Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives.

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses. Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g. In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene. Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

Charting host-microbe co-metabolism in skin aging and application to metagenomics data.

During aging of human skin, a number of intrinsic and extrinsic factors cause the alteration of the skin's structure, function and cutaneous physiology. Many studies have investigated the influence of the skin microbiome on these alterations, but the molecular mechanisms that dictate the interplay between these factors and the skin microbiome are still not fully understood. To obtain more insight into the connection between the skin microbiome and the human physiological processes involved in skin aging, we performed a systematic study on interconnected pathways of human and bacterial metabolic processes that are known to play a role in skin aging. The bacterial genes in these pathways were subsequently used to create Hidden Markov Models (HMMs), which were applied to screen for presence of defined functionalities in both genomic and metagenomic datasets of skin-associated bacteria. These models were further applied on 16S rRNA gene sequencing data from skin microbiota samples derived from female volunteers of two different age groups (25-28 years ('young') and 59-68 years ('old')). The results show that the main bacterial pathways associated with aging skin are those involved in the production of pigmentation intermediates, fatty acids and ceramides. This study furthermore provides evidence for a relation between skin aging and bacterial enzymes involved in protein glycation. Taken together, the results and insights described in this paper provide new leads for intervening with bacterial processes that are associated with aging of human skin.

Multi-Nutrient Fortified Dairy-Based Drink Reduces Anaemia without Observed Adverse Effects on Gut Microbiota in Anaemic Malnourished Nigerian Toddlers: A Randomised Dose-Response Study.

Prevalence of anaemia among Nigerian toddlers is reported to be high, and may cause significant morbidity, affects brain development and function, and results in weakness and fatigue. Although, iron fortification can reduce anaemia, yet the effect on gut microbiota is unclear. This open-label randomised study in anaemic malnourished Nigerian toddlers aimed to decrease anaemia without affecting pathogenic gut bacteria using a multi-nutrient fortified dairy-based drink. The test product was provided daily in different amounts (200, 400 or 600 mL, supplying 2.24, 4.48 and 6.72 mg of elemental iron, respectively) for 6 months. Haemoglobin, ferritin, and C-reactive protein concentrations were measured to determine anaemia, iron deficiency (ID) and iron deficiency anaemia (IDA) prevalence. Faecal samples were collected to analyse gut microbiota composition. All three dosages reduced anaemia prevalence, to 47%, 27% and 18%, respectively. ID and IDA prevalence was low and did not significantly decrease over time. Regarding gut microbiota, Enterobacteriaceae decreased over time without differences between groups, whereas Bifidobacteriaceae and pathogenic E. coli were not affected. In conclusion, the multi-nutrient fortified dairy-based drink reduced anaemia in a dose-dependent way, without stimulating intestinal potential pathogenic bacteria, and thus appears to be safe and effective in treating anaemia in Nigerian toddlers.

Enumeration and Identification of Bacterial Spores in Cocoa Powders.

ABSTRACT: The presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genera Bacillus and Geobacillus are typically present. Spores are a concern in heat-treated foods when they survive heat treatments and the finished product supports germination, growth, and potentially toxin production. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS, respectively) were evaluated, leading to the recommendation of one global method specifically for cocoa powders. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. The method includes dilution of cocoa powder using buffered peptone water, heating at 80°C for 10 min for TMS and TTS counts, and heating at 100°C for 30 min for a heat-resistant (HR) spore count. Tryptic soy agar is used as a recovery medium with a maximal concentration of cocoa powder of 2.5 mg/mL (to prevent growth inhibition) and a nonnutrient agar overlay to prevent swarming of bacteria. Plates are incubated for at least 72 h at 30°C for recovery of mesophilic bacteria and 55°C for thermophilic bacteria. Suitable alternatives to specific method parameters are provided. Median values of total spore concentrations are low (<400 CFU/g for TMS and <75 CFU/g for TTS), and concentrations of HR spores are very low (<5 CFU/g). Importantly, the relation between concentrations of HR spores in cocoa powder and incidence of spoilage of heat-treated beverages containing cocoa is currently unclear. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore-forming species identified (49 and 39%, respectively). Both species are known for high variability in spore heat resistance. The development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders.

Correction: Comparative genomic analysis of the multispecies probiotic-marketed product VSL#3.

