Investigating the biocontrol and anti-biofilm potential of a three phage cocktail against Cronobacter sakazakii in different brands of infant formula

Abstract

In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4°C-37°C, and pH6-8, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3×10⁸pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells (≤10⁴cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (<10cfu/mL). This was observed following a 20h challenge in different brands of infant formula, where samples in the absence of the phage cocktail reached concentrations of ~10⁹cfu/mL. The phage cocktail also demonstrated promise in preventing the establishment of biofilm, as biofilm formation could not be detected for up to 48h post treatment. These results highlight the potential application of this phage preparation for biocontrol of C. sakazakii contamination in reconstituted infant formula and also as a preventative agent against biofilm formation.

Keywords: Bacteriophages; Biocontrol; Biofilm; Cronobacter sakazakii; Infant formula.