Role of Bacillus species in biofilm persistence and emerging antibiofilm strategies in the dairy industry.

Biofilm-forming Bacillus species are often involved in persistent contamination and spoilage of dairy products. They therefore present a major microbiological challenge in the field of dairy food quality and safety. Due to their substantial physiological versatility, Bacillus species can survive in various parts of dairy manufacturing plants, leading to a high risk of product spoilage and potential dissemination of foodborne diseases. Furthermore, biofilm and heat-resistant spore formation make these bacteria challenging to eliminate. Thus, some strategies have been employed to remove, prevent, or delay the formation of Bacillus biofilms in the dairy industry, but with limited success. Lack of understanding of the Bacillus biofilm structure and behavior in conditions relevant to dairy-associated environments could partially account for this situation. The current paper reviews dairy-associated biofilm formation by Bacillus species, with particular attention to the role of biofilm in Bacillus species adaptation and survival in a dairy processing environment. Relevant model systems are discussed for the development of novel antimicrobial approaches to improve the quality of dairy food. © 2020 Society of Chemical Industry.

Adaptation of Bacillus species to dairy associated environment facilitates their biofilm forming ability.

Biofilm-forming Bacillus species are often involved in contamination of dairy products and therefore present a major microbiological challenge in the field of food quality and safety. In this study, we sequenced and analyzed the genomes of milk- and non-milk-derived Bacillus strains, and evaluated their biofilm-formation potential in milk. Unlike non-dairy Bacillus isolates, the dairy-associated Bacillus strains were characterized by formation of robust submerged and air-liquid interface biofilm (pellicle) during growth in milk. Moreover, genome comparison analysis revealed notable differences in putative biofilm-associated determinants between the dairy and non-dairy Bacillus isolates, which correlated with biofilm phenotype. These results suggest that biofilm formation by Bacillus species might represent a presumable adaptation strategy to the dairy environment.

Bacillus strain BX77: a potential biocontrol agent for use against foodborne pathogens in alfalfa sprouts.

Despite regulatory and technological measures, edible sprouts are still often involved in foodborne illness and are considered a high-risk food. The present study explored the potential of spore-forming Bacillus isolates to mitigate Salmonella and Escherichia coli contamination of alfalfa sprouts. Food-derived Bacillus strains were screened for antagonistic activity against S. enterica serovar Typhimurium SL1344 (STm) and enteropathogenic E. coli O55:H7. Over 4 days of sprouting, levels of STm and E. coli on contaminated seeds increased from 2.0 log CFU/g to 8.0 and 3.9 log CFU/g, respectively. Treatment of the contaminated seeds with the most active Bacillus isolate, strain BX77, at 7 log CFU/g seeds resulted in substantial reductions in the levels of STm (5.8 CFU/g) and E. coli (3.9 log CFU/g) in the sprouted seeds, compared to the control. Similarly, co-culturing STm and BX77 in sterilized sprout extract at the same ratio resulted in growth inhibition and killed the Salmonella. Confocal-microscopy experiments using seeds supplemented with mCherry-tagged Salmonella revealed massive colonization of the seed coat and the root tip of 4-day-old sprouted seeds. In contrast, very few Salmonella cells were observed in sprouted seeds grown with BX77. Ca-hypochlorite disinfection of seeds contaminated with a relatively high concentration of Salmonella (5.0 log CFU/g) or treated with BX77 revealed a mild inhibitory effect. However, disinfection followed by the addition of BX77 had a synergistic effect, with a substantial reduction in Salmonella counts (7.8 log CFU/g) as compared to untreated seeds. These results suggest that a combination of chemical and biological treatments warrants further study, toward its potential application as a multi-hurdle strategy to mitigate Salmonella contamination of sprouted alfalfa seeds.

A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling.

