Sporulation of Bacillus spp. within biofilms: a potential source of contamination in food processing environments.

Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures.

Quantification of the extracellular matrix of the Listeria monocytogenes biofilms of different phylogenic lineages with optimization of culture conditions.

AIMS: The purpose of this study was to quantify the extracellular matrix of Listeria monocytogenes biofilm. A preliminary study was carried out to establish a relationship between phylogenetic lineage of 27 strains and their ability to form biofilm in various conditions. METHODS AND RESULTS: Biofilm formation on microtitre plates of 27 strains of L. monocytogenes belonging to lineages I or II was evaluated in different conditions [two temperatures (37 and 22°C) and two media (tryptone soy broth yeast extract medium (TSBYE) and MCDB 202 defined medium)] using crystal violet assay. Lineage II strains produced significantly more biofilm than lineage I strains. In microtitre plates assay, biofilm quantities were greater in MCDB 202 vs TSBYE medium [confirmed by scanning electron microscopy (SEM) analysis] and at 37 vs 22°C. Cultivable bacteria from biofilm population on Petri dishes were enumerated in greater quantities in TSBYE than in MCDB 202 medium. The SEM investigation established that L. monocytogenes biofilms produce extracellular matrix in both media at 37°C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain that presented similar pH values and exopolymer contents in both media. Proteins were the most abundant exopolymer components, followed by DNA and polysaccharides. CONCLUSIONS: The interpretation of results of biofilm quantification was depending on the growth conditions, the viability of the bacteria and the analysis method. The quantities of proteins, DNA and polysaccharides were different according to the strains and the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened the potential of a wide panel of L. monocytogenes strains to synthesize exopolymers in biofilm growing condition. The characterization of L. monocytogenes biofilm composition may help to develop new strategies to prevent the formation of biofilms and to remove the biofilms.

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures.

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry.

Viability and surface properties of spores subjected to a cleaning-in-place procedure: consequences on their ability to contaminate surfaces of equipment.

This study was designed to evaluate how conditions encountered by spores during cleaning-in-place (CIP) procedures affected their surface properties, their viability and ability to contaminate materials. Spores from five Bacillus cereus strains were treated with NaOH at high temperature. Results revealed that high temperatures (exceeding 60 degrees C) and NaOH concentrations (over 0.5%) were required to significantly decrease spore viability (3-5log decrease). In these conditions, modifications were also clearly observed by microscopy to various surface structures of spores (appendages, exosporium, and especially to the hair-like nap) but also to their coat. Therefore, the ability of culturable spores to adhere decreased for the majority of strains tested. We then demonstrated that spores in suspension in NaOH could adhere to surfaces of a CIP rig and that the contamination level was controlled by flow pattern. Consequently, re-adhesion along the processing line might occur during CIP procedures and this phenomenon must be taken into account when defining cleaning strategies.

Physico-chemical and hygienic property modifications of stainless steel surfaces induced by conditioning with food and detergent.

The effect of repeated conditioning procedures (25 runs), consisting of soiling (milk and meat products) and cleaning steps, on the hygienic status, physico-chemical properties and surface chemical composition of stainless steel (SS) surfaces, was investigated. Five SSs differing in grade and finish were used. Both soiling and surface cleaning/conditioning procedures resulted in a similar increase in the surface contamination with carbon, while the changes in the basic component of the surface free energy depended on the conditioning procedure. The passive film was also affected, the Fe/Cr ratio in particular. The hygienic status was also changed, especially with milk as shown by monitoring the number of residual adhering Bacillus cereus spores after contaminating the surface with spores followed by cleaning. The results show that in food environments, the presence and the nature of conditioning molecules play a major role in the hygienic status of SS surfaces.

Occurrence of Bacillus cereus spores with a damaged exosporium: consequences on the spore adhesion on surfaces of food processing lines.

This study was designed to evaluate the occurrence of Bacillus cereus spores with a damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8-agar, 30 degrees C) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (Spo8-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40 degrees C on Spo8-agar or at 30 degrees C in Spo8-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through a peristaltic pump, the exosporium of a significant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was significantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P < or = 0.004) and enhanced resistance to cleaning (P < or = 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spores adherent to food processing line surfaces.

Comparative evaluation of adhesion, surface properties, and surface protein composition of Listeria monocytogenes strains after cultivation at constant pH of 5 and 7.

