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Aims This study explored how dairy farm operating systems with free-stall or tie-stall housing and cow hygiene score influence the occurrence of zoonotic bacteria in raw milk. Methods and Results Samples from bulk tank milk (BTM), milk filters, faeces, feed, teats and teat milk were collected from 11 farms with loose housing and seven farms with tie-stall housing every second month over a period of 11 months and analysed for the presence of STEC by culturing combined with polymerase chain reaction and for Campylobacter spp. and L. monocytogenes by culturing only. Campylobacter spp., L. monocytogenes and STEC were present in samples from the farm environment and were also detected in 4%, 13% and 7% of the milk filters, respectively, and in 3%, 0% and 1% of BTM samples. Four STEC isolates carried the eae gene, which is linked to the capacity to cause severe human disease. L. monocytogenes were detected more frequently in loose housing herds compared with tie-stalled herds in faeces (p = 0.02) and feed (p = 0.03), and Campylobacter spp. were detected more frequently in loose housing herds in faeces (p < 0.01) and teat swabs (p = 0.03). An association between cow hygiene score and detection of Campylobacter spp. in teat milk was observed (p = 0.03). Conclusion Since some samples collected from loose housing systems revealed a significantly higher (p < 0.05) content of L. monocytogenes and Campylobacter spp. than samples collected from tie-stalled herds, the current study suggests that the type of housing system may influence the food safety of raw milk. Significance and Impact of the Study This study highlights that zoonotic bacteria can be present in raw milk independent of hygienic conditions at the farm and what housing system is used. Altogether, this study provides important knowledge for evaluating the risk of drinking unpasteurized milk.

Listeria monocytogenes is a ubiquitous environmental bacterium associated with a wide variety of natural and human-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation of L. monocytogenes from various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole-genome sequencing (WGS) was performed for the collected isolates and for L. monocytogenes from Norwegian dairy farms and slugs (218 isolates in total). The data were compared to available data sets from clinical and food-associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0 to 8 whole-genome multilocus sequence typing (wgMLST) allelic differences were collected over time in the same location, demonstrating persistence of L. monocytogenes in natural, urban, and farm environments. Furthermore, several clusters with 6 to 20 wgMLST allelic differences containing isolates collected across different locations, times, and habitats were identified, including nine clusters harboring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, and CC1) and food (CC9, CC121, CC7, and CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases.

Shiga toxin (Stx) is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor. CI maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and the three already described Erus, ten novel Erus (named Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 seem to be widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain.IMPORTANCEEHEC is an emerging health challenge worldwide and outbreaks caused by this pathogen tend to be more frequent and severe. Increased knowledge on how characteristics of the replication region influence the virulence of E. coli may be used for more precise identification of high-risk EHEC strains.

Listeria monocytogenesis a ubiquitous environmental bacterium associated with a wide variety of natural and man-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation ofL. monocytogenesfrom various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole genome sequencing (WGS) was performed for the collected isolates and forL. monocytogenesfrom Norwegian dairy farms and slugs, in total 218 isolates. The data was compared with available datasets from clinical and food associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0-8 wgMLST allelic differences were collected over time in the same location, demonstrating persistence ofL. monocytogenesin natural, urban and farm environments. Furthermore, several clusters with 6-20 wgMLST allelic differences containing isolates collected across different locations, times and habitats were identified, including nine clusters harbouring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, CC1) and food (CC9, CC121, CC7, CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases.ImportanceListeria monocytogenesis a deadly foodborne pathogen that is widespread in the environment. For effective management, both public health authorities and food producers need reliable tools for source tracking, surveillance, and risk assessment. For this, whole genome sequencing (WGS) is regarded as the present and future gold standard. In the current study, we use WGS to show thatL. monocytogenescan persist for months and years in natural, urban and dairy farm environments. Notably, clusters of almost identical isolates, with genetic distances within the thresholds often suggested for defining an outbreak cluster, can be collected from geographically and temporally unrelated sources. The work highlights the need for a greater knowledge of the genetic relationships between clinical isolates and isolates ofL. monocytogenesfrom a wide range of environments, including natural, urban, agricultural, livestock, food production, and food processing environments, in order to correctly interpret and use results from WGS analyses.

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Aims: This study explores how different dairy farm operating systems influence the occurrence of zoonotic bacteria in raw milk. Methods and Results: Samples from bulk tank milk, milk filters, feces, feed, teats and teat milk were collected from eleven farms with loose housing and seven with tie-stall housing every second month over a period of 11 months and analyzed for the presence of Campylobacter spp., L. monocytogenes and STEC. Campylobacter spp., L. monocytogenes and STEC were abundant in samples from the farm environment and were also detected in 4%, 13% and 7% of the milk filters, respectively, and in 3%, 0% and 1% of bulk tank milk samples. Four STEC isolates carried the eae gene, which is linked to the capacity to cause more severe human disease. Conclusion: The results indicate a higher prevalence of L. monocytogenes and Campylobacter spp. in samples collected from loose housed herds compared to tie-stalled herds suggesting that the operating system can influence the food safety of raw milk. Significance and Impact of the study: This study highlights that zoonotic bacteria can be present in raw milk independent of hygienic conditions at the farm and what hosing system is used. Altogether, this study provides an important knowledge base for evaluating the risk of drinking unpasteurized milk.

Shiga toxin is the major virulence factor of enterohemorrhagicEscherichia coli(EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack theOandPgenes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to aserugenes (afterEHEC phagereplicationunit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.

Food business operators are responsible for food safety and assessment of shelf lives for their ready-to-eat products. For assisting them, a customized software based on predictive models, ListWare, is being developed. The aim of this study was to develop and validate a predictive model for the growth of Listeria monocytogenes in sliced roast beef. A challenge study was performed comprising 51 different combinations of variables. The growth curves followed the Baranyi and Roberts model with no clear lag phase and specific growth rates in the range

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H(2)O(2)), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H(2)O(2) or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H(2)O(2) and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest. IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

SummaryBacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies. By using cryo-EM and 3D helical reconstruction we show that Bacillus Enas form a novel class of Gram-positive pili. Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought and chemical damage. Phylogenomic analysis reveals the presence of defined ena clades amongst different eco- and pathotypes. We propose Enas to represent a novel class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Spore forming bacteria comprise a large part of the human gut microbiota. However, study of the endospores in gut microbiota is limited due to difficulties of culturing and numerous unknown germination factors. In this study we propose a new method for culture-independent characterization of endospores in stool samples. We have enriched DNA of spore-forming bacterial species from stool samples of 40 mother-child pairs from a previously described mother-child cohort. The samples were exposed to a two-step purification process comprising ethanol and ethidium monoazide (EMA) treatment to first kill vegetative cells and to subsequently eliminate their DNA from the samples. The composition of the ethanol-EMA resistant DNA was characterized by 16S rRNA marker gene sequencing. Operational taxonomic units (OTUs) belonging to the Clostridia class (OTU1: Romboutsia, OTU5: Peptostreptococcaceae and OTU14: Clostridium senso stricto) and one belonging to the Bacillus class (OTU20: Turicibacter) were significantly more abundant in the samples from mothers and children after ethanol-EMA treatment than in those treated with ethanol only. No correlation was observed between ethanol-EMA resistant OTUs detected in children and in their mothers, which indicates that a low level of spore-forming species are shared between mothers and their children. Anaerobic ethanol-resistant bacteria were isolated from all mothers and all children over 1 year of age. Generally, in 70% of the ethanol-treated samples used for anaerobic culturing, 16S rRNA gene sequences of bacterial isolates corresponded to OTUs detected in these samples after EMA treatment. We report a new DNA-based method for the characterization of endospores in gut microbiota. Our method has high degree of correspondence to the culture-based method, although it requires further optimization. Our results also indicate a high turnover of endospores in the gut during the first two years of life, perhaps with a high environmental impact. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Listeria monocytogenes, the causative agent of the serious food-borne disease listeriosis, can rapidly adapt to a wide-range of environmental stresses, including visible light. This study shows that exposure of the L. monocytogenes EGDe strain to low-intensity broad spectrum visible light inhibited bacterial growth and caused altered multicellular behavior during growth on semi-solid agar as compared to when the bacteria were grown in complete darkness. These light-dependent changes were observed regardless of the presence of the blue light receptor (Lmo0799) and the stressosome regulator Sigma B (SigB), which have been suggested to be important for the ability of L. monocytogenes to respond to blue light. A genome wide transcriptional analysis revealed that exposure of L. monocytogenes EGDe to broad-spectrum visible light caused altered expression of 2409 genes belonging to 18 metabolic pathways, as compared to bacteria grown in darkness. The light-dependent differentially expressed genes are involved in functions such as glycan metabolism, cell wall synthesis, chemotaxis, flagellar synthesis and resistance to oxidative stress. Exposure to light conferred reduced bacterial motility in semi-solid agar, which correlates well with the light-dependent reduction in transcript levels of flagellar and chemotaxis genes. Similar light-induced reduction in growth and motility was also observed in two different L. monocytogenes food isolates, suggesting that these responses are typical for L. monocytogenes. Together, the results show that even relatively small doses of broad-spectrum visible light causes genome-wide transcriptional changes, reduced growth and motility in L. monocytogenes. IMPORTANCE Despite major efforts to control L. monocytogenes, this pathogen remains a major problem to the food industry where it poses a continuous risk of food contamination. The ability of L. monocytogenes to sense and adapt to different stressors in the environments enables it to persist in many different niches, including food production facilities and in food products. The present study shows that exposure of L. monocytogenes to low-intensity broad-spectrum visible light, reduces its growth and motility as well as alters its multicellular behavior. Light exposure also caused genome-wide changes in transcript levels, affecting multiple metabolic pathways, which are likely to influence the bacterial physiology and lifestyle. In practical terms, the data presented in this study suggest that broad spectrum visible light is an important environmental variable to consider as a strategy to improve food safety by reducing L. monocytogenes contamination in food production environments.