The spore-forming bacterium Bacillus subtilis forms matrix-enclosed biofilms in response to environmental cues that to date remain poorly defined. Biofilm formation depends on the synthesis of an extracellular matrix, which is indirectly regulated by the transcriptional regulator Spo0A. The activity of Spo0A depends on its phosphorylation state. The level of phosphorylated Spo0A (Spo0A~P) is controlled by a network of kinases and phosphatases, which respond to environmental and physiological signals. In spite of significant progress in understanding biofilm development, the fundamental question of how cells sense the environmental cues that trigger biofilm formation has largely remained unaddressed. Here, we report that biofilm formation of B. subtilis in LB medium is triggered by a combination of glycerol and manganese (GM). Moreover, LB medium with GM significantly stimulates biofilm-associated sporulation and production of an undefined brown pigment. We further show that transcription of the major operons responsible for matrix production and biofilm formation is dramatically enhanced in response to GM. We also establish that KinD is a principal histidine kinase responsible for sensing the presence of GM exclusively by its extracellular CACHE domain. Finally, we show that GM has a similar biofilm-promoting effect in two related Bacillus species, B. licheniformis and B. cereus, indicating that the biofilm-promoting effect of GM is conserved in Bacillus species.

The role of milk fat globule size in modulating the composition of postbiotics produced by Bacillus subtilis and their effect on mammary epithelial cells.

Milk lipids are secreted into the milk collecting ducts as milk fat globule (MFG) where they are exposed to microflora of the udder. We hypothesized that MFG size modulates the metabolic fingerprint of B. subtilis. Accordingly, small and large (2.3 and 7.0 µm, respectively) MFG were isolated from cow milk and used as a substrate for B. subtilis. Small MFG enhanced growth, whereas large MFG enhanced biofilm formation. Bacteria incubated with small MFG had increased concentration of metabolites related to energy production whereas metabolome of the bacteria incubated with large MFG had reduced concentrations of metabolites important for biofilm formation. Postbiotics from bacteria grown on large MFG exacerbated the proinflammatory response of MEC to LPS, and changed the expression of key enzymes involved in lipid and protein synthesis. Our results suggest that MFG size modulate growth trajectories and metabolome of B. subtilis, and consequently the stress response of host cells.

Upregulation of ica Operon Governs Biofilm Formation by a Coagulase-Negative Staphylococcus caprae.

Staphylococcus caprae is a Gram-positive, coagulase-negative staphylococci (CoNS), which appears as commensal in the skin, as well as a prevalent mastitis pathogen of goats. Occasionally, it is also associated with infections in humans. Biofilm formation has been identified as a putative virulence factor in S. caprae. Biofilms are multicellular communities protected by a self-produced extracellular matrix (ECM), which facilitates the resistance of bacterial cells to antimicrobial treatments. The ECM is constructed by exopolysaccharides, including the major exopolysaccharide-polysaccharide intercellular adhesion (PIA), regulated by the ica operon in Staphylococcus species. The aim of this study was to characterize the expression of the ica operon in relation to biofilm formation in S. caprae. Results showed that within a few hours of growth, S. caprae could adhere to polystyrene surfaces, start to accumulate, and form biofilm. Peak biofilm biomass and maturation were reached after 48 h, followed by a reduction in biomass after 72 h. Confocal laser scanning microscopy showed the expression of matrix-associated proteins and polysaccharides at various time points. The expression dynamics of the ica operon were investigated using real-time reverse transcriptase PCR (RT)-qPCR, which showed elevated expression during the early stages of biofilm formation and subsequent downregulation throughout the biofilm aging process. In conclusion, our results show that the ica operon is essential in regulating biofilm formation in S. caprae, similar to other Staphylococcus species. Furthermore, the robustness of the observed biofilm phenotype could account for the successful intramammary colonization and may explain disease persistence caused by this pathogenic bacterium.

Pulcherrimin protects Bacillus subtilis against oxidative stress during biofilm development.