AIMS: To analyse the cellular mechanisms that influence Listeria monocytogenes adhesion onto inert surfaces under acidic growth conditions. METHODS AND RESULTS: The adhesion capability of all the strains was significantly reduced after cultivation at constant pH 5 than at constant pH 7 and the cell surface was significantly less hydrophobic at pH 5 than at 7. At pH 5, the analyses of surface protein composition revealed that the flagellin was downregulated for all strains, which was confirmed by the absence of flagella and the P60 protein was upregulated for L. monocytogenes EGD-e, X-Li-mo 500 and 111. The use of L. monocytogenes EGD mutants revealed that flagellin could be involved in the adhesion process, but not P60 protein. It was also observed that the hydrophobic character was not linked to the presence or the absence of flagellin or P60 protein at the cell surface of L. monocytogenes. CONCLUSIONS: The decrease of L. monocytogenes adhesion at pH 5 could be attributed to the downregulation of the flagellin synthesis under the acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Conservation of food product at pH 5 will delay bacterial adhesion and biofilm formation during food processing on inert surfaces when the product is contaminated with L. monocytogenes.

Influence of surface chemistry on the hygienic status of industrial stainless steel.

Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning. Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2 h. Surfaces were rinsed and then covered with whole milk and allowed to dry. Surfaces were then cleaned in an experimental flow system that mimics an industrial application. After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined. Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily. There was a relation between the polar component and ease of cleaning. The higher the polar component the more easily the surface cleaned. The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.

Adhesion of Escherichia coli, Citrobacter freundii and Klebsiella pneumoniae isolated from milk: consequence on the efficiency of sanitation procedures.

The effects of adhesion and of biofilm development on the efficiency of cleaning and disinfection procedures were investigated on three strains belonging to the E. coli, C. freundii and K. pneumoniae species, which were able to raise more or less complex biofilms. Resistance to a rinsing procedure was strain dependent but not related to the biofilm structure: E. coli was poorly adherent although embedded in an organic matrix. Conversely, a similar increase in the heat- and disinfectant-resistance was observed, regardless of the complexity of the biofilm (more or less significant organic matrix). These result suggested the essential role of the bacterial physiological state in resistance to sanitation procedures.

Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments.

AIMS: The effect of salt and acid on the growth and surface properties of two strains of Listeria monocytogenes was investigated. METHODS AND RESULTS: Medium acidification and NaCl supplementation induced a marked increase in the lag and growth times (up to fivefold higher) and a decrease in the maximal optical density. Due to a strong synergic effect of pH and NaCl, growth was only detected after 280 h incubation for Scott A and not detected after 600 h for LO28 at pH 5.0 and 10% NaCl. Furthermore, the addition of NaCl in acidic conditions gave rise to cell filamentation and cell surfaces became strongly hydrophilic. CONCLUSIONS: Some L. monocytogenes strains subjected to high NaCl concentrations in acidic conditions are able to grow but may present altered adhesion properties due to modification of their surface properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted that L. monocytogenes do represent a hazard in acid and salted foods, such as soft cheese.

Potential occurrence of adhering living Bacillus spores in milk product processing lines.

AIMS: The hygienic risk associated with microbial soil on surfaces of milk processing lines was evaluated, based on experimental results. METHODS AND RESULTS: From a panel of Bacillus spores isolated from milk products, B. cereus CUETM 98/4, was found to be highly resistant to heat (D100=3.32 min in whole milk) and oxidant disinfectant (70% lethality of adherent spores with Ikalin 2%). From adhesion trials, up to 1.1 x 10(7) spores cm(-2) were found to be adherent to solid surfaces when suspended in saline or in custard (10(5) and 10(7) cfu ml(-1)), and over 10% of these adherent spores would resist the cleaning procedure. CONCLUSION: A highly contaminated milk (10(5) cfu ml(-1)) subjected to a current sterilization process (8 log reduction) led to a residual contamination of less than 1 cfu in the representative processing line after a complete production run. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that under appropriate processing conditions (efficient sterilization and cleaning procedures), even disinfection would be sufficient to eliminate any contamination risk. Conversely, the disinfection procedure becomes an essential step under inappropriate processing conditions.

Communication during the recruitment phase of a multicenter trial: the recruitment hotline.

The Bypass Angioplasty Revascularization Investigation (BARI) is a randomized trial that compares the safety and efficacy of angioplasty and bypass surgery in selected patients with multivessel coronary disease. During recruitment, the Clinical Coordinating Center (CC) required an organized manner of responding to the many questions expected from the 18 clinical sites. Thus a dedicated telephone line was established to provide the clinical sites with information quickly and ensure consistent dissemination of information. In addition, the hotline functioned as a backup mechanism for patient randomization in the event of a computer failure at one of the sites. During the first 13 months of recruitment, 1332 calls were received. The average number of daily calls peaked at 7.3 with 14 calls being the highest in any one day. Calls gradually declined as the clinical sites became more familiar with the protocol, data collection forms, and computer systems. Most questions were answered by the data management staff; however a substantial number (37%) required faculty level input. For questions that could not be answered immediately, the median time for a return call was 25 min. The BARI hotline was an efficient way to provide accurate and consistent feedback to all sites and to identify areas that required protocol clarification. It allowed rapid identification of differences in protocol interpretation across sites so that these variations could be addressed. Review of specific questions by the Operations Committee resulted in decisions on how to apply the protocol to particularly difficult or exceptional cases. While the system was labor-intensive, its benefits outweighed this disadvantage. Recommended modifications to lower costs would result in a system that could be easily adapted for use in other clinical trials.

