Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production, adhesion properties and physiology of Bacillus cereus vegetative cells

Abstract

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25-100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.

The growth, physiology and toxigenic potential of Bacillus cereus in cooked rice during storage temperature abuse

“…The reasons for doing so were 3-fold; firstly, little is known concerning the toxin in terms of when it is expressed during the growth of B. cereus and what conditions favour its production (Ghelardi et al, 2002;Jaaskelainen, Haggblom, Andersson, Vanne, & Salkinoja-Salonen, 2003;Titball, 1998), secondly, PC-PLC activity may give an indication of the broad toxigenic potential of a given B. cereus strain growing under a certain set of conditions (Del Torre, Della Corte, & Stecchini, 2001) and thirdly, the fluorescent assay used in this study was found by the authors to be a quantitative and convenient method of measuring the activity of this toxin in batch cultures (Cronin & Wilkinson, 2008c) and hence it was of interest to evaluate its usefulness in a food matrix.…”

“…The activity of phosphatidylcholine-specific phospholipase C (PC-PLC -a toxin with an unknown role in food borne illness; Rowan, Caldow, Gemmell, & Hunter, 2003) was also monitored during these trials in order to relate toxin activity to growth as measured using plate counting and physiology as measured using FCM. A relationship was previously noted between PC-PLC activity and acquisition by the majority of B. cereus cells in batch culture of both intracellular esterase and redox activity (Cronin & Wilkinson, 2008c).…”

“…Measurement of PC-PLC activity was carried out using the Amplex Ò Red Assay Kit (Invitrogen) as described previously (Cronin & Wilkinson, 2008c). This enzyme-coupled assay involves the detection of the fluorescent product of the Amplex Ò Red reagent, (10-acetyl-3,7-dihydrophenoxazine) resorufin.…”

Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes

“…Also, measurement of internal pH and how it changes with culture changes. [62,63] Measurements of physical or structural parameters of a cell, including surface proteins…”

The potential of flow cytometry in the study of Bacillus cereus

“…2000). There are many examples of workers utilizing FCM to enumerate micro‐organisms in suspension, including estimation of growth rates of bacteria under various conditions and construction of growth curves (Comas and Vives‐Rego 2002; see Cronin and Wilkinson 2008c). Recently, Leser et al.…”