Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to simulated food processing treatments, determined using fluorescence-activated cell sorting and plate counting

Abstract

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.

Fig. 1.

Flow cytometric analysis and sorting strategy used for various microbial species.

Fig. 2.

Mean log reduction of exponential-phase cells of S. aureus, E. coli, and L. monocytogenes exposed to various simulated food processing treatments compared with controls. For all samples, n = 6. Black bars are results for S. aureus, light-gray bars are results for E. coli, and dark-gray bars indicate results for L. monocytogenes. Treatments were as follows: 80°C, cells heated to 80°C for 10 min; 60°C, cells heated to 60°C for 20 min; pH4, cells exposed to broth at pH 4; 0.1% Tween 80, 0.1% (wt/vol) Tween 80 solution; 0.1% Sorbate, 0.1% (wt/vol) potassium sorbate solution; 10% NaCl, 10% (wt/vol) sodium chloride solution; 70% IPA, 70% isopropyl alcohol; 0.1% SDS, 0.1% (wt/vol) sodium dodecyl sulfate solution; 0.5 mM CTAB, 0.5 mM cetrimonium bromide.

Fig. 3.

Cytometric profiles of E. coli cells stained with PI/Syto 9 after heat treatment at >95°C (a) or treatment with 0.5 mM CTAB (b).

Fig. 4.

Cytometric profiles of S. aureus cells stained with DiOC₂(3) after exposure to heating at 80°C (a) or heating to 60°C (b).

Fig. 5.

Cytometric profiles of live Listeria monocytogenes control cells stained with Syto 9/PI (a), Listeria monocytogenes treated with IPA and stained with Syto 9/PI (b), or Listeria monocytogenes treated with NaCl and stained with DiOC₂(3) (c).