Flow Cytometry to Assess the Counts and Physiological State of Cronobacter sakazakii Cells after Heat Exposure

Abstract

Cronobacter sakazakii is an opportunistic pathogen that is associated with outbreaks of neonatal necrotizing enterocolitis, septicaemia, and meningitis. Reconstituted powdered infant formulae is the most common vehicle of infection. The aim of the present study is to gain insight into the physiological states of C. sakazakii cells using flow cytometry to detect the compromised cells, which are viable but non-culturable using plate-based methods, and to evaluate the impact of milk heat treatments on those populations. Dead-cell suspensions as well as heat-treated and non-heat-treated cell suspensions were used. After 60 or 65 °C treatments, the number of compromised cells increased as a result of cells with compromised membranes shifting from the heat-treated suspension. These temperatures were not effective at killing all bacteria but were effective at compromising their membranes. Thus, mild heat treatments are not enough to guarantee the safety of powered infant formulae. Flow cytometry was capable of detecting C. sakazakii’s compromised cells that cannot be detected with classical plate count methods; thus, it could be used as a screening test to decrease the risk derived from the presence of pathogenic viable but non-culturable cells in this food that is intended for newborns’ nutrition.