Pulcherrimin is an iron-binding reddish pigment produced by various bacterial and yeast species. In the soil bacterium Bacillus subtilis, this pigment is synthesized intracellularly as the colorless pulcherriminic acid by using two molecules of tRNA-charged leucine as the substrate; pulcherriminic acid molecules are then secreted and bind to ferric iron extracellularly to form the red-colored pigment pulcherrimin. The biological importance of pulcherrimin is not well understood. A previous study showed that secretion of pulcherrimin caused iron depletion in the surroundings and growth arrest on cells located at the edge of a B. subtilis colony biofilm. In this study, we identified that pulcherrimin is primarily produced under biofilm conditions and provides protection to cells in the biofilm against oxidative stress. We presented molecular evidence on how pulcherrimin lowers the level of reactive oxygen species (ROS) and alleviates oxidative stress and DNA damage caused by ROS accumulation in a mature biofilm. We also performed global transcriptome profiling to identify differentially expressed genes in the pulcherrimin-deficient mutant compared with the wild type, and further characterized the regulation of genes by pulcherrimin that are related to iron homeostasis, DNA damage response (DDR), and oxidative stress response. Based on our findings, we propose pulcherrimin as an important antioxidant that modulates B. subtilis biofilm development.

The Bacillary Postbiotics, Including 2-Undecanone, Suppress the Virulence of Pathogenic Microorganisms.

Secreted molecules from probiotic Bacilli have often been considered potential pharmaceuticals to fight infections caused by bacterial or yeast pathogens. In the present study, we investigated the antagonistic potential of secreted probiotic filtrates (hereafter, postbiotics) derived from Lactobacillus plantarum cells against pathogenic microorganisms, such as Escherichia coli, Staphylococcus aureus, and Candida albicans. We found that the postbiotics mitigate the biofilms of the tested pathogens with no notable effect on their planktonic growth. In addition, the postbiotics suppressed some virulence traits, for instance, the dendrite swarming motility of E. coli and yeast-to-hyphal switch in C. albicans. Further assays with an active constituent produced by the L. plantarum cells-2-undecanone revealed two significant findings: (i) 2-undecanone inhibits C. albicans biofilms and hyphae in vitro and in a Caenorhabditis elegans model, and (ii) it interacts specifically with Gln 58 amino acid residue of hyphal wall protein-1 (Hwp-1) in molecular docking analysis. The results suggest the targeted mode of antagonistic action of 2-undecanone against C. albicans biofilm. In total, the findings of the study depict an appealing strategy to use postbiotics, including specific ketone molecules, produced by L. plantarum for developing novel antibiofilm and anti-hyphal pharmaceuticals.

Spatiotemporal bio-shielding of bacteria through consolidated geometrical structuring.

The probiotic bacterium Lactobacillus plantarum is often reckoned as a 'generalist' for its ability to adapt and survive in diverse ecological niches. The genomic signatures of L. plantarum have shown its intricate evolutionary ancestry and dynamic lifestyles. Here, we report on a unique geometrical arrangement of the multicellular population of L. plantarum cells. Prominently, a phenomenon of the cone-shaped colony formation and V-shaped cell chaining are discovered in response to the acidic-pH environment. Moreover, subsequent cold stress response triggers an unusual cellular arrangement of consolidated bundles, which appeared to be independently governed by a small heat shock protein (HSP 1). We further report that the V-shaped L. plantarum chaining demonstrates potent antagonistic activity against Candida albicans, a pathogenic yeast, both in vitro and in a Caenorhabditis elegans co-infection model. Finally, we deduce that the multifaceted traits manifested by this probiotic bacterium is an outcome of its dynamic flexibility and cellular heterogeneity.

The milk fat globule size governs a physiological switch for biofilm formation by Bacillus subtilis.

