The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 μM DPP) and iron-limited (75 μM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO 4, FeCl 3, Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.
We thank Professor Ian Poxton at University of Edinburgh for providing C. difficile VPI 10463 and NCTC 11223 strains, Dr. Peter Mullany at University College London for providing C. difficile strain 630 and Professor Harry Flint for his critical review of the manuscript.
This work was supported by The European Commission (grant number PIEF-GA-2008-220815). The Rowett Institute of Nutrition and Health receives support from the Scottish Government (Rural and Environment Science and Analytical Services; RESAS).
- Clostridium difficile
- Iron requirements
- Iron utilization bioassays
- Microtiter growth assay