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 (3×10pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells (≤10cfu/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 ~10cfu/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.

Characteristics of lytic phage vB_EcoM-ECP26 and reduction of shiga-toxin producing Escherichia coli on produce romaine

“…To overcome BIM emergence, phage cocktail techniques or a combination with other natural antibacterial agents could be used [16]. Endersen et al [17] reported that a combined-phage preparation possessed a strong bactericidal effect on Cronobacter sakazakii. Therefore, for more efficient control of pathogenic E. coli, lytic phages with a broad host spectrum should be screened and used as a cocktail with vB_EcoM-ECP26.…”

Biopelículas y persistencia microbiana en la industria alimentaria

“…Así, varios estudios han pretendido en la última década identificar nuevos bacteriófagos efectivos en la eliminación de biopelículas de los principales patógenos transmitidos por los alimentos, y, de hecho, algunos fagos han sido postulados como agentes de control biológico contra las biopelículas de Cronobacter sakazakii, E. coli, S. aureus, Salmonella spp. y L. monocytogenes (Chaitiemwong et al, 2014;Endersen et al, 2017;González et al, 2017;Sadekuzzaman, Yang, Mizan y Ha, 2017;Shafique, Alvi, Abbas y ur Rehman, 2017). Además, algunas enzimas líticas derivadas de fagos, como las endolisinas, también han demostrado actividad contra las biopelículas bacterianas (Gutiérrez, Ruas-Madiedo, Martínez, Rodríguez y García, 2014).…”

The use of bacteriophages to control and detect pathogens in the dairy industry

“…Similar results were reported by Endersen et al . who used a three‐phage mix against four brands of artificially contaminated reconstituted IF and reported a reduction in C. sakazakii levels to below the detection limit in <5 h. Importantly, this phage mix also displayed lethal effects against Cronobacter in established biofilms (Endersen et al ).…”