Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair.

Alicja K Warda,Marcel H Tempelaars,Masja N Nierop Groot,Tjakko Abee

doi:10.3389/fmicb.2016.01096

Alicja K Warda, Marcel H Tempelaars + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2016.01096

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Abstract

The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

Highlights

An increased demand for food with improved freshness, sensorial, and nutritional values has directed food processing toward the use of milder heat treatments that require secondary mild preservation hurdles to assure stability and safety of the products (Pasha et al, 2014)
Reduction of the heat treatment intensity may lead to subpopulations of spores that are Recovery of Bacillus cereus Spores sublethally damaged rather than inactivated resulting in increased heterogeneity in the population (Cazemier et al, 2001; Warda et al, 2015)
Bacillus cereus ATCC 14579 obtained from the American Type Culture Collection (ATCC), and its mutant derivatives used in this study (Table 1) were cultured in Bacto Brain Hart Infusion broth (BHI, Beckton Dickinson) at 30◦C with aeration at 200 rpm

Summary

Introduction

An increased demand for food with improved freshness, sensorial, and nutritional values has directed food processing toward the use of milder heat treatments that require secondary mild preservation hurdles to assure stability and safety of the products (Pasha et al, 2014). As a result, these products are challenged by resistant microbial spores, that survive heat and other preservation hurdles used in food processing (Postollec et al, 2012; Stecchini et al, 2013; Checinska et al, 2015). The presence of superdormant spores may further increase heterogeneity (Ghosh and Setlow, 2010), and this conceivably results in less accurate prediction of spore outgrowth behavior

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