Inactivation kinetics of Geobacillus stearothermophilus spores by a peracetic acid or hydrogen peroxide fog in comparison to the liquid form.

The aim of this study was to compare the sporicidal effect of the disinfectants peracetic acid (PAA) or hydrogen peroxide (H2O2) applied as a fog or as a liquid. The efficacy of fogging of the disinfectants was tested in a closed isolator cabinet using highly heat and chemical-resistant spores of Geobacillus stearothermophilus. Fogging of a 0.06% solution of PAA resulted in over 5-log reduction of spores in 10 min, whereas for PAA used in liquid form the same reduction was achieved in 4.5 min. The inactivation curves for fog and liquid were fitted using three different models (Linear with shoulder, Weibull, Gauss-Eyring). This showed a shoulder for the fog with an estimated length of 4.1 min, but the D values, calculated for the linear parts of the curves, were not significantly different (1.1 and 0.8 min for the PAA fog and solution, respectively). Similar results were obtained for a 12% H2O2 solution, albeit that H2O2 was less effective compared to PAA, requiring 60 min to reach 3-log reduction when applied as a fog, with an estimated shoulder of 18.5 min. Fogging of a 0.06% peracetic acid solution effectively inactivated G. stearothermophilus spores. Overall, the data show that fogging can be an effective method of applying disinfectants but that a shoulder in the inactivation curves should be considered in process design. This study provides inactivation kinetics for disinfection using PAA or H2O2-based fog, which can aid in selection and validation of process parameters for disinfection of contained areas by fogging.

Inactivation of conidia from three Penicillium spp. isolated from fruit juices by conventional and alternative mild preservation technologies and disinfection treatments.

Fungi are able to grow on diverse food products and contribute to food spoilage worldwide causing food loss. Consumers prefer freshly squeezed fruit juices, however, the shelf life of these juices is limited due to outgrowth of yeast and fungi. The shelf life of pulsed electric field (PEF) treated juice can be extended from 8 days up to a few weeks before spoilage by moulds becomes apparent. Conidia produced by three Penicillium ssp. (Penicillium expansum, Penicillium buchwaldii and Penicillium bialowiezense), previously isolated from spoiled PEF treated fruit juice and smoothie, were characterized for resistance towards selected mild physical processing techniques in orange juice and toward sanitizers on surfaces. The results show that Penicillium spp. conidia are susceptible to mild heat, high pressure pasteurization (HPP), PEF, cold atmospheric plasma (CAP), UV, and chemical sanitizers chlorine dioxide and hypochlorite albeit with different susceptibility. Treatment with mild heat, HPP, PEF, or chlorine dioxide reduced conidia by more than 5 log. For hypochlorite, UV, and CAP the reduction was between 1 and 3 log. Together, this study provides data for the development of intervention strategies to eliminate spoilage mould conidia in fruit juices.

Role of cell surface composition and lysis in static biofilm formation by Lactobacillus plantarum WCFS1.

Next to applications in fermentations, Lactobacillus plantarum is recognized as a food spoilage organism, and its dispersal from biofilms in food processing environments might be implicated in contamination or recontamination of food products. This study provides new insights into biofilm development by L. plantarum WCFS1 through comparative analysis of wild type and mutants affected in cell surface composition, including mutants deficient in the production of Sortase A involved in the covalent attachment of 27 predicted surface proteins to the cell wall peptidoglycan (ΔsrtA) and mutants deficient in the production of capsular polysaccharides (CPS1-4, Δcps1-4). Surface adhesion and biofilm formation studies revealed none of the imposed cell surface modifications to affect the initial attachment of cells to polystyrene while biofilm formation based on Crystal Violet (CV) staining was severely reduced in the ΔsrtA mutant and significantly increased in mutants lacking the cps1 cluster, compared to the wild-type strain. Fluorescence microscopy analysis of biofilm samples pointed to a higher presence of extracellular DNA (eDNA) in cps1 mutants and this corresponded with increased autolysis activity. Subsequent studies using Δacm2 and ΔlytA derivatives affected in lytic behaviour revealed reduced biofilm formation measured by CV staining, confirming the relevance of lysis for the build-up of the biofilm matrix with eDNA.

Characterization of four Paenibacillus species isolated from pasteurized, chilled ready-to-eat meals.

Food spoilage is often caused by microorganisms. The predominant spoilage microorganisms of pasteurized, chilled ready-to-eat (RTE) mixed rice-vegetable meals stored at 7°C were isolated and determined as Paenibacillus species. These sporeforming psychrotrophic bacteria are well adapted to grow in the starch-rich environment of pasteurized and chilled meals. Growth of the Paenibacillus isolates appeared to be delayed by decreased (<7°C) temperature or chilled temperature (7°C) combined with decreased pH (<5), increased sodium chloride (>5.5%, corresponding with an aw<0.934), or decreased aw (<0.931; using sucrose). To gain insight in the effect of the pasteurization processing of the meal on spore inactivation, heat-inactivation kinetics were determined and D-values were calculated. According to these kinetics, pasteurization up to 90°C, necessary for inactivation of vegetative spoilage microorganisms and pathogens, does not significantly contribute to the inactivation of Paenibacillus spores in the meals. Furthermore, outgrowth of pasteurized spores was determined in the mixed rice-vegetable meal at several temperatures; P. terrae FBR-61 and P. pabuli FBR-75 isolates did not substantially increase in numbers during storage at 2°C, but had a significant increase within a month of storage at 4°C or within several days at 22°C. Overall, this work shows the importance of Paenibacillus species as spoilage microorganisms of pasteurized, chilled RTE meals and that the meals' matrix, processing conditions, and storage temperature are important hurdles to control microbial meal spoilage.

