Antimicrobial Bacillus: Metabolites and Their Mode of Action

The agricultural industry utilizes antibiotic growth promoters to promote livestock growth and health. However, the World Health Organization has raised concerns over the ongoing spread of antibiotic resistance transmission in the populace, leading to its subsequent ban in several countries, especially in the European Union. These restrictions have translated into an increase in pathogenic outbreaks in the agricultural industry, highlighting the need for an economically viable, non-toxic, and renewable alternative to antibiotics in livestock. Probiotics inhibit pathogen growth, promote a beneficial microbiota, regulate the immune response of its host, enhance feed conversion to nutrients, and form biofilms that block further infection. Commonly used lactic acid bacteria probiotics are vulnerable to the harsh conditions of the upper gastrointestinal system, leading to novel research using spore-forming bacteria from the genus Bacillus. However, the exact mechanisms behind Bacillus probiotics remain unexplored. This review tackles this issue, by reporting antimicrobial compounds produced from Bacillus strains, their proposed mechanisms of action, and any gaps in the mechanism studies of these compounds. Lastly, this paper explores omics approaches to clarify the mechanisms behind Bacillus probiotics.

Control of Bacillus weihenstephanensis in Pasteurized Liquid Whole Eggs Formulated with Nisin

Bacillus weihenstephanensis can grow at refrigeration temperature and cause food poisoning. It has been isolated from liquid whole egg products. The moderate heat used for pasteurization of liquid egg products is ineffective for killing spore-forming bacteria including Bacillus. Available predictive models and a pretrial study in broth suggested the potential for growth of Bacillus spp. under the tested conditions. Hence, hurdles such as storage of product below 4°C or use of preservatives would be needed to ensure the food safety of pasteurized egg products. This study evaluated the growth inhibition of B. weihenstephanensis in pasteurized liquid whole egg product formulated with 6.25 ppm nisin during storage at refrigerated and abuse refrigerated temperatures for a total 13 weeks, in three replicate trials. At day 0, the product had a pH of 7.52±0.29, while background microflora such as aerobic plate counts, presumptive B. cereus, and yeast and molds were <10 CFU/g. Product inoculated with target 2.5 log10 CFU/g of B. weihenstephanensis, stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks exhibited no growth in all three replicate trials. Average counts reduced (p<0.05) by at least one-log10 in six weeks in all samples stored at either 7 or 10°C. Similarly, growth of total plate counts, presumptive Bacillus spp., yeast and mold counts was not observed in uninoculated controls stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks. Visual and odor evaluation performed at each sampling time point showed no abnormalities. This study assessed the efficacy of maximum allowed level of nisin for use in pasteurized liquid whole eggs and validated the inhibition of B. weihenstephanensis in the product for an extended shelf life of up to 13 weeks.

Morphological and physiological-biochemical variability of spore-forming bacteria isolated from the agrocoenosis of winter wheat

From an agrocoenosis of winter wheat (Triticum aestivum L.; phylloplane and rhizosphere of the root system; typical chernozem, soil column measuring up to 40 cm), using the classical microbiological methods, we had isolated soil bacteria and characterized them according to the morphological features as representatives of Gram-positive and spore-forming bacteria of Bacillus sp. genus. In the earing-swelling phase of grain, the screening studies found non-pigmented forms of colonies of bacterial isolates, 19 of which were classified to colonial-morphological diversity of R-type with the diameter of 7 to 13 mm. The analysis of physiological condition of cells of populations of soil isolates revealed technologic specificity according to parameters of spore formation in different conditions and incubation time (up to 48–72 h). We observed 90.0% of free spores in axenic cultures as early as after 72 h of cultivation and no more than 10.0% of prospores in the studied monoisolates with stable morphologic traits. Isolates Н10 and Н45 demonstrated the ability to grow in higher cultivation temperatures (+37…+40 °С). According to environmental рН, isolates were able to grow in рН ranging 4.5–8.0. Differential diagnostic testing revealed that as the source of carbon, with formation of acid, soil isolates used arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch, rhamnose and dulcite (with development of alkaline). There was observed active use of mineral forms of nitrogen: ammonium salt and nitrates, aminoacids and proteins. The isolates hydrolyzed casein, gelatin, starch, and litmus was being reduced in the young during growth in milk with litmus. They also exerted catalase activity and were oxidase-positive. Biochemical testing using API test system determined that the studied isolated bacteria differed by a range of fermentation carbohydrates, reduction of nitrates. In the conditions of submerged fermentation, isolates Н38 and Н40 grew in heightened temperature ranges of cultivation (40 °С) for 48 h (according to fact of spore development). Therefore, according to the key morphologic and biochemical traits, strains Н3, Н10, Н13, Н36, Н38, Н40, Н43, Н45 were similar to such of reference strain B. subtilis 8A, and were identified to Bacillus sp., species B. subtilis.