Germination of Bacillus spores is triggered by the binding of specific nutrients to germinant receptors (GRs) located in the spore’s inner membrane. The GRs typically consist of A, B, and C subunits, encoded by tricistronic ger operons. The Bacillus licheniformis genome contains the gerA family operons gerA, ynd, and gerK. In contrast to the ABC(D) organization that characterizes gerA operons of many Bacillus species, B. licheniformis genomes contain a pentacistronic ynd operon comprising the yndD, yndE(3), yndE(2), yndF(1), and yndE(1) genes encoding A, B, B, C, and B GR subunits, respectively (subscripts indicate paralogs). Here we show that B. licheniformis spores can germinate in the absence of the Ynd and GerK GRs, although cooperation between all three GRs is required for optimal germination with amino acids. Spores carrying an incomplete set of Ynd B subunits demonstrated reduced germination efficiencies, while depletion of all three Ynd B subunits restored germination of the spore population to levels only slightly lower than those of wild-type spores at high germinant concentrations. This suggests that the presence of an incomplete set of Ynd B subunits exhibits a dominant negative effect on germination and that the A and C subunits of the Ynd GR are sufficient for the cooperative functionality between Ynd and GerA. In contrast to the B subunits of Ynd, the B subunit of GerA was essential for amino acid-induced germination. This study provides novel insights into the role of individual GR subunits in the cooperative interaction between GRs in triggering spore germination. IMPORTANCE Spore-forming bacteria are problematic for the food industry, as spores can survive decontamination procedures and subsequently revive in food products, with the risk of food spoilage and foodborne disease. The Ynd and GerA germination receptors (GRs) cooperate in triggering efficient germination of Bacillus licheniformis spores when nutrients are present in the surrounding environment. This study shows that the single B subunit of GerA is essential for the cooperative function between Ynd and GerA, while the three B subunits of the Ynd GR are dispensable. The ability of GRs lacking individual subunits to stimulate germination together with other GRs could explain why ger operons lacking GR subunit genes are maintained in genomes of spore-forming species.

Bacillus licheniformis is frequently associated with food spoilage due to its ability to form highly resistant endospores. The present study reveals that B. licheniformis spore peptidoglycan shares a similar structure to spores of other species of Bacillus. Two enzymatic activities associated with depolymerisation of the cortical peptidoglycan, which represents a crucial step in spore germination, were detected by muropeptide analysis. These include lytic transglycosylase and N-acetylglucosaminidase activity, with non-lytic epimerase activity also being detected. The role of various putative cortex-lytic enzymes that account for the aforementioned activity was investigated by mutational analysis. These analyses indicate that SleB is the major lysin involved in cortex depolymerisation in B. licheniformis spores, with CwlJ and SleL having lesser roles. Collectively, the results of this work indicate that B. licheniformis spores employ a similar approach for cortical depolymerisation during germination as spores of other Bacillus species.