[This corrects the article DOI: 10.1371/journal.pone.0192452.].

Comparative genomic analysis of the multispecies probiotic-marketed product VSL#3.

Several probiotic-marketed formulations available for the consumers contain live lactic acid bacteria and/or bifidobacteria. The multispecies product commercialized as VSL#3 has been used for treating various gastro-intestinal disorders. However, like many other products, the bacterial strains present in VSL#3 have only been characterized to a limited extent and their efficacy as well as their predicted mode of action remain unclear, preventing further applications or comparative studies. In this work, the genomes of all eight bacterial strains present in VSL#3 were sequenced and characterized, to advance insights into the possible mode of action of this product and also to serve as a basis for future work and trials. Phylogenetic and genomic data analysis allowed us to identify the 7 species present in the VSL#3 product as specified by the manufacturer. The 8 strains present belong to the species Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium breve and B. animalis subsp. lactis (two distinct strains). Comparative genomics revealed that the draft genomes of the S. thermophilus and L. helveticus strains were predicted to encode most of the defence systems such as restriction modification and CRISPR-Cas systems. Genes associated with a variety of potential probiotic functions were also identified. Thus, in the three Bifidobacterium spp., gene clusters were predicted to encode tight adherence pili, known to promote bacteria-host interaction and intestinal barrier integrity, and to impact host cell development. Various repertoires of putative signalling proteins were predicted to be encoded by the genomes of the Lactobacillus spp., i.e. surface layer proteins, LPXTG-containing proteins, or sortase-dependent pili that may interact with the intestinal mucosa and dendritic cells. Taken altogether, the individual genomic characterization of the strains present in the VSL#3 product confirmed the product specifications, determined its coding capacity as well as identified potential probiotic functions.

Dynamic sporulation gene co-expression networks for Bacillus subtilis 168 and the food-borne isolate Bacillus amyloliquefaciens: a transcriptomic model.

Sporulation is a survival strategy, adapted by bacterial cells in response to harsh environmental adversities. The adaptation potential differs between strains and the variations may arise from differences in gene regulation. Gene networks are a valuable way of studying such regulation processes and establishing associations between genes. We reconstructed and compared sporulation gene co-expression networks (GCNs) of the model laboratory strain Bacillus subtilis 168 and the food-borne industrial isolate Bacillus amyloliquefaciens. Transcriptome data obtained from samples of six stages during the sporulation process were used for network inference. Subsequently, a gene set enrichment analysis was performed to compare the reconstructed GCNs of B. subtilis 168 and B. amyloliquefaciens with respect to biological functions, which showed the enriched modules with coherent functional groups associated with sporulation. On basis of the GCNs and time-evolution of differentially expressed genes, we could identify novel candidate genes strongly associated with sporulation in B. subtilis 168 and B. amyloliquefaciens. The GCNs offer a framework for exploring transcription factors, their targets, and co-expressed genes during sporulation. Furthermore, the methodology described here can conveniently be applied to other species or biological processes.

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis.

Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA2mob required higher HA temperatures for efficient germination than spores lacking spoVA2mob The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers.IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

SpoVT: From Fine-Tuning Regulator in Bacillus subtilis to Essential Sporulation Protein in Bacillus cereus.

Sporulation is a highly sophisticated developmental process adopted by most Bacilli as a survival strategy to withstand extreme conditions that normally do not support microbial growth. A complicated regulatory cascade, divided into various stages and taking place in two different compartments of the cell, involves a number of primary and secondary regulator proteins that drive gene expression directed toward the formation and maturation of an endospore. Such regulator proteins are highly conserved among various spore formers. Despite this conservation, both regulatory and phenotypic differences are observed between different species of spore forming bacteria. In this study, we demonstrate that deletion of the regulatory sporulation protein SpoVT results in a severe sporulation defect in Bacillus cereus, whereas this is not observed in Bacillus subtilis. Although spores are initially formed, the process is stalled at a later stage in development, followed by lysis of the forespore and the mother cell. A transcriptomic investigation of B. cereus ΔspoVT shows upregulation of genes involved in germination, potentially leading to premature lysis of prespores formed. Additionally, extreme variation in the expression of species-specific genes of unknown function was observed. Introduction of the B. subtilis SpoVT protein could partly restore the sporulation defect in the B. cereus spoVT mutant strain. The difference in phenotype is thus more than likely explained by differences in promoter targets rather than differences in mode of action of the conserved SpoVT regulator protein. This study stresses that evolutionary variances in regulon members of sporulation regulators can have profound effects on the spore developmental process and that mere protein homology is not a foolproof predictor of similar phenotypes.