Effect of Calcium and Manganese Supplementation on Heat Resistance of Spores of Bacillus Species Associated With Food Poisoning, Spoilage, and Fermentation

Bacterial spores often survive thermal processing used in the food industry, while heat treatment leads not only to a decrease in the nutritional and organoleptic properties of foods, but also to a delay in fermentation of fermented foods. Selective reduction of undesirable spores without such impediments is an ongoing challenge for food scientists. Thus, increased knowledge of the spore-forming bacteria is required to control them. In this study, the heat resistance results (D100°C) of the spores of four Bacillus species were determined and compared to previous literature, and found that B. cereus has significantly lower heat resistance than the other Bacillus species, B. coagulans, B. subtilis, and B. licheniformis. Using the spores of these strains, this study also evaluated the effects of single and combined supplementation of calcium (0.00–2.00 mM) and manganese (0.00–0.50 mM) on heat resistance (D100°C). The results revealed that the spores of B. licheniformis and B. cereus displayed the smallest heat resistance when sporulated on media rich in calcium. Conversely, B. coagulans spores and B. subtilis spores exhibited the greatest heat resistance when sporulated under calcium-rich conditions. The opposite results (stronger heat resistance for B. licheniformis spores and B. cereus spores, and smaller heat resistance for B. coagulans spores and B. subtilis spores) were obtained when the spores were formed on media poor in the minerals (particularly calcium). Based on the results, the Bacillus species were divided into two groups: B. licheniformis and B. cereus; and B. coagulans and B. subtilis. The study provides valuable insight to selectively reduce spores of undesirable Bacillus species in the food industry.

Distribution of Soil Microorganisms in Field under Potatoes due to Fertilizer and Organics

Background: Using organic manure derived from animal or vegetable matter is often associated with balanced crop production. The influence of organics and fertilizers on soil sustainability during cultivation of potatoes in field was studied. Methods: Vegetable experiment comprising 10 years (2011-2021), having a control variant (no fertilization), independent mineral, organics (compost) and their combination (50:50%). Changes in the amount and distribution of microbial groups in experimental variants and associated untreated controls were evaluated. Total bacterial number, spore-forming bacteria, fungi, nitrogen-utilizing bacteria and actinomycetes were assessed. Result: Applied organics and fertilizers had statistically proven a positive effect on growth parameters of potatoes. Organics and fertilizers supplied with mineral and foliar feeding registered taller plants with increased number of branches, number and mass of leaves and stem and root masses, compared to potatoes with organic and organic-mineral combination. Application of compost led to a significant increase in populations of all physiological groups of microorganisms excluding spore-forming bacteria and bacteria utilizing mineral nitrogen, which is a good indicator of increased soil microbial activity in respective treatments. Mineral fertilization had a positive effect on total number of bacteria and on absorption of mineral nitrogen. The treatment in variant (50:50 %) at rhizosphere soil was associated with organic manure application and at non-rhizosphere soil with mineral fertilizer.