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signalling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.

The food industry is under pressure to reduce the NaCl content in food, but the consequences on the ability of L. monocytogenes to survive in the human host and cause listeriosis is not known. In this study, a recently developed internationally harmonized static in vitro digestion (IVD) model was used to investigate the survival of L. monocytogenes in the gastric and intestinal phases after exposure to 5 or 0.5% NaCl. Six isolates from three Scandinavian foodborne listeriosis outbreaks, all related to NaCl containing foods, the EGDe reference strain and an EGDe mutant, deleted for the major stress regulator gene, sigB, were included. A ten-fold reduction of NaCl in the cultivation media significantly reduced the survival fraction of the EGDe strain in the IVD model while one of the clinical outbreak isolates showed a significantly increased survival fraction. Finally, the EGDe strain was able to attach and invade cultured HT-29 cells after passage through the IVD model. Altogether, these results suggest that a reduction of the NaCl content from 5 to 0.5% prior to exposure to the IVD model has the potential to cause a change in the relative survival fraction and that the effect is strain dependent.

When nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/DSM13 genome exhibits three gerA family operons (gerA, gerK, and ynd) encoding germinant receptors. We show that spores of B. licheniformis germinate efficiently in response to a range of different single l-amino acid germinants, in addition to a weak germination response seen with d-glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with single l-amino acid germinants, whereas the GerK germination receptor is essential for germination with d-glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with single l-amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present in B. licheniformis. We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination.

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at 300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process.

Benzyl alcohol (BnOH) is widely used as a component of foods, cosmetics, household products and medical products. It is generally considered to be safe for human use, however, it has been connected to a number of adverse effects, including hypersensitivity reactions and neonatal deaths. BnOH is a membrane fluidizing agent that can affect membrane protein activity and cellular processes such as ligand binding to cell surface receptors, endocytosis and degradation of lysosomal cargo. In this study, we examined the effects of BnOH on intracellular transport using Shiga toxin (Stx), diphtheria toxin (DT) and ricin. BnOH caused reduced toxicity of all three toxins at BnOH concentrations that cause membrane fluidization. The reduced toxicity of Stx and ricin was mainly due to inhibition of retrograde transport between endosomes and the trans-Golgi network as BnOH had small effects on cell association and endocytosis of ricin and Stx. Strikingly, BnOH also induced a reversible fragmentation of the Golgi apparatus.

Shiga toxin-producing Escherichia coli bacteria cause hemorrhagic colitis and hemolytic uremic syndrome in humans. Currently, only supportive treatment is available for diagnosed patients. We show here that 24-h pretreatment with an ether lipid precursor, the alkylglycerol sn-1-O-hexadecylglycerol (HG), protects HEp-2 cells against Shiga toxin and Shiga toxin 2. Also the endothelial cell lines HMEC-1 and HBMEC are protected against Shiga toxins after HG pretreatment. In contrast, the corresponding acylglycerol, DL-α-palmitin, has no effect on Shiga toxicity. Although HG treatment provides a strong protection (~30 times higher IC₅₀) against Shiga toxin, only a moderate reduction in toxin binding was observed, suggesting that retrograde transport of the toxin from the plasma membrane to the cytosol is perturbed. Furthermore, endocytosis of Shiga toxin and retrograde sorting from endosomes to the Golgi apparatus remain intact, but transport from the Golgi to the endoplasmic reticulum is inhibited by HG treatment. As previously described, HG reduces the total level of all quantified glycosphingolipids to 50-70% of control, including the Shiga toxin receptor globotriaosylceramide (Gb3), in HEp-2 cells. In accordance with this, we find that interfering with Gb3 biosynthesis by siRNA-mediated knockdown of Gb3 synthase for 24 h causes a similar cytotoxic protection and only a moderate reduction in toxin binding (to 70% of control cells). Alkylglycerols, including HG, have been administered to humans for investigation of therapeutic roles in disorders where ether lipid biosynthesis is deficient, as well as in cancer therapy. Further studies may reveal if HG can also have a therapeutic potential in Shiga toxin-producing E. coli infections.