Milk lipids are organized in the form of milk fat globules (MFG), ranging in size from 0. 1 to 15 µm. The MFG size is closely associated with the composition of fatty acids, polar lipids, sphingolipids, cholesterol and the content of the MFG membrane (MFGM). Also, the MFGM integral proteins and glycoconjugates differ in composition and structure between different MFG size groups. These compositional differences may modulate the functionality of the MFG and its interaction with microbial cells. We report that small (2.3 µm) MFG facilitates the growth of the Gram-positive bacterium Bacillus subtilis whereas induction of biofilm formation was found in the presence of large (7.0 µm) MFG. Attempting to distinguish between the role played by the size from that played by the composition of the MFG, we compared phospholipid composition between treatments. We found that adjusting the phosphatidylethanolamine (PE) level to the concentration found in the small MFG, increased growth but suppressed biofilm formation in the presence of large MFG. The same normalization protocol for phosphatidylinositol (PI) or sphingomyeline (SM) did not exert a similar effect, suggesting a specific role for PE in regulating bacteria proliferation. We suggest that the content of MFGM, affected by MFG size, governs the ability of B. subtilis to utilize lipids from milk fat. This process might affect the bacterial decision-making toward biofilm formation or growth.

Role of Probiotic Bacilli in Developing Synbiotic Food: Challenges and Opportunities.

The human body is inhabited by a vast diversity of probiotic microorganisms that could positively affect human physiology. Besides, prebiotic food substances may induce symbiotic relationship among probiotic species through the successful establishment of commensal microbiota, whose connections with the host are multifaceted and multidirectional. As deliberated throughout this review, prebiotic and synbiotic foods contain the capability to stimulate numerous health characteristics in host organisms through various means. Predominantly, the normal microbiota fosters the digestion of food and may boost the innate and adaptive immune system's functionalities. Therefore, live probiotic bacteria, for instance, probiotic Bacilli obtained together with prebiotic food, can help stimulate healthiness in humans. Thus, we discuss how certain dietary fibers may preserve the probiotic efficacy by serving as the scaffold for probiotic Bacilli to colonize them through forming symbiotic interactions. The fibers can essentially promote protection by encapsulating probiotic Bacilli against various environmental and physical stresses that might kill the free-living bacterial cells. Besides, these fibers would serve as prebiotic substances that would eventually be utilized for the proliferation of probiotic cells. It is believed that applying this conceptual idea will provide a novel platform toward developing probiotic and synbiotic foods, as discussed in this review.

Antimicrobial Effect of Zn2+ Ions Governs the Microbial Quality of Donor Human Milk.

Donor human milk (HM) obtained at HM banks is exceptionally crucial for the feeding and treatment of preterm infants. Bacterial contaminations of HM in various stages of its handling are very common and can lead to disqualification of donations or severe infections in worse cases. Hence, HM donations are subject to strict bacteriological evaluations pre- and post-pasteurization. The main contaminating species vary between countries, banks and donors and even exhibit inter-individual variation. We initiated an assessment of the bacteriological composition of HM donated by women hospitalized in a neonatal intensive care unit in Israel. The most common bacterium identified was Staphylococcus epidermidis, found in all but one of the HM samples; the presence of several species of coagulase-negative Staphylococci was also noted. Next, we sought to develop a platform towards antibacterial treatment using Zn2+ ions that have recently been found to be potent against contaminants isolated from bovine milk. Zn2+ efficiently inhibited the growth of viable aerobic population and S. epidermidis in HM. Growth was also inhibited in other Gram-positive bacteria such as Bacillus cereus, a well-known food-borne pathogen. S. epidermidis and B. cereus cells grown in the presence of zinc were taken for microscopic evaluation, aiming to demonstrate zinc's antimicrobial mode of action morphologically. Images obtained using scanning electron microscopy indicated leakage of cellular content and cell lysis in S. epidermidis. Besides, B. cereus cells showed abnormalities in their cell surface and complete loss of flagella upon treatment with zinc. Along with the above findings, it should be noted that this was a pilot study that tested how high doses of Zn2+ affect breast milk as a product. Further research is likely needed on the safety of consumption of Zn2+-treated HM in infants and older children.

Chickpea-Derived Prebiotic Substances Trigger Biofilm Formation by Bacillus subtilis.

