Study of the molecular bases governing biofilm formation in Bacillus cereus

Conclusiones: Entre las conclusiones obtenidas, cabe destacar: - B. cereus produce fibras tipo amiloides necesarias para el correcto desarrollo estructural del biofilm. - El exopolisacárido denominado EPS1 está implicado en la movilidad tipo swarming de la colonia, mientras que el EPS2 tiene un papel determinante en la adhesión y en la formación de biofilm. Ambos polisacáridos tienen un papel sinérgico en la protección de la colonia. - Los individuos que forman parte del biofilm sobrexpresan los elementos relacionados con la defensa frente al ataque del huésped o competidores, mientras que las células en estado planctónico muestran un estado de ataque, sobrexpresando toxinas y elementos para un ataque rápido y efectivo. Fecha de lectura de Tesis: 6 de julio 2018.Introducción o motivación de la tesis: B. cereus es un patógeno humano responsable de múltiples enfermedades, destacando por ser más comunes las intoxicaciones alimentarias por el consumo de alimentos contaminados en origen. La formación de biofilms está directamente relacionada con la contaminación de alimentos, bien por favorecer la adhesión de las bacterias sobre la superficie de los alimentos o como reservorio de esporas y células vegetativas en la industria alimentaria, contaminando lotes completos de productos comerciales. El estudio de las bases moleculares de la formación de biofilm en esta bacteria es escaso, lo que ha motivado la realización de esta tesis doctoral. Desarrollo teórico y metodología: Para el desarrollo de la tesis doctoral se ha utilizado un triple enfoque genético, transcriptómico y proteómico. Se ha estudiado la formación de fibras de proteínas tipo amiloides como elemento estructural indispensable para la formación de biofilm y se han caracterizado bioquímicamente dos proteínas implicadas directamente en la formación de estas fibras. De forma general, los exopolisacáridos también constituyen un elemento principal en la formación de biofilm. Se han estudiado dos regiones génicas implicadas en la síntesis de exopolisacáridos, siendo uno de ellos imprescindible para el correcto desarrollo del biofilm. Además, el enfoque transcriptómico y proteómico ha permitido la identificación de múltiples funciones y adaptaciones de B. cereus al pasar al estilo de vida sésil y en comunidad que supone la formación de un biofilm

Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired biofilms can be formed by spoilage and pathogenic microorganisms in food production environments. Bacillus cereus is a foodborne human pathogen able to cause two types of food poisoning, emetic and diarrheal. B. cereus can persist in factory environments in the form of biofilms, which can become a source of food contamination. This thesis adds to the knowledge about (a)biotic factors and conditions that affect B. cereus biofilm formation, including the effect of type of substratum such as polystyrene and stainless steel, with the latter supporting the highest biofilm formation for all tested strains including two reference strains and 20 food isolates. The ability of B. cereus to use a variety of iron sources was subsequently studied in these 22 strains and linked to the genes encoding iron transport systems present in the respective genomes, revealing significant diversity in the capacity to use complex and non-complex iron sources for growth and biofilm formation. For spore forming Bacilli, biofilm formation and sporulation are two intertwined cellular processes and studies in wet and dry (air-exposed) biofilms revealed differences in sporulation rate and efficacy, with biofilm-derived spores showing higher heat resistance than their planktonic counterparts. Additionally, comparative phenotype and transcriptome analysis of B. cereus wild type and a Sigma 54 deletion mutant provided insight into the pleiotropic role of this transcriptional regulator in B. cereus biofilm formation and physiology in general. Taken together, this knowledge improves our understanding of the biofilm lifecycle of this notorious food-borne human pathogen and provides clues which can help to reduce the domestication of this microorganism in production environments.</p

Poultry and Beef Meat as Potential Seedbeds for Antimicrobial Resistant Enterotoxigenic Bacillus Species: A Materializing Epidemiological and Potential Severe Health Hazard

