Bacterial growth depends on NTBI. (A-E) Y enterocolitica and (F-I) V vulnificus. (A) NTBI and (B) serum iron measurements in WT, iron-depleted HKO, iron-loaded WT, and iron-loaded HKO mice. Presence of NTBI correlates with the severity of Y enterocolitica infection. (C) Minihepcidin treatment abolished NTBI in serum of iron-loaded WT and HKO mice. (D) Microscopy images (magnification ×4) of bacterial growth 24 hours after plating Y enterocolitica O9 (10⁶ CFU/mL) on agar plates made of human plasma supplemented with 0 to 60 μM FAC. Bacterial growth was observed only in the samples with measurable NTBI (dark gray section). (E) Transferrin (Tf) saturation (sat), NTBI concentration, and bacterial growth on agar plates made from plasma of 3 different donors. Statistical analysis (A,C): Mann-Whitney U test. (F-I) V vulnificus (1 × 10³ CFU/mL) were grown in vitro in human plasma supplemented with (F) 0 to 100 μM FAC, (G) 0 to 100 μM holo-Tf, or (H) 0 to 100 μM apo-Tf. V vulnificus growth was initiated only when 40 to 100 μM FAC was added to the plasma at which point transferrin saturation reached 100%. Bacteria did not grow in plasma supplemented with holo-Tf or apo-Tf. Each line represents mean (n = 3) ± standard deviation. (I) V vulnificus growth was measured in 6 different human plasma samples supplemented with a range of FAC. Black circle indicates the iron concentration at which V vulnificus growth was initiated. White bar shows plasma iron concentration at which transferrin saturation reached 100% for each sample. Dashed line indicates baseline plasma iron concentration for each human sample. V vulnificus growth in vitro in human plasma occurred only when transferrin was nearly completely saturated. OD, optical density.