Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

• Anopore allows quantification of heterogeneity in bacterial spore outgrowth.
• Heat treatment introduces a distinct phenotype during spore outgrowth at mild acid stress.
• Initial outgrowth is an important determinant of heterogeneous behavior in spore populations.

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90 °C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH 7 and pH 5.5, without and with added NaCl or sorbic acid (HSA). At pH 7 non-heated and heat-treated germinated spores required 6 h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH 7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH 5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6 h at pH 5.5, which increased to 12 h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH 5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24 h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH 5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth.