Cereus Biofilm Research Articles

Impact of the Isolation Source on the Biofilm Formation Characteristics of Bacillus cereus.

The human pathogen and food spoiler Bacillus cereus can form biofilms that act as a persistent source of contamination, which is of public health concern. This study aimed to understand how the source of isolation might affect the behavior of biofilm formation. Biofilm formation abilities of 56 strains of B. cereus isolated from different environments, including human food poisoning, farm, and food, were determined. Crystal violet assay results revealed significant (p < 0.05) differences in biofilm formation abilities among the strains isolated from different sources only at an early stage of incubation. However, strain origin showed no impact on later stage of biofilm formation. Next, correlation of the group of isolates on the basis of their biofilm-forming abilities with the number of sessile cells, sporulation, and extracellular polymeric substance (EPS) formation was determined. The number of sessile cells and spores in biofilms was greatly influenced by the groups of isolates that formed dense, moderate, and weak biofilms. The contribution of extracellular DNA and/or proteins to EPS formation was also positively correlated with biofilm formation abilities. Our results that the source of isolation had significant impact on biofilm formation might provide important information to develop strategies to control B. cereus biofilm formation.

Optimisation of Bacillus cereus biofilm removal in the dairy industry using an in vitro model of cleaning‐in‐place incorporating serine protease

This article addresses a major hygienic issue in the dairy industry, namely biofilm removal. Response surface methodology was deployed to optimise Bacillus cereus biofilm removal conditions using serine protease. The currently practiced alkali cleaning‐in‐place (CIP) method was also optimised, and while the optimised protease CIP resulted in complete removal of biofilm cells, the reference CIP (with alkali) resulted in a reduction of 4.08 log biofilm cells/cm2 and the optimised alkali CIP resulted in a reduction of ∼4.92 log biofilm cells/cm2. Furthermore, the amount of the biofilm matrix removed in the optimised protease CIP was significantly higher than that of alkali CIP. It was concluded that the optimised protease CIP has better applicability.

Biofilm formation of Bacillus cereus under food-processing-related conditions.

This study aims to understand the biofilm formation abilities of eight Bacillus cereus strains under food-industry-related conditions. Biofilms were grown in microtiter plates in tryptic soy broth (TSB) or brain heart infusion (BHI) at 30°C for 24 or 48h and quantified via the crystal violet assay. A significantly larger of biofilm was formed in TSB than in BHI after 48h. Selected strains were used to test biofilm formation under food-related conditions produced by different surfaces (e.g., stainless steel, plastic, or glass), temperatures (25 or 30°C), carbon sources, (glucose or glycerol) and NaCl. Biofilm formation appeared to be affected by surface properties, temperature, and carbon sources. A larger biofilm was formed on stainless steel at 30°C compared to plastic and glass surfaces at 25 and 30°C. Moreover, addition of glucose in combination with NaCl in TSB produced significantly larger biofilm than glucose, glycerol and/or NaCl. These results indicate that food-industry-related conditions could promote B. cereus biofilm formation, which is relevant to food safety.

Genome-Wide Investigation of Biofilm Formation in Bacillus cereus.

Bacillus cereus is a soil-dwelling Gram-positive bacterium capable of forming structured multicellular communities, or biofilms. However, the regulatory pathways controlling biofilm formation are less well understood in B. cereus In this work, we developed a method to study B. cereus biofilms formed at the air-liquid interface. We applied two genome-wide approaches, random transposon insertion mutagenesis to identify genes that are potentially important for biofilm formation, and transcriptome analyses by RNA sequencing (RNA-seq) to characterize genes that are differentially expressed in B. cereus when cells were grown in a biofilm-inducing medium. For the first approach, we identified 23 genes whose disruption by transposon insertion led to altered biofilm phenotypes. Based on the predicted function, they included genes involved in processes such as nucleotide biosynthesis, iron salvage, and antibiotic production, as well as genes encoding an ATP-dependent protease and transcription regulators. Transcriptome analyses identified about 500 genes that were differentially expressed in cells grown under biofilm-inducing conditions. One particular set of those genes may contribute to major metabolic shifts, leading to elevated production of small volatile molecules. Selected volatile molecules were shown to stimulate robust biofilm formation in B. cereus Our studies represent a genome-wide investigation of B. cereus biofilm formation.IMPORTANCE In this work, we established a robust method for B. cereus biofilm studies and applied two genome-wide approaches, transposon insertion mutagenesis and transcriptome analyses by RNA-seq, to identify genes and pathways that are potentially important for biofilm formation in B. cereus We discovered dozens of genes and two major metabolic shifts that seem to be important for biofilm formation in B. cereus Our study represents a genome-wide investigation on B. cereus biofilm formation.

