Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.

Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh "reducing" conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures.IMPORTANCE Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly.

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment.

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut.

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II.

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

Identification of a novel botulinum neurotoxin gene cluster in Enterococcus.

The deadly neurotoxins of Clostridium botulinum (BoNTs) comprise eight serotypes (A-G; X). The neurotoxin gene cluster encoding BoNT and its accessory proteins includes an operon containing an ntnh gene upstream of the boNT gene. Another operon contains either ha (haemagglutinin) or orfX genes (of unknown function). Here we describe a novel boNT gene cluster from Enterococcus sp. 3G1_DIV0629, with a typical ntnh gene and an uncommon orfX arrangement. The neurotoxin (designated putative eBoNT/J) contains a metallopeptidase zinc-binding site, a translocation domain and a target cell attachment domain. Structural properties of the latter suggest a novel targeting mechanism with consequent implications for application by the pharmaceutical industry. This is the first complete boNT gene cluster identified in a non-clostridial genome.

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes.

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease - Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25-30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

Diversity of the Germination Apparatus in Clostridium botulinum Groups I, II, III, and IV.

Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favorable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified [encoded by various combinations of gerA, gerB, and gerC genes (gerX)]. There are three gene clusters with an ABC-like configuration; ABC [gerX1], ABABCB [gerX2] and ACxBBB [gerX4], and a single CA-B [gerX3] gene cluster. Subtypes have been identified for most germinant receptor types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2, and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in dipicolinic acid release. The cortex-lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.

Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell.

Clostridium sporogenes forms highly heat resistant endospores, enabling this bacterium to survive adverse conditions. Subsequently, spores may germinate, giving rise to vegetative cells that multiply and lead to food spoilage. Electron microscopy was used to visualise changes in spore structures during germination, emergence and outgrowth. C. sporogenes spores were surrounded by an exosporium that was oval in shape and typically 3 µm in length. An aperture of 0.3-0.4 µm was observed at one end of the exosporium. The rupture of the spore coats occurs adjacent to the opening in the exosporium. The germinated cell emerges through this hole in the spore coat and then through the pre-existing aperture in the exosporium, before eventually being released, leaving behind a largely intact exosporium with an enlarged aperture (0.7-1.0 µm) and coat shell. The formation of this aperture, its function and its alignment with the spore coat is discussed.

Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems.

Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.

Rapid affinity immunochromatography column-based tests for sensitive detection of Clostridium botulinum neurotoxins and Escherichia coli O157.

Existing methods for detection of food-borne pathogens and their toxins are frequently time-consuming, require specialized equipment, and involve lengthy culture procedures and/or animal testing and are thus unsuitable for a rapid response to an emergency public health situation. A series of simple and rapid affinity immunochromatography column (AICC) assays were developed to detect Clostridium botulinum neurotoxin types A, B, E, and F and Escherichia coli O157 in food matrices. Specifically, for milk, grape juice with peach juice, and bottled water, the detection limit for the botulinum neurotoxin type A complex was 0.5 ng. Use of this method with a 10-ml sample would therefore result in a detection limit of 50 pg ml(-l). Thus, this assay is approximately 2 orders of magnitude more sensitive than a comparable lateral-flow assay. For botulinum neurotoxin complex types B, E, and F, the minimum detection limit was 5 ng to 50 ng. Sensitive detection of E. coli O157 was achieved, and the detection limit was 500 cells. The AICC test was also shown to be specific, rapid, and user friendly. This test takes only 15 to 30 min to complete without any specialized equipment and thus is suitable for use in the field. It has the potential to replace existing methods for presumptive detection of botulinum neurotoxin types A, B, E, and F and E. coli O157 in contaminated matrices without a requirement for preenrichment.

Proteomic analysis of rainbow trout (Oncorhynchus mykiss, Walbaum) serum after administration of probiotics in diets.

The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.