Although Bacillus cereus is of particular concern in food safety and public health, the role of other Bacillus species was overlooked. Therefore, we investigated the presence of eight enterotoxigenic genes, a hemolytic gene and phenotypic antibiotic resistance profiles of Bacillusspecies in retail meat samples. From 255 samples, 124 Bacillus isolates were recovered, 27 belonged to B. cereusand 97 were non-B. cereus species. Interestingly, the non-B. cereus isolates carried the virulence genes and exhibited phenotypic virulence characteristics as the B. cereus. However, correlation matrix analysis revealed the B. cereus group positively correlates with the presence of the genes hblA, hblC, and plc, and the detection of hemolysis (p \u3c 0.05), while the other Bacillus sp. groups are negatively correlated. Tests for antimicrobial resistance against ten antibiotics revealed extensive drug and multi-drug resistant isolates. Statistical analyses didn’t support a correlation of antibiotic resistance to tested virulence factors suggesting independence of these phenotypic markers and virulence genes. Of special interest was the isolation of Paenibacillus alvei and Geobacillus stearothermophilus from the imported meat samples being the first recorded. The isolation of non-B. cereus species carrying enterotoxigenic genes in meat within Egypt, suggests their impact on food safety and public health and should therefore not be minimised, posing an area that requires further research

Detection and characterization of Bacillus cereus isolated from the dialysis fluid

In this study, B. cereus was detected in dialysis fluids within international parameters (ultrapure – maximum limit of 0.1 CFU/mL for heterotrophic bacteria count) by analyzing the pellet obtained through the centrifugation method. We also investigated the ability of the B. cereus isolate to form a biofilm at different temperatures, the production of virulence factors, and the susceptibility to commercial antimicrobial agents. This study demonstrated a high ability of B. cereus to persist in the hemodialysis system, which can be explained by its broad ability to produce a biofilm at 25 °C, its relevant production of virulence factors, such as β-hemolysin, lecithinase and cereulide, and its important resistance pattern to antimicrobial drugs. In conclusion, these new findings expand the understanding that this microorganism should not be neglected and new methods for tracking it should be considered

The spore formation and toxin production in biofilms of Bacillus cereus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Listed in Dean's List of Exceptional Theses 2022Bacillus cereus (B. cereus) is a foodborne pathogen causing diarrhoea and emesis which are the consequences of enterotoxin and emetic toxin production, respectively. Sporulation and biofilm formation are used as survival strategies by B. cereus protecting cells from harsh environments. However, these survival strategies also make B. cereus more difficult to control in the food industry. The aim of this study is to investigate the spore formation and toxin production in the biofilm of B. cereus. In this study, higher sporulation and higher spore heat resistance were demonstrated in biofilms grown on stainless-steel (SS) compared to planktonic populations. The structure of coat in spores isolated from biofilms, the upregulated germination genes in planktonic cells and upregulated sigma factor B in biofilm cells are possible explanations for these observations. The levels of dipicolinic acid (DPA) did not affect the heat resistance of spores harvested from biofilms in this study. Haemolytic toxin (Hbl) was mainly secreted by cells into surrounding media while emetic toxin (cereulide) was associated with cells. Higher Hbl toxin was observed in the presence of biofilms grown on SS compared to either planktonic culture or biofilm grown on glass wool (GW) using the Bacillus cereus Enterotoxin Reverses Passive Latex Agglutination test (BCET-RPLA). This was supported by the significant (P < 0.05) increase in HblACD expression in biofilm cells on SS, using both real-time quantitative PCR (RT-qPCR) and RNA sequencing. The transcriptomic analysis also revealed that biofilms grown on SS had an upregulated secretion pathway, suggesting biofilms of B. cereus grown on SS are more pathogenic than planktonic cells. Unlike the Hbl toxin, cereulide was associated with biofilm cells/structures and attached to the biofilm-forming substrates including SS and GW used in this study. The expression of cerA and cerB was similar between biofilms and planktonic cells using RT-qPCR. This project highlights the importance of biofilms by B. cereus in food safety through the enhanced heat resistance of spores, the higher Hbl toxin production and attached cereulide toxin

The Use of Ozone as an Eco-Friendly Strategy against Microbial Biofilm in Dairy Manufacturing Plants: A Review