Specific antibody response to oligomannosidic epitopes in Crohn's disease.

Elevated antibody levels against the yeast Saccharomyces cerevisiae have been reported in sera from patients with Crohn's disease and not with ulcerative colitis. The aim of the study was to identify the nature of the epitopes supporting this antibody response. Whole cells from different S. cerevisiae strains were selected in immunofluorescence assay for their ability to differentiate the antibody responses of patients with Crohn's disease and ulcerative colitis. Their cell wall phosphopeptidomannans were then tested as antigen in enzyme-linked immunosorbent assay (ELISA) against sera from 42 patients with Crohn's disease, 20 patients with ulcerative colitis, and 34 healthy controls. Graded chemical degradations were performed on the most reactive strain phosphopeptidomannan. The discriminating epitope was determined through gas-liquid chromatography-mass spectrometry. The greatest discrimination among patients with Crohn's disease, ulcerative colitis, and controls was obtained with Su1, a S. cerevisiae strain used in brewing of beer. ELISA directed against phosphopeptidomannan of this strain was 64% sensitive and 77% specific for discriminating Crohn's disease versus ulcerative colitis and 71% sensitive and 89% specific for Crohn's disease versus controls. Periodate oxidation and selective degradation demonstrated that the most important polysaccharide epitope was shared by both the acid-stable and the alkali-labile domains of the phosphopeptidomannan. The determination of oligomannose sequences of S. cerevisiae Su1 phosphopeptidomannans suggested that a mannotetraose, Man (1 --> 3)Man(1 --> 2)Man(1 --> 2)Man, supported the serological response seen in Crohn's disease. Further identification of the immunogen eliciting this antibody response as a marker of the disease may help to understand its etiology.

Probable presence of beta(1-2)-linked oligomannosides that act as human immunoglobulin G3 epitopes and are distributed over a Candida albicans 14- to 18-kilodalton antigen.

Kinetic analysis of candidosis patients' immunoglobulin G3 response has shown that reactivity towards beta(1-2)-linked mannan-derived oligomannosides was associated with the recognition through metaperiodate-sensitive epitopes of a 14- to 18-kDa Candida albicans antigen unreactive with concanavalin A.

Qualitative and quantitative differences in recognition patterns of Candida albicans protein and polysaccharide antigens by human sera.

Cytoplasmic and cell wall proteins and glycoproteins extracted from Candida albicans germ tubes were screened by Western blotting for their ability to differentiate between the serological responses of patients with candidosis and healthy individuals. Molecules of 114, 74 and 65 kDa were not recognized by any sera. Qualitative differences were observed for responses to proteins and glycoproteins from 29 to 60 kDa. Conversely, only quantitative differences were found to high molecular mass glycoproteins. Their recognition by control sera was invariably associated with reactivity against a 14-18 kDa antigen. However, despite a high level of antibodies against high molecular mass mannoproteins, some patients sera failed to react with the 14-18 kDa antigen, or lost this reactivity during the course of the disease.

Complete 1H- and 13C-resonance assignments for D-mannooligosaccharides of the beta-D-(1-->2)-linked series released from the phosphopeptidomannan of Candida albicans VW.32 (serotype A).

D-Mannooligosaccharides (dp 1 to > 17) were released by mild acid hydrolysis from the phosphopeptidomannan of a Candida albicans strain of A serotype (VW.32). Among these, mannooligosaccharides ranging from bi- to hepta-ose, which were obtained in appreciable amounts, were structurally investigated and found to belong to the beta-D-(1-->2)-linked series. The occurrence of such compounds has already been reported in other Candida albicans strains. The complete 1H- and 13C-resonance assignments for manno-tri- to manno-hepta-ose are reported and general rules applicable for the 1NMR spectrum analysis of linear mannooligosaccharide of the general structure, beta-D-Man p-(1-->2)-[beta-D-Man p-(1-->2)]n-beta-D-Man p are proposed.

Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies.