Chickpea-based foods are known for their low allergenicity and rich nutritional package. As an essential dietary legume, chickpea is often processed into milk or hummus or as an industrial source of protein and starch. The current study explores the feasibility of using the chickpea-derived prebiotic substances as a scaffold for growing Bacillus subtilis (a prospective probiotic bacterium) to develop synbiotic chickpea-based functional food. We report that the chickpea-derived fibers enhance the formation of the B. subtilis biofilms and the production of the antimicrobial pigment pulcherrimin. Furthermore, electron micrograph imaging confirms the bacterial embedding onto the chickpea fibers, which may provide a survival tactic to shield and protect the bacterial population from environmental insults. Overall, it is believed that chickpea-derived prebiotic substances provide a staple basis for developing functional probiotics and synbiotic food.

Biofilm formation onto starch fibres by Bacillus subtilis governs its successful adaptation to chickpea milk.

Beneficial biofilms may confer effective adaptation to food matrices that assist bacteria in enduring hostile environmental conditions. The matrices, for instance, dietary fibres of various food products, might serve as a natural scaffold for bacterial cells to adhere and grow as biofilms. Here, we report on a unique interaction of Bacillus subtilis cells with the resistant starch fibresof chickpea milk (CPM), herein CPM fibres, along with the production of a reddish-pink pigment. Genetic analysis identified the pigment as pulcherrimin, and also revealed the involvement of Spo0A/SinI pathway in modulating the observed phenotypes. Besides, through successful colonization of the CPM fibres, the wild-type cells of B. subtilis displayed enhanced survivability and resilience to environmental stress, such as heat and in vitro gastrointestinal treatments. In total, we infer that the biofilm formation on CPM fibres is an adaptation response of B. subtilis for strategic survival.

The biocide chlorine dioxide stimulates biofilm formation in Bacillus subtilis by activation of the histidine kinase KinC.

Bacillus subtilis forms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (ClO(2)), an extremely effective and fast-acting biocide. ClO(2) accelerated biofilm formation in B. subtilis as well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production in B. subtilis, epsA-epsO and yqxM-sipW-tasA, was enhanced by ClO(2), in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of ClO(2) to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to ClO(2), indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide.

Genetic adaptation of Streptococcus mutans during biofilm formation on different types of surfaces.

BACKGROUND: Adhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces. RESULTS: Growth analysis of the immobilized bacterial populations generated on the different surfaces shows that the bacteria constructed a more confluent and thick biofilms on a hydroxyapatite surface compared to the other tested surfaces. Using DNA-microarray technology we identified the differentially expressed genes of S. mutans, reflecting the physiological state of biofilms formed on the different biomaterials tested. Eight selected genes were further analyzed by real time RT-PCR. To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans embedded on those biofilms. Comparative transcriptome analyses indicated on changes in the S. mutans genome in biofilms formed onto different types of surfaces and enabled us to identify genes most differentially expressed on those surfaces. In addition, the levels of autoinducer-2 in biofilms from the various tested surfaces were different. CONCLUSIONS: Our results demonstrate that gene expression of S. mutans differs in biofilms formed on tested surfaces, which manifest the physiological state of bacteria influenced by the type of surface material they accumulate onto. Moreover, the stressful circumstances of adjustment to the surface may persist in the bacteria enhancing intercellular signaling and surface dependent biofilm formation.

Efficiency of Bacillus subtilis metabolism of sugar alcohols governs its probiotic effect against cariogenic Streptococcus mutans.

Bacillus subtilis is a Gram-positive probiotic bacterium that successfully colonises plant roots due to its ability to utilise various sugars. The vast probiotic potential of B. subtilis has been recently demonstrated in numerous host organisms under different environmental conditions. We examined the probiotic potential of B. subtilis against the pathogenic bacterium Streptococcus mutans, which is involved in various oral disorders due to its robust biofilm-forming capability. B. subtilis cells attenuated biofilm formation by S. mutans during their dual growth in the presence of sugar alcohols. Transcription of genes encoding key enzymes in the metabolism of sugar alcohols by B. subtilis were highly induced. Moreover, growth-curve analysis suggested that B. subtilis is more efficient at early utilising sugar alcohols than S. mutans, as supported by the bacterial metabolic activity rates. Similarly, a comparison of secondary metabolites of mono and mixed cultures of B. subtilis and S. mutans indicated that B. subtilis is more active metabolically in the dual culture. Finally, knock-out mutations of the genes encoding key enzymes in the central metabolic pathway significantly reduced B. subtilis' ability to mitigate biofilm formation by S. mutans. We conclude that effective metabolism of sugar alcohols by B. subtilis reinforces the probiotic potential of this bacterium against pathogenic species such as S. mutans.