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at 300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis , a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry.

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signalling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.

When nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/DSM13 genome exhibits three gerA family operons ( gerA , gerK , and ynd ) encoding germinant receptors. We show that spores of B. licheniformis germinate efficiently in response to a range of different single l -amino acid germinants, in addition to a weak germination response seen with d -glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with single l -amino acid germinants, whereas the GerK germination receptor is essential for germination with d -glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with single l -amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present in B. licheniformis . We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination. IMPORTANCE To ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning. Bacillus licheniformis is a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the mechanisms of spore germination are poorly characterized in this species. This study provides novel knowledge on germination of B. licheniformis spores. We characterize the germinant recognition profiles of the three germinant receptors present in B. licheniformis spores and demonstrate that the GerA germinant receptor cooperates with the Ynd and GerK germinant receptors to enable an effective germination response to l -amino acids. We also demonstrate that GerK is required for germination in response to the single germinant glucose. This study demonstrates the complex interactions between germinant receptors necessary for efficient germination of B. licheniformis spores.

In response to nutrient limitation in the environment, the global transcriptional regulator CodY modulates various pathways in low G+C Gram-positive bacteria. In Bacillus subtilis CodY triggers adaptation to starvation by secretion of proteases coupled to the expression of amino acid transporters. Furthermore, it is involved in modulating survival strategies like sporulation, motility, biofilm formation, and CodY is also known to affect virulence factor production in pathogenic bacteria. In this study, the role of CodY in Bacillus cereus ATCC 14579, the enterotoxin-producing type strain, is investigated. A marker-less deletion mutant of codY (ΔcodY) was generated in B.cereus and the transcriptome changes were surveyed using DNA microarrays. Numerous genes involved in biofilm formation and amino acid transport and metabolism were upregulated and genes associated with motility and virulence were repressed upon deletion of codY. Moreover, we found that CodY is important for efficient production of toxins and for adapting from nutrient-rich to nutrient-limited growth conditions of B.cereus. In contrast, biofilm formation is highly induced in the ΔcodY mutant, suggesting that CodY represses biofilm formation. Together, these results indicate that CodY plays a crucial role in the growth and persistence of B.cereus in different environments such as soil, food, insect guts and the human body.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted β-tongue were replaced with glycine (NheC cys− ) or where the entire hydrophobic stretch was deleted (NheC hr− ), the predicted hydrophobic β-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

Bacillus cereus ATCC 10987 and ATCC 14579 can be induced to high levels of resistance to tetracycline. The chromosomal B. cereus gene bctl encodes a transmembrane protein with homology to Gram-positive tetracycline efflux proteins and relation to other members of the major facilitator superfamily of transport proteins. A mutant strain containing an insertionally inactivated bctl gene did not show impaired tetracycline resistance. No additional altered phenotype was observed in the mutant. Accumulation studies suggested that the resistance mechanism involves a reduced sensitivity to intracellular tetracycline.

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli. Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

The Bacillus cereus group comprises seven species: Bacillus cereus sensu stricto, Bacillus thuringiensis, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus cytotoxicus. Three members of the B. cereus group B. anthracis, B.cereus, and B. cytotoxicus are well-known human pathogens. The members of the B. cereus group produce endospores that are highly resistant to disinfectants, radiation, desiccation, and heat. All members of the group express a wide range of protein toxins such as phospholipases, hemolysins, and enterotoxins. In addition, B. anthracis, the causative agent of anthrax, produces three different proteins necessary for the development of anthrax and B. thuringiensis produce endotoxins active against insects. Members of the B. cereus group are widespread in nature and are easily spread to foods, where they may cause several types of food-associated illnesses. Due to the highly resistant spores, they are becoming increasingly important in the food industry and especially during production of sous-vide foods. In this chapter, the protein toxins expressed in B. cereus sensu stricto and B. thuringiensis are described.