Cleaning and Disinfection of Bacillus cereus Biofilm.

Methodology has been evolving for the testing of disinfectants against bacterial single-species biofilms, as the difficulty of biofilm remediation continues to gain much-needed attention. Bacterial single-species biofilm contamination presents a real risk to good manufacturing practice-regulated industries. However, mixed-species biofilms and biofilms containing bacterial spores remain an even greater challenge for cleaning and disinfection. Among spore-forming microorganisms frequently encountered in pharmaceutical manufacturing areas, the spores of Bacillus cereus are often determined to be the hardest to disinfect and eradicate. One of the reasons for the low degree of susceptibility to disinfection is the ability of these spores to be encapsulated within an exopolysachharide biofilm matrix. In this series of experiments, we evaluated the disinfectant susceptibility of B. cereus biofilms relative to disassociated B. cereus spores and biofilm from a non-spore-forming species. Further, we assessed the impact that pre-cleaning has on increasing that susceptibility.

Comparative analysis of biofilm formation by Bacillus cereus reference strains and undomesticated food isolates and the effect of free iron

Biofilm formation of Bacillus cereus reference strains ATCC 14579 and ATCC 10987 and 21 undomesticated food isolates was studied on polystyrene and stainless steel as contact surfaces. For all strains, the biofilm forming capacity was significantly enhanced when in contact with stainless steel (SS) as a surface as compared to polystyrene (PS). For a selection of strains, the total CFU and spore counts in biofilms were determined and showed a good correlation between CFU counts and total biomass of these biofilms. Sporulation was favoured in the biofilm over the planktonic state. To substantiate whether iron availability could affect B. cereus biofilm formation, the free iron availability was varied in BHI by either the addition of FeCl3 or by depletion of iron with the scavenger 2,2-Bipyridine. Addition of iron resulted in increased air–liquid interface biofilm on polystyrene but not on SS for strain ATCC 10987, while the presence of Bipyridine reduced biofilm formation for both materials. Biofilm formation was restored when excess FeCl3 was added in combination with the scavenger. Further validation of the iron effect for all 23 strains in microtiter plate showed that fourteen strains (including ATCC10987) formed a biofilm on PS. For eight of these strains biofilm formation was enhanced in the presence of added iron and for eleven strains it was reduced when free iron was scavenged. Our results show that stainless steel as a contact material provides more favourable conditions for B. cereus biofilm formation and maturation compared to polystyrene. This effect could possibly be linked to iron availability as we show that free iron availability affects B. cereus biofilm formation.

In vitro model study for biofilm formation by Bacillus cereus in dairy chilling tanks and optimization of clean-in-place (CIP) regimes using response surface methodology

An in vitro model study was carried out to check biofilm formation by a selected strain of Bacillus cereus isolated from a chilling tank where pasteurized milk was stored. The strain was able to form biofilm even at 4 °C. From the study of different simulated conditions it was found that B. cereus cell count reached up to 106 cfu/cm2 of the surface of stainless steel tank if inadequately cleaned tanker is left to stand empty at room temperature. Prevalence of biofilm in dairy environment is a matter of concern as it can be an important reservoir for food spoilage and can lead to foodborne outbreaks. In the present study response surface methodology (RSM) was used to optimize parameters influencing biofilm cell removal. The RSM results were used to design an optimized clean-in-place (CIP) regime which consisted of 1.5% NaOH at 65 °C for 30 min – water rinse – 1% HNO3 at 65 °C for 10 min – water rinse. Effectiveness of reference CIP (1% NaOH at 65 °C for 10 min – water rinse – 1% HNO3 at 65 °C for 10 min – water rinse) and optimized CIP was assessed against 24 h-old biofilm. While the reference CIP caused 3.29 log reduction/cm2 in B. cereus biofilm cells, the optimized CIP achieved 4.77 log reduction/cm2 in biofilm cell count. Thus, the optimized CIP regime designed in the present study was found to be significantly (p < 0.05) more effective in biofilm cell removal as compared to the reference CIP.

