Although outbreaks caused by Cronobacter spp. (Enterobacter sakazakii) are rare, infections by this organism have a case-fatality rate which may reach 80%. Powdered infant milk formula (PIMF) is considered a major source for human infection with Cronobacter spp. The organism has the capability to survive in dry environments for long periods (approximately 2 years). Current interest in the use of natural antimicrobials including lactoferrin (LF) and nisin has developed because of the desire for preservative-free food products. The objective of the present study was to evaluate the antimicrobial activity of bovine LF or nisin against undesiccated and desiccated Cronobacter spp. cells in 0.2% peptone water (PW) and reconstituted PIMF at different temperatures. In 0.2% PW, 2.5 mg/ml LF was able to inactivate 4 log(10) CFU/ml of undesiccated cells of Cronobacter spp. in 4 h at 37 degrees C but at lower temperatures, higher concentrations of LF as well as longer exposure were needed to achieve the same effect as at 37 degrees C. Similarly, the effect of nisin against undesiccated cells of Cronobacter spp. was concentration and temperature dependent in 0.2% PW. It was found that 1500 IU/ml caused a 4 log(10) CFU/ml reduction of undesiccated cells of Cronobacter spp. at 21 degrees C and 37 degrees C. Desiccated Cronobacter spp. cells in 0.2% PW were more sensitive to LF action than were undesiccated cells. A 4 log(10) CFU/ml reduction was obtained with 2.5 mg/ml LF after 1 h at 21 and 37 degrees C or 8 h at 10 degrees C. In contrast, desiccated cells of Cronobacter spp. were more resistant to nisin. Furthermore, neither LF nor nisin had detectable antimicrobial activity against desiccated or undesiccated Cronobacter spp. in reconstituted PIFM. Heating at 55 degrees C for 5 min with nisin in reconstituted PIFM did not enhance the antimicrobial activity of nisin. Unexpectedly, nisin appeared to protect Cronobacter spp. from the damaging effects of heat treatment. The reduced antimicrobial activity of LF and nisin in reconstituted PIMF was potentially explained by the higher concentration of Ca(2+), Mg(2+) and Fe(3+) in the latter.