Mitigating Milk-Associated Bacteria through Inducing Zinc Ions Antibiofilm Activity.

Dairy products are a sector heavily impacted by food loss, often due to bacterial contaminations. A major source of contamination is associated with the formation of biofilms by bacterial species adopted to proliferate in milk production environment and onto the surfaces of milk processing equipment. Bacterial cells within the biofilm are characterized by increased resistance to unfavorable environmental conditions and antimicrobial agents. Members of the Bacillus genus are the most commonly found spoilage microorganisms in the dairy environment. It appears that physiological behavior of these species is somehow depended on the availability of bivalent cations in the environment. One of the important cations that may affect the bacterial physiology as well as survivability are Zn2+ ions. Thus, the aim of this study was to examine the antimicrobial effect of Zn2+ ions, intending to elucidate the potential of a zinc-based antibacterial treatment suitable for the dairy industry. The antimicrobial effect of different doses of ZnCl2 was assessed microscopically. In addition, expression of biofilm related genes was evaluated using RT-PCR. Analysis of survival rates following heat treatment was conducted in order to exemplify a possible applicative use of Zn2+ ions. Addition of zinc efficiently inhibited biofilm formation by B. subtilis and further disrupted the biofilm bundles. Expression of matrix related genes was found to be notably downregulated. Microscopic evaluation showed that cell elongation was withheld when cells were grown in the presence of zinc. Finally, B. cereus and B. subtilis cells were more susceptible to heat treatment after being exposed to Zn2+ ions. It is believed that an anti-biofilm activity, expressed in downregulation of genes involved in construction of the extracellular matrix, would account for the higher sensitivity of bacteria during heat pasteurization. Consequently, we suggest that Zn2+ ions can be of used as an effective antimicrobial treatment in various applications in the dairy industry, targeting both biofilms and vegetative bacterial cells.

Proteolytic Activity of Bacillus subtilis upon κ-Casein Undermines Its "Caries-Safe" Effect.

Milk is believed to be a relatively "caries-safe" food. This belief relies on the fact that caseins, which constitute around 80% of milk's protein content, were found to inhibit the adhesion of Streptococcus mutans to enamel and, therefore, decrease biofilm formation. While S. mutans is considered a leading cause of dental disorders, Bacillus subtilis is a non-pathogenic foodborne bacterium, frequently contaminating milk and its products. This study aimed to investigate the effects of dairy-associated foodborne bacteria such as B. subtilis on biofilm formation by S. mutans in the presence of casein proteins. Our results indicate that there is a significant decrease in total biofilm formation by S. mutans exposed to a casein protein mixture in a mono-species culture, whereas, in the co-culture with B. subtilis, an inhibitory effect of the caseins mixture on S. mutans biofilm formation was observed. Proteolytic activity analysis suggested that B. subtilis is capable of breaking down milk proteins, especially κ-casein, which enables biofilm formation by S. mutans in the presence of milk caseins. Therefore, these findings may challenge the assumption that milk is "caries-safe", especially in a complex microbial environment.

Analyzing movement trajectories using a Markov bi-clustering method.

In this study we treat scribbling motion as a compositional system in which a limited set of elementary strokes are capable of concatenating amongst themselves in an endless number of combinations, thus producing an unlimited repertoire of complex constructs. We broke the continuous scribblings into small units and then calculated the Markovian transition matrix between the trajectory clusters. The Markov states are grouped in a way that minimizes the loss of mutual information between adjacent strokes. The grouping algorithm is based on a novel markov-state bi-clustering algorithm derived from the Information-Bottleneck principle. This approach hierarchically decomposes scribblings into increasingly finer elements. We illustrate the usefulness of this approach by applying it to human scribbling.