Managing spoilage and pathogenic bacteria contaminations represents a major challenge for the food industry, especially for the dairy sector. Biofilms formed by these microorganisms in food processing environment continue to pose concerns to food manufacturers as they may impact both the safety and quality of processed foods. Bacteria inside biofilm can survive in harsh environmental conditions and represent a source of repeated food contamination in dairy manufacturing plants. Among the novel approaches proposed to control biofilm in food processing plants, the ozone treatment, in aqueous or gaseous form, may represent one of the most promising techniques due to its antimicrobial action and low environmental impact. The antimicrobial effectiveness of ozone has been well documented on a wide variety of microorganisms in planktonic forms, whereas little data on the efficacy of ozone treatment against microbial biofilms are available. In addition, ozone is recognized as an eco-friendly technology since it does not leave harmful residuals in food products or on contact surfaces. Thus, this review intends to present an overview of the current state of knowledge on the possible use of ozone as an antimicrobial agent against the most common spoilage and pathogenic microorganisms, usually organized in biofilm, in dairy manufacturing plants

The antimicrobial effect of red wine on Bacillus cereus in simulated gastrointestinal conditions

Diversos estudos têm vindo a descrever uma panóplia de efeitos benéficos na saúde humana, potencialmente atribuíveis ao consumo de vinho, incluindo efeito anti-oxidante, anti-carcinogénico, anti-inflamatório, anti-cardiovascular, assim como propriedades antimicrobianas. Este estudo foi conduzido com o objectivo de avaliar a actividade antimicrobiana do vinho sobre Bacillus cereus, células vegetativas e esporos. Os resultados apresentados neste trabalho indicam claramente, via testes in vitro, que o vinho inactiva com eficácia as células vegetativas das duas estirpes de B. cereus utilizadas. O vinho tinto inactivou as células vegetativas em fase estacionária, atingindo-se números de colónias não detectáveis (< 500 CFU mL-1), em menos de 10 s de exposição. Como tal, os ensaios de inactivação subsequentes foram efectuados com vinho diluído com água (diluição de 1:4 e 1:8). O vinho diluído 1:4 causou uma redução de 4.5 ciclos logarítmicos nas contagens de células viáveis, em 20 s de ensaio. No entanto, os esporos de B. cereus apresentaram uma elevada resistência à exposição directa ao vinho, com reduções nas contagens inferiores a 1.0 ciclo logarítmico, em 3 h. A influência de componentes do vinho (etanol, ácidos orgânicos, baixo pH e compostos fenólicos) também foi contemplada neste estudo, em células vegetativas. A combinação de ácidos orgânicos e etanol resultou numa actuação sinergética, que provocou padrões de inibição de viabilidade celular similares aos do vinho. Os compostos fenólicos testados não causaram inactivação das células (nas concentrações utilizadas). Relativamente aos resultados obtidos em condições gástricas simuladas, em contexto de refeição simulada, podemos concluir que o consumo de vinho ao longo de uma refeição pode diminuir consideravelmente o número de células de B. cereus que poderá persistir no tracto gastrointestinal. O queijo fresco pasteurizado conferiu maior protecção às células do B. cereus, quando comparado com a matriz arroz com frango. Nesta investigação também foi avaliado o comportamento de esporos de B. cereus quando submetidos a condições gastrointestinais na presença e na ausência de vinho. A presença de vinho inibe a multiplicação das células resultantes da germinação de esporos no fluido intestinal sintético, dando origem a contagens totais (células vegetativas e esporos) de B. cereus mais baixas do que na ausência de vinho. Esta tese gerou resultados que indicam que o consumo de vinho durante uma refeição conduz à redução do número de células viáveis de B. cereus no tracto gastrointestinal, assim como à diminuição do impacto da eventual germinação de esporos que pode ocorrer no intestino, reduzindo, consequentemente, o risco de infecção que o referido patogénico pode causar.Several studies describe the burgeoning health benefits of red wine consumption, including antioxidative, anti-carcinogenic, anti-inflammatory, anti-cardiovascular and antimicrobial properties. This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results of this work clearly show, via in vitro tests, that wine exerts a strong inactivation effect against vegetative cells of two B. cereus strains. The red wine tested inactivated B. cereus stationary phase vegetative cells to undetectable numbers (< 500 CFU mL-1) in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Wine diluted 1:4 caused a reduction of 4.5 log cycles on viable cell counts, in 20 s. Nevertheless, B. cereus spores were found to be highly resistant to the wine exposure, with decreases in the counts lower than 1.0 log cycles, after 3 h. The influence of wine components (ethanol, organic acids, low pH and phenolic compounds) was investigated on vegetative cells. Organic acids, when combined with ethanol, acted synergistically and conduced to a similar inhibition pattern as that of wine. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations studied. Regarding data obtained in simulated gastric conditions, in a simulated meal context, we can conclude that the ingestion of wine during a meal diminishes considerably the number of B. cereus cells persisting in the alimentary tract. Pasteurized fresh cheese was found to be more protective to the cells than the chicken-rice matrix. We also evaluated the behavior of B. cereus spores under gastrointestinal conditions. In a consumption-like scenario, the treatment SGF (synthetic gastric fluid)- SIF (synthetic intestinal fluid) +Food+Wine, when compared to the system SGF-SIF+Food+Water, led to lower total counts of B. cereus in the intestine, showing that wine inhibits the multiplication of the cells obtained from the germination of spores. This work provides evidence that drinking wine with meals leads to a reduction of the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of infection the aforementioned pathogen may cause

