Abstract
Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.
Highlights
Spore forming bacteria are commonly present in the environment and difficult to eradicate because they produce highly resistant spores that may remain dormant for years until germination
The impact of heat-treatment on germination and outgrowth of B. cereus American Type Culture Collection (ATCC) 14579 spores was assessed using spores in suspension buffer heated for one min at 95°C
Germination and outgrowth of untreated and heat-treated spores was monitored by microscopic observation and by the relative change in optical density at 595 nm (OD595) that reflects the transition from phase bright to phase dark spores
Summary
Spore forming bacteria are commonly present in the environment and difficult to eradicate because they produce highly resistant spores that may remain dormant for years until germination. We assessed the germination and outgrowth performance of untreated and heat-damaged B. cereus spores using optical density measurements and microscopy analysis at selected time points. Primers (S3 Table) were designed targeting the 21 selected genes that were expressed in outgrowth of heat-damaged spores and five candidate genes for normalisation (BC0257, BC0544, BC1409, BC4471, and BC4743) were selected based on their constant expression levels across the different conditions used on the DNA microarrays.
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