Quantitative assessment of viable cells of Lactobacillus plantarum strains in single, dual and multi-strain biofilms.

Biofilms of Lactobacillus plantarum are a potential source for contamination and recontamination of food products. Although biofilms have been mostly studied using single species or even single strains, it is conceivable that in a range of environmental settings including food processing areas, biofilms are composed of multiple species with each species represented by multiple strains. In this study six spoilage related L. plantarum strains FBR1-FBR6 and the model strain L. plantarum WCFS1 were characterised in single, dual and multiple strain competition models. A quantitative PCR approach was used with added propidium monoazide (PMA) enabling quantification of intact cells in the biofilm, representing the viable cell fraction that determines the food spoilage risk. Our results show that the performance of individual strains in multi-strain cultures generally correlates with their performance in pure culture, and relative strain abundance in multi-strain biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The total biofilm quantified by CV staining of the dual and multi-strain biofilms formed was mainly correlated to CV values of the dominant strain obtained in single strain studies. However, the combination of strain FBR5 and strain WCFS1 showed significantly higher CV values compared to the individual performances of both strains indicating that total biofilm formation was higher in this specific condition. Notably, L. plantarum FBR5 was able to outgrow all other strains and showed the highest relative abundance in dual and multi-strain biofilms. All the dual and multi-strain biofilms contained a considerable number of viable cells, representing a potential source of contamination.

Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains.

Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerRI (sub)clusters that possibly affected the functionality of these GRs. IMPORTANCE: Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair.

The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

Draft Genome Sequences of Four Thermophilic Spore Formers Isolated from a Dairy-Processing Environment.

Spores of thermophilic spore-forming bacteria are a common cause of contamination in dairy products. Here, we report draft genome sequences of four thermophilic strains from a milk-processing plant or standard milk, namely, a Geobacillus thermoglucosidans isolate (TNO-09.023), Geobacillus stearothermophilus TNO-09.027, and two Anoxybacillus flavithermus isolates (TNO-09.014 and TNO-09.016).

Comparative Genomics of Iron-Transporting Systems in Bacillus cereus Strains and Impact of Iron Sources on Growth and Biofilm Formation.

Iron is an important element for bacterial viability, however it is not readily available in most environments. We studied the ability of 20 undomesticated food isolates of Bacillus cereus and two reference strains for capacity to use different (complex) iron sources for growth and biofilm formation. Studies were performed in media containing the iron scavenger 2,2-Bipyridine. Transcriptome analysis using B. cereus ATCC 10987 indeed showed upregulation of predicted iron transporters in the presence of 2,2-Bipyridine, confirming that iron was depleted upon its addition. Next, the impact of iron sources on growth performance of the 22 strains was assessed and correlations between growth stimulation and presence of putative iron transporter systems in the genome sequences were analyzed. All 22 strains effectively used Fe citrate and FeCl3 for growth, and possessed genes for biosynthesis of the siderophore bacillibactin, whereas seven strains lacked genes for synthesis of petrobactin. Hemoglobin could be used by all strains with the exception of one strain that lacked functional petrobactin and IlsA systems. Hemin could be used by the majority of the tested strains (19 of 22). Notably, transferrin, ferritin, and lactoferrin were not commonly used by B. cereus for growth, as these iron sources could be used by 6, 3, and 2 strains, respectively. Furthermore, biofilm formation was found to be affected by the type of iron source used, including stimulation of biofilms at liquid-air interphase (FeCl3 and Fe citrate) and formation of submerged type biofilms (hemin and lactoferrin). Our results show strain variability in the genome-encoded repertoire of iron-transporting systems and differences in efficacy to use complex iron sources for growth and biofilm formation. These features may affect B. cereus survival and persistence in specific niches.

Draft Whole-Genome Sequences of Three Lactobacillus plantarum Food Isolates.

Lactobacillus plantarum is a widespread member of the Lactobacillus genus and frequently isolated from spoiled acidified food products. Here, we report the draft genome sequences of three L. plantarum food isolates.

Draft Whole-Genome Sequences of 11 Bacillus cereus Food Isolates.

Bacillus cereus is a foodborne pathogen causing emetic and diarrheal-type syndromes. Here, we report the whole-genome sequences of 11 B. cereus food isolates.

Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity.

We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.

Bacterial Spores in Food: Survival, Emergence, and Outgrowth.

Spore-forming bacteria are ubiquitous in nature. The resistance properties of bacterial spores lie at the heart of their widespread occurrence in food ingredients and foods. The efficacy of inactivation by food-processing conditions is largely determined by the characteristics of the different types of spores, whereas food composition and storage conditions determine the eventual germination and outgrowth of surviving spores. Here, we review the current knowledge on variation in spore resistance, in germination, and in the outgrowth capacity of spores relevant to foods. This includes novel findings on key parameters in spore survival and outgrowth obtained by gene-trait matching approaches using genome-sequenced Bacillus spp. food isolates, which represent notorious food spoilage and pathogenic species. Additionally, the impact of strain diversity on heat inactivation of spores and the variability therein is discussed. Knowledge and quantification of factors that influence variability can be applied to improve predictive models, ultimately supporting effective control of spore-forming bacteria in foods.

Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores.

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.

Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production.

Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.

Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates.

Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry.

Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores.

Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH 5.5) meat-based BHI. Rice, broccoli and BHI pH 5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH 7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1 min at 95 °C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH 7.4 was more pronounced than on rice, broccoli and BHI pH 5.5 conceivably reflecting that conditions in BHI pH 7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, pH of BHI and the effect of food matrix highlighting the impact of different (model) food recovery media on outgrowth efficiency and heterogeneity of non-heat-treated and heat-damaged B. cereus spores.

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.

Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores.

Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.