Exosporium Morphogenesis in Bacillus Cereus and Bacillus Anthracis

Bacillus cereus is a Gram-positive spore-forming bacteria that can cause food poisoning and its close relative, Bacillus anthracis is the etiological agent of anthrax. In both cases, the spore, a differentiated cell type in a dormant state, starts the infection process. Thus, the exosporium, which constitutes the surface of the spore, plays an important role during natural infection of both B. cereus and B. anthracis. Proteins from the exosporium of B. cereus ATCC 4342, a B. anthracis-like strain, were extracted with 2% β-mercaptoethanol under alkaline conditions and identified by liquid chromatography coupled with tandem mass spectrometry. A novel cell surface protein, reticulocyte binding protein (Rbp), was identified in this sample. Inactivation of rbp by insertional mutagenesis resulted in spores devoid of the hair-like nap typical of the exosporium, which suggests that Rbp could be a new component of the exosporium nap or could have a role in its assembly. GerQ, a protein that is crosslinked by transglutaminase in the spore coat of Bacillus subtilis, was also identified in the B. cereus ATCC 4342 exosporium.Absence of GerQ during sporulation resulted in a brittle exosporium in both B. cereus ATCC 4342 and B. anthracis ΔSterne spores. This suggests that transglutaminase participates in exosporium assembly and maturation by crosslinking small proteins and processed peptides, providing structural stability and resistance to degradation to the spore surface. In addition, a novel exosporium protein, ExsM, was characterized in this study. Subcellular localization of an ExsM-GFP fusion protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutants presented a tightly wrapped exosporium resulting in smaller and rounder spores than wild-type spores that have an extended exosporium. Thin-section electron microscopy revealed that exsM spores were encased by a double layer exosporium, both of which were composed of a basal layer and a hair-like nap. Compared to wild-type spores, exsM spores were more resistant to lysozyme treatment, germinated with a higher efficiency, and had a delay in outgrowth. Insertional mutagenesis of exsM in B. anthracis ΔSterne rendered spores with a partial second exosporium that were also smaller in size. These findings suggest that ExsM plays a critical role in the formation of the exosporium. B.cereus, but not B. anthracis, spores possess long appendages projecting from their surface. B. cereus ATCC 14579 appendages were isolated from spores by extraction with 2% β-mercaptoethanol under alkaline conditions, followed by CsCl gradient ultracentrifugation. Mass spectrometry analysis revealed that camelysin (CalY) and spore coat-associated protein N (CotN) were associated with the appendages sample. Both proteins are homologous to TasA, the main component of B. subtilis biofim extracellular matrix. TasA forms amyloids fibrils that hold the cells together and provide structure to the extracellular matrix. Therefore, spore appendages may have a role in biofilm formation, acting like a scaffold for the biofilm matrix and holding the spores together