Six monoclonal antibodies (MAbs) from various laboratory sources (EB-CA1, EB-CA2, H5, AF1, C6, and 5B2), reacting with the polysaccharidic moieties of Candida albicans mannoproteins, were used for epitope mapping by an enzyme-linked immunosorbent assay (ELISA) with neoglycolipids and by Western blotting (immunoblotting) of a C. albicans germ tube extract. The ELISA involved neoglycolipids constructed from three families of oligomannosides released by sequential depolymerization of C. albicans phosphopeptidomannan by acid hydrolysis (NGLH), beta-elimination (NGLO), and acetolysis (NGLA). All of the MAbs exhibited low reactivities against NGLO. MAbs EB-CA1, EB-CA2, and H5 reacted mainly against NGLA, and MAbs C6 and AF1 recognized mainly NGLH, whereas MAb 5B2 reacted with both families of neoantigens. When this method was compared with Western blotting, strong reactivity to NGLA was associated with the presence of epitopes shared by high-molecular-weight mannoproteins, whereas strong reactivity to NGLH was associated with a reactivity to a family of 14- to 18-kDa antigens. The reactivity of MAb 5B2 was associated with both high-molecular-weight mannoproteins and the 14- to 18-kDa antigens. In relation to the present knowledge about the structure of the C. albicans phosphopeptidomannan oligomannosidic repertoire, these results provide preliminary data concerning the molecular basis of the recognition of mannopyranosyl sequences by MAbs and their distribution among C. albicans mannoproteins.

Evaluation of an enzyme immunoassay using neoglycolipids constructed from Candida albicans oligomannosides to define the specificity of anti-mannan antibodies.

In order to study the respective roles of oligomannoside sequences in the antigenicity of Candida albicans phosphopeptidomannan, a method was developed for constructing neoglycolipids from oligomannosides released by depolymerisation of this molecule. Oligomannosides released by acetolysis were converted to neoglycolipids by coupling them to 4-hexadecylaniline in an equimolar reaction checked by thin layer chromatography. When coated onto microEIA plates, the neoglycolipids exhibited strong reactions which were dose dependent and were saturable with concanavalin A. Reactivity of neoglycolipids with immunoglobulins were then tested with a panel of monoclonal and polyclonal antibodies reacting with epitopes present in the original phosphopeptidomannan. One of two IgM monoclonal antibodies and two of five monospecific rabbit polyclonal IgG reacted strongly with neoglycolipids therefore providing evidence of the presence of structures mimicking epitopes within the pool of neoglycolipids. When 38 sera from 18 hospital inpatients with various levels of antibodies to Candida albicans were tested, a correlation was observed between the EIA to detect neoglycolipids and the EIA to detect phosphopeptidomannan. Successive sera from all patients showing seroconversion in the immunofluorescence assay had increased EIA signals for neoglycolipids.

Presence of human antibodies reacting with Candida albicans O-linked oligomannosides revealed by using an enzyme-linked immunosorbent assay and neoglycolipids.

In order to study the presence of antibodies directed against Candida albicans O-linked oligomannosides (oligomannosides O) in patient sera, we have developed an enzyme-linked immunosorbent assay (ELISA) involving neoglycolipids constructed with these residues (NGLO). Oligomannosides O released by mild alkaline degradation of the C. albicans cell wall phosphopeptidomannan (PPM) contained one to seven mannose residues, among which the quantitatively major components, mannobiose and mannotriose, were shown by 1H nuclear magnetic resonance to contain exclusively alpha (1-2) linkages. The pool of oligomannosides was converted to neoglycolipids by coupling them to 4-hexadecylaniline in an equimolar reaction checked by thin-layer chromatography. We have tested against these neoantigens, coated on ELISA plates, 15 pairs of sera corresponding to individual seroconversions observed in 15 patients during the course of a mycological and serological survey of candidiasis. For all patients, seroconversions resulted in an increased level of antibodies against NGLO. A significant correlation was observed between the results of ELISA-NGLO, ELISA involving the original PPM molecule, and routine antibody detection tests, indirect immunofluorescence assay, and cocounterimmunoelectrophoresis. These results therefore demonstrate the synthesis of human antibodies reactive with oligomannosides O constitutive of the C. albicans mannan molecule which have been previously described as exhibiting an inhibitory effect on human lymphocytic proliferation.

1H-NMR spectroscopy of manno-oligosaccharides of the beta-1,2-linked series released from the phosphopeptidomannan of Candida albicans VW-32 (serotype A).

Manno-oligosaccharides (DP 2 to greater than 15) were released by mild acid hydrolysis from the phosphopeptidomannan of a Candida albicans strain of A serotype (VW-32). Manno-oligosaccharides ranging from biose to heptaose were obtained in appreciable amount. Structural investigation of these oligosaccharides showed them to be of the beta-1,2-linked series. The occurrence of such compounds has already been reported in other strains of Candida albicans. We here report the assignment of the structural reporter groups of each of them, and general rules applicable for the 1H-NMR spectrum analysis of linear manno-oligosaccharide of general structure: Man(beta 1-2) [Man(beta 1-2)]nMan