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Abstract Background Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated. Results Sec-type signal peptides were identified in all toxin components, and secretion of Hbl component B was shown to be dependent on an intact Sec-type signal peptide sequence. Further indication that secretion of Hbl, Nhe and CytK is dependent on the Sec translocation pathway, the main pathway on which bacterial secretion relies, was suggested by the observed intracellular accumulation and reduced secretion of the toxins in cultures supplemented with the SecA inhibitor sodium azide. Although a FEA deficient strain (a flhA mutant) showed reduced toxin expression and reduced cytotoxicity, it readily secreted overexpressed Hbl B, showing that the FEA is not required for Hbl secretion. Thus, the concurrent lack of flagella and reduced toxin secretion in the FEA deficient strain may point towards the presence of a regulatory link between motility and virulence genes, rather than FEA-dependent toxin secretion. Conclusions The Hbl, Nhe and CytK toxins appear to be secreted using the Sec pathway, and the reduced Hbl expression of a FEA deficient strain was shown not to be due to a secretion defect.

Abstract Background Children with attention-deficit/hyperactivity disorder (ADHD) often experience problems with education, interaction with others and emotional disturbances. Families of ADHD children also suffer a significant burden, in terms of strain on relationships and reduced work productivity. This parent survey assessed daily life for children with ADHD and their families. Method This pan-European survey involved the completion of an on-line questionnaire by parents of children (6–18 years) with ADHD (ADHD sample) and without ADHD (normative population sample). Parents were questioned about the impact of their child's ADHD on everyday activities, general behaviour and family relationships. Results The ADHD sample comprised 910 parents and the normative population sample 995 parents. 62% of ADHD children were not currently receiving medