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

Paloma Cal-Sabater,María J. Cao,María J. Castro,Emiliano J. Quinto,Javier Mateo,Irma Caro

doi:10.3390/foods8120688

Paloma Cal-Sabater, María J. Cao + Show 4 more

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https://doi.org/10.3390/foods8120688

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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.

Highlights

During the last decade, scientific interest has turned to Cronobacter sakazakii, formerly Enterobacter sakazakii, as a human pathogen
A small number of live cells were detected in TCS60 (Figure 2 (B1)) but in TCS65 or TCS100 (Figure 2(B2,B3), respectively), showing that the 60 ◦ C treatment does not kill all bacteria, resulting in a high number of Compromised cells (CC)
The results were different in non-heat-treated cell suspension (nTCS):TCS60 and nTCS:TCS65 mixtures, i.e., the number of CC increased as a result of cells, with compromised membranes shifting from TCS, i.e., the used temperatures were not completely effective at killing all bacteria but were effective at compromising their membranes and subsequently, their capacity to grow on agar forming colonies and be detected with conventional analyses

Summary

Introduction

Scientific interest has turned to Cronobacter sakazakii, formerly Enterobacter sakazakii, as a human pathogen. C. sakazakii is an opportunistic pathogen that is associated with outbreaks of neonatal necrotizing enterocolitis, septicemia, and meningitis [1,2,3]. Raghav and Aggarwal [5] identified a 66kDa toxin which was most active at pH 6, with the ability to be stable at 90 ◦ C for 30 min, and potent cell toxicity (LD50 = 56 pg). This organism has a high case fatality rate in vulnerable infants in neonatal intensive care units and in surviving patients, severe neurological sequelae have occurred including hydrocephalus, quadriplegia, and developmental delay [2,6]. The International Commission for Microbiological Specifications for Foods [7] ranked Cronobacter spp. as a “severe hazard for restricted populations causing life-threatening or substantial chronic sequelae of long duration.” The FAO/WHO [8] noted that infants are the group at particular risk

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