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Abstract
Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains (high producers B4094 and B4123, and type strain DSM13T) was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4–20 times lower in milk than in LB broth (maximum 36 μg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11–C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 μg/ml for Caco-2 human intestinal epithelial cells and 16.8 μg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested.
Highlights
Bacillus licheniformis is a facultative anaerobic endosporeproducing bacterium that is ubiquitously found in the environment, plant material, and soil
Bacillus licheniformis is not considered to be a pathogen and has not been shown to be able to invade the outer barriers of the body without previous lesions, but sporadic cases of human infection related to B. licheniformis have been reported (Sugar and McCloskey, 1977; Park et al, 2006; Haydushka et al, 2012; Padhi et al, 2012)
The presence of high salt concentrations of 1M NaCl in Luria Bertani (LB) broth did not have a substantial impact on the final OD600 that was reached by the cultures of all 11 tested isolates at 37◦C, while at 55◦C the additional salt stress resulted in lower cell densities (Supplementary Figure S1)
Summary
Bacillus licheniformis is a facultative anaerobic endosporeproducing bacterium that is ubiquitously found in the environment, plant material, and soil. B. licheniformis has occasionally been reported as a causative agent of foodborne intoxication after the consumption of cooked meat, vegetables, milk powders, and dairy products (Salkinoja-Salonen et al, 1999; Pavic et al, 2005; Logan, 2012; Rønning et al, 2015). The agent causing foodborne illness was identified as the surfactant lichenysin (Salkinoja-Salonen et al, 1999; Mikkola et al, 2003) with one fatal case linked to the consumption of infant formula containing lichenysin (Salkinoja-Salonen et al, 1999)
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