Spore-forming bacteria in the dairy chain

Spore-forming bacteria form the most diverse and most complex group of bacteria in terms of their elimination from the dairy chain, due to their ability to form highly resistant spores. As ubiquitous microorganisms, spore-formers can enter the product along the milk-processing continuum from different sources, and subsequently cause spoilage in various types of dairy products. The most important classes of spore-forming bacteria relevant to the dairy industry are Bacilli and Clostridia. Bacilli are responsible mainly for the spoilage and decreased shelf-life of fluid milk, while Clostridia cause late gas blowing in cheese. Spore-forming microorganisms contaminate raw milk primarily at the farm level, with potential for recontamination to occur at various points along the dairy production continuum. The most effective measure in reducing spore load at the farm level is adequate pre-milking teat preparation, while at the dairy plant level, bactofugation and microfiltration are applied. Understanding the ecology of spore-formers can improve application of systematic approaches for controlling the spoilage bacteria in dairy processing systems. Also, novel technologies, such as high-pressure processing, ultrasound treatment, irradiation etc., could provide the dairy industry with the powerful tools to eliminate these bacteria from the dairy chain.

Farm Silage Facilities and Their Management for the Prevention of Anaerobic Bacteria Spore Contamination in Raw Milk

At feed-out, aerobic spoilage of silage enables an increase in anaerobic spore-forming bacteria (ANSB) that may enter the total mixed ration (TMR). The aim of our study was to understand whether in hot summers the silage structures and management may affect the level of ANSB in milk for long-ripening cheese production. A survey of silage facilities, management, and their relationships with silage, TMR, feces, and milk ANSB most probable number (MPN) content was conducted in the Po Valley during summer months. Silo type did not affect the mean ANSB, but only the wideness of their value distributions, with a narrow range for bags and a wider range for bunkers. The unloading equipment affected the ANSB count; the front-end loader with cutter was associated with a lower ANSB count—probably as a result of the reduced surface left after daily silage removal. Silo length and daily removed face width were the main factors affecting contamination of silage by spore-forming bacteria during summer, with longer silos and wider surface removal reducing ANSB contamination—probably as a consequence of reduced aerobic spoilage at the silage surface. The silage contamination by spore-forming bacteria within a log10 2 MPN g−1 allowed a low concentration of spore-forming bacteria at the farm bulk milk tank level. Fecal ANSB levels did not factor into the regression that explains the ANSB in farm milk. It has been found that silage facilities’ features and their management are an important first step to reduce the extent of ANSB contamination at the farm level.

Recent Advances in the Application of the Antimicrobial Peptide Nisin in the Inactivation of Spore-Forming Bacteria in Foods

Conventional thermal and chemical treatments used in food preservation have come under scrutiny by consumers who demand minimally processed foods free from chemical agents but microbiologically safe. As a result, antimicrobial peptides (AMPs) such as bacteriocins and nisin that are ribosomally synthesised by bacteria, more prominently by the lactic acid bacteria (LAB) have appeared as a potent alternative due to their multiple biological activities and represent a powerful strategy to prevent the development of spore-forming microorganisms. Unlike thermal methods, they are natural without an adverse impact on food organoleptic and nutritional attributes. AMPs such as nisin and bacteriocins are generally effective in eliminating spore-forming bacteria compared to the more resilient spore forms. However, in combination with other non-thermal treatments, such as high pressure, supercritical carbon dioxide, electric pulses, AMPs such as nisin has proven that the synergistic effect is effective in the inactivation of microbial spores through the disruption of the spore structure and prevention of spore outgrowth. The control of microbial spores in foods is essential in maintaining food safety and extension of shelf-life. Thus, exploration of the mechanisms of action of AMPs such as nisin is critical in their design and effective application in food industry. This review harmonises mechanisms of inactivation based information from published literature on utilising AMPs in the control of microbial spores in food. It highlights future perspectives in research and application in food processing.