Impact of Lactococcus lactis spp. lactis Bio Adhesion on Pathogenic Bacillus cereus Biofilm on Silicone Flowing System.

Bacillus cereus is a food pathogen that can attach on most of the surfaces and form biofilms, which facilitate the persistence and resistance toward antimicrobials. The aims of this study were (i) to characterize the structural dynamics of B. cereus sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the impact of bio adhesion of Lactococcus lactis on B. cereus biofilm. Significantly greater biofilm volume and thickness were observed under dynamic conditions than under static conditions after 48h and B. cereus biofilm was highly organized. The variation of physico-chemical characteristics of silicone by B. cereus bio adhesion favours the adhesion of hydrophilic Lc. lactis on the surface adhered by biofilm. Lc. lactis was able to adhere to silicone surface and produce biofilm obviously exhibited a significant reduction of B. cereus adhered cells up to nine orders of magnitude after 48h of contact with competitive activity for nutrient and oxygen. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone with lactococcal protective biofilm.

The Ability of Bacillus licheniformis Protease to Remove Bacillus cereus and Pseudomonas aeruginosa biofilm

Microbial biofilms are active communities of microorganisms attached to a surface and surrounded by a self-produced matrix composed of extracellular polymeric substances, acting as a shield against desiccation, cleaning procedures and antimicrobial substances. No matter which biomolecule is most responsible in the matrix formation, all these extracellular polymeric substances can nevertheless be broken down with different enzymes leading to a disruption of the biofilm matrix and the removal of the biofilm. Enzyme treatments can be used to break down extracellular polymeric substances as an alternative when standard cleaning agents do not give satisfactory results in removing biofilms. The aim of the study was to analyze the activity of a protease from B. licheniformis against the biofilm formed by B. cereus and P. aeruginosa. Fluorescent microscopy and confocal laser scanning microscopy was performed in order to assess the effect of the enzyme on the biofilm structure and to characterize the biofilm formed by both microorganisms. The results show that the enzyme used was more active against the B. cereus biofilm matrix compared to the activity on the P. aeruginosa biofilm matrix.

Involvement of motility and flagella in Bacillus cereus biofilm formation

Bacillus cereus is a food-borne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of the B. cereus flagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to glass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation. In static conditions, reaching the air-liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bottom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flow cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm. Finally, motility is involved in the spreading of the biofilm on glass surfaces.

Influence of biofilm composition on the resistance to detachment

Bacillus cereus and Pseudomonas fluorescens were used to develop monoculture biofilms in a bioreactor rotating system using a stainless steel cylinder for biofilm formation. The biofilms were allowed to grow for 7 days, exposed continuously to a Reynolds number of agitation (ReA) of 2,400. Afterwards, the biofilms were characterised in terms of respiratory activity, amount of biomass, cellular density, cellular size and total and extracellular proteins and polysaccharides. The biofilm mechanical stability was assessed by sequential submission of the biofilms to increasing ReA, respectively, 4,000, 8,100, 12,100 and 16,100. The results showed that P. fluorescens biofilms were five times more active, had a higher amount of biomass, cellular density, a reduced cellular size and a four-fold higher amount of extracellular proteins and polysaccharides than B. cereus biofilms. The application of shear stress forces higher than the one under which the biofilm was formed (ReA = 2,400) caused biomass removal. The high percentage of removal occurred with the implementation of a ReA of 8,100 for both B. cereus and P. fluorescens biofilms. The total series of ReA did not give rise to total biofilm removal, as only about 76% of P. fluorescens biofilm mass and 53% of B. cereus biofilm mass were detached from the cylinders. This latter result evidences that B. cereus had a higher mechanical stability than P. fluorescens biofilms. The overall results demonstrate that P. fluorescens and B. cereus formed physiologically distinct biofilms, B. cereus biofilms mainly being constituted by cells and P. fluorescens biofilms largely constituted by extracellular proteins and polysaccharides. B. cereus biofilms had a substantially higher mechanical stability than P. fluorescens biofilms.

