Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Abstract

The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sublethal heat; exposure to organic acids, salt, and sodium hydroxide; and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/propidium iodide (PI), DiOC2(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labeling by commercial anti-S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensities of stained cells to those of unstained cells [MFI/MFI(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, resulted in significantly lower fluorescence values compared to controls. IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting, should be undertaken. Researchers should be aware of physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

Protein A-Mediated Binding ofStaphylococcusspp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent

“…This was in the context of S. aureus NCTC 8325 losing its ability to bind antibody. It could be that the manner of S. lentus and S. xylosus binding to antibody was similar to the damage-mediated, nonspecific binding found in a previous study by this group, where cell damage was shown to increase antibody signal above that of healthy control cells ( 15 ). S. lentus and S. xylosus also displayed the smallest log reductions after incubation on stainless steel compared to the other strains, as measured by plate counting, and FCM data showed that ∼50% of the cells maintained esterase activity.…”

“…In general, and similar to the data yielded by the surface spiking experiment, permeabilized cells showed greater antibody binding. This points toward a conclusion reached in the Kennedy et al ( 15 ) study, where it was recommended that a cell membrane permeability dye such as PI be used to remove membrane-damaged cells from the final count in FCM antibody-mediated detection assays in order to reduce the problem of false positives.…”

“…A number of simulated food-processing treatments were applied to exponentialphase cultures of the S. aureus chicken ready-meal isolate. A previous study by Kennedy et al (15) found that certain simulated food-processing treatments produced changes in the intensity of binding of a pair of commercial mouse IgM anti-S. aureus antibodies to an S. aureus food isolate and S. aureus ATCC 8325. These alterations depended on the nature of the treatment; the organic acids citric acid and oxalic acid resulted in a significant decrease in antibody binding, possibly through damage to the cell membrane and intracellular enzymes, whereas heat treatments to simulate mild to more severe heat processing treatments (from 50 to 80°C) resulted in only small differences in the degree of antibody binding.…”

“…We first wanted to gain an understanding of the phenomenon and so, randomly choosing an anti-human IgG2a antibody generated in mice, we tested the ability of various Staphylococcus strains to bind this. In a previous study, we demonstrated that the physiological state and types of treatments to induce cellular damage or death affect the extent of antibody binding of S. aureus ( 15 ). In the present study, we investigated the effect of heat killing on the SpA-mediated binding of antibody to S. aureus .…”

Potential of Flow Cytometric Approaches for Rapid Microbial Detection and Characterization in the Food Industry—A Review

“…Furthermore, specific targets such as food pathogens [ 51 ], infectious bacteria [ 52 ], and environmental contaminants are easily detected by FCM combined with phylogenetic labeling. Flow-FISH is a phylogenetic labeling method in which specific nucleic acid sequences inside intact viable cells are labeled [ 53 ], as further discussed in Section 4 .…”