Haemolysin BL (HBL) is a Bacillus cereus toxin composed of a binding component, B, and two lytic components, L1 and L2. HBL is also the enterotoxin responsible for the diarrhoeal food poisoning syndrome caused by several strains of B. cereus. The three genes encoding the HBL components constitute an operon and are transcribed from a promoter 608 bp upstream of the hblC translational start site. The first gene of the hbl operon, hblC, in the B. cereus type strain, ATCC 14579, was inactivated in this study. Inactivation of hblC strongly reduced both the enterotoxigenic activity of B. cereus ATCC 14579 and the haemolytic activity against sheep erythrocytes, while maintaining full haemolytic activity against human erythrocytes.

The opportunistic pathogen Bacillus cereus is the genetically stable member of a group of closely related bacteria including the insect pathogen Bacillus thuringiensis and the mammalian pathogen Bacillus anthracis. Physical maps of B. cereus and B. thuringiensis strains show considerable variations in discrete parts of the chromosome, suggesting that certain genome regions are more prone to rearrangements. B. cereus belongs to the same subgroup of Bacillus species as Bacillus subtilis, by both phenotypic and rRNA sequence classification. The analysis of 80 kb of genome sequence sampled from different regions of the B. cereus ATCC 10987 chromosome is reported. Analysis of the sequence and comparison of the localization of the putative genes with that of B. subtilis orthologues show the following: (1) gene organization is not conserved between B. cereus and B. subtilis; (2) several putative genes are more closely related to genes from other bacteria and archaea than to B. subtilis, or may be absent in B. subtilis 168; (3) B. cereus contains a 155 bp repetitive sequence that is not present in B. subtilis. By hybridization, this repeat is present in all B. cereus and B. thuringiensis strains so far investigated.

Background L-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13. Results In this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3ΔgerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800. Conclusions A wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.

The complete sequence of a Bacillus cereus member of the SNF2 family of putative helicases showed conservation of all seven motifs typical of this family. Bcsnf predicted a protein of 1064 aa where the conserved SNF2 domain was located at the carboxy terminus, whereas the 633 amino-terminal aa showed no homology to any protein in the databases. A putative transcriptional start was identified by primer extension, indicating that Bcsnf is not a part of a larger operon. No phenotypical changes were observed after insertional inactivation of Bcsnf. The completely sequenced genomes of Mycoplasma genitalium and Haemophilus influenzae contain one ORF each with similarity to the SNF2 family: MG018 and HI0616, respectively. A phylogenetic tree of the SNF2 family showed that BcSNF and MG018 were most closely related, and appeared closer to the eukaryotic members of the SNF2 family than to the two other bacterial members of the family, HepA from Escherichia coli and HI0616.

Aims The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted to see whether cytotoxicity reflected the measured levels of lichenysin. Methods and Results Fifty-three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lchAA gene fragments were detected in all strains by polymerase chain reaction (PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC-MS/MS (liquid chromatography–tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells. Conclusions Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 μg ml−1, which is in accordance with previous studies. Significance and Impact of Study This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive.

The structure of NheA, a component of the Bacillus cereus Nhe tripartite toxin, has been solved at 2.05 A resolution using selenomethionine multiple-wavelength anomalous dispersion (MAD). The structure shows it to have a fold that is similar to the Bacillus cereus Hbl-B and E. coli ClyA toxins, and it is therefore a member of the ClyA superfamily of α-helical pore forming toxins (α-PFTs), although its head domain is significantly enlarged compared with those of ClyA or Hbl-B. The hydrophobic β-hairpin structure that is a characteristic of these toxins is replaced by an amphipathic β-hairpin connected to the main structure via a β-latch that is reminiscent of a similar structure in the β-PFT Staphylococcus aureus α-hemolysin. Taken together these results suggest that, although it is a member of an archetypal α-PFT family of toxins, NheA may be capable of forming a β rather than an α pore.

The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml(-1), but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

The nonhaemolytic enterotoxin (Nhe) of Bacillus cereus plays a key role in cases of B. cereus food poisoning. The toxin is comprised of three different proteins: NheA, NheB and NheC. Here, the expression in Escherichia coli, purification and crystallization of the NheA protein are reported. The protein was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The crystals of NheA diffracted to 2.05 Å resolution and belonged to space group C2, with unit-cell parameters a = 308.7, b = 58.2, c = 172.9 Å, β = 110.6°. Calculation of V(M) values suggests that there are approximately eight protein molecules per asymmetric unit.