeast cultures. 1 ml of cell culture was collected at indicated time points, and processed for flow cytometry. Detailed information regarding strain construction is provided as Supplementary Methods.