Draft Genome Sequences of Seven Thermophilic Spore-Forming Bacteria Isolated from Foods That Produce Highly Heat-Resistant Spores, Comprising Geobacillus spp., Caldibacillus debilis, and Anoxybacillus flavithermus.

Here, we report the draft genomes of five strains of Geobacillus spp., one Caldibacillus debilis strain, and one draft genome of Anoxybacillus flavithermus, all thermophilic spore-forming Gram-positive bacteria.

A transposon present in specific strains of Bacillus subtilis negatively affects nutrient- and dodecylamine-induced spore germination.

Spore germination shows a large inter-strain variability. Spores of certain Bacillus subtilis strains, including isolates from spoiled food products, exhibit different germination behavior from spores of the well-studied model organism Bacillus subtilis 168, often for unknown reasons. In this study, we analyzed spore germination efficiencies and kinetics of seventeen B. subtilis strains with previously sequenced genomes. A subsequent gene-trait matching analysis revealed a correlation between a slow germination phenotype and the presence of a mobile genetic element, i.e., a Tn1546-like transposon. A detailed investigation of the transposon elements showed an essential role of a specific operon (spoVA2mob ) in inhibiting spore germination with nutrients and with the cationic surfactant dodecylamine. Our results indicate that this operon negatively influences release of Ca-DPA by the SpoVA channel and may additionally alter earlier germination events, potentially by affecting proteins in the spore inner membrane. The spoVA2mob operon is an important factor that contributes to inter-strain differences in spore germination. Screening for its genomic presence can be applied for identification of spores that exhibit specific properties that impede spore eradication by industrial processes.

Genome Sequences of 12 Spore-Forming Bacillus Species, Comprising Bacillus coagulans, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Bacillus vallismortis, Isolated from Foods.

Here, we report the draft genomes of twelve isolates of five different Bacillus species, all spore-forming, Gram-positive bacteria.

Draft Genome Sequences of 10 Bacillus subtilis Strains That Form Spores with High or Low Heat Resistance.

Here, we report the draft genome sequences of 10 isolates of Bacillus subtilis, a spore forming Gram-positive bacterium. The strains were selected from food products and produced spores with either high or low heat resistance.

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.

Next-Generation Whole-Genome Sequencing of Eight Strains of Bacillus cereus, Isolated from Food.

Bacillus cereus can contaminate food and cause emetic and diarrheal foodborne illness. Here, we report whole-genome sequences of eight strains of B. cereus, isolated from different food sources.

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid.

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.

Draft Genome Sequences of Four Bacillus thermoamylovorans Strains Isolated from Milk and Acacia Gum, a Food Ingredient.

The thermophilic bacterium Bacillus thermoamylovorans produces highly heat-resistant spores that can contaminate food products, leading to their spoilage. Here, we present the whole-genome sequences of four B. thermoamylovorans strains, isolated from milk and acacia gum.

Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.

SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis.

Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information. To facilitate obtaining a complete overview as well as new insights concerning this complex and tightly regulated process, we have developed a database-driven knowledge platform called SporeWeb (http://sporeweb.molgenrug.nl) that focuses on gene regulatory networks during sporulation in the Gram-positive bacterium Bacillus subtilis. Dynamic features allow the user to navigate through all stages of sporulation with review-like descriptions, schematic overviews on transcriptional regulation and detailed information on all regulators and the genes under their control. The Web site supports data acquisition on sporulation genes and their expression, regulon network interactions and direct links to other knowledge platforms or relevant literature. The information found on SporeWeb (including figures and tables) can and will be updated as new information becomes available in the literature. In this way, SporeWeb offers a novel, convenient and timely reference, an information source and a data acquisition tool that will aid in the general understanding of the dynamics of the complete sporulation cycle.

Live-cell imaging tool optimization to study gene expression levels and dynamics in single cells of Bacillus cereus.

Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium Bacillus cereus specifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of single B. cereus cells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression in B. cereus. With a case study concerning cotD expression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show that cotD is not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.