Lethality of Chlorine, Chlorine Dioxide, and a Commercial Produce Sanitizer to Bacillus cereus and Pseudomonas in a Liquid Detergent, on Stainless Steel, and in Biofilm

Many factors that are not fully understood may influence the effectiveness of sanitizer treatments for eliminating pathogens and spoilage microorganisms in food or detergent residues or in biofilms on food contact surfaces. This study was done to determine the sensitivities of Pseudomonas cells and Bacillus cereus cells and spores suspended in a liquid dishwashing detergent and inoculated onto the surface of stainless steel to treatment with chlorine, chlorine dioxide, and a commercial produce sanitizer (Fit). Cells and spores were incubated in a liquid dishwashing detergent for 16 to 18 h before treatment with sanitizers. At 50 μg/ml, chlorine dioxide killed a significantly higher number of Pseudomonas cells (3.82 log CFU/ml) than did chlorine (a reduction of 1.34 log CFU/ml). Stainless steel coupons were spot inoculated with Pseudomonas cells and B. cereus cells and spores, with water and 5% horse serum as carriers. Chlorine was more effective than chlorine dioxide in killing cells and spores of B. cereus suspended in horse serum. B. cereus biofilm on stainless steel coupons that were treated with chlorine dioxide or chlorine at 200 μg/ml had total population reductions (vegetative cells plus spores) of ≥4.42 log CFU per coupon; the number of spores was reduced by ≥3.80 log CFU per coupon. Fit (0.5%) was ineffective for killing spot-inoculated B. cereus and B. cereus in biofilm, but treatment with mixtures of Fit and chlorine dioxide caused greater reductions than did treatment with chlorine dioxide alone. In contrast, when chlorine was combined with Fit, the lethality of chlorine was completely lost. This study provides information on the survival and sanitizer sensitivity of Pseudomonas and B. cereus in a liquid dishwashing detergent, on the surface of stainless steel, and in a biofilm. This information will be useful for developing more effective strategies for cleaning and sanitizing contact surfaces in food preparation and processing environments.

Inactivation and removal of Bacillus cereus by sanitizer and detergent

This study was conducted to compare the susceptibility of planktonic cells, attached single cells and biofilm cells of Bacillus cereus to sodium hypochlorite and Spartec, a quaternary ammonium compound (QAC). Removal of B. cereus biofilm by simulating clean-in-place (CIP) procedures employing 1.5% Spec-Tak 1000, an alkaline detergent; 0.65% Dilac, an acid detergent and 1.5% Diverform Plus, a sodium hypochlorite containing alkaline detergent was also examined. The results show that B. cereus cells in a biofilm were most resistant to chemical sanitizers followed by the attached single cell and cells in a planktonic state. When B. cereus cells were in a planktonic state, 25 ppm hypochlorite or 100 ppm QAC induced a more than 5.0 log CFU/ml reduction of cell numbers within 15 s. However, the same sanitizer used in this study showed little effect on cells in a biofilm even when the exposure time was extended to 5 min. The sanitizers tested were less effective against cells in biofilm formed on milk pre-soiled than on unsoiled stainless steel chips. It was also noted that Spec-Tak 1000 treatment at 70 °C for 10 min and water rinse in the first step of the long-hot CIP procedure can effectively remove the biofilm B. cereus cells from the milk pre-soiled stainless chip. With this long-hot CIP procedure, the biofilm cells of B. cereus were removed by ca. 6.10 log CFU/chip compared to ca. 1.47 log CFU/chip noted in the control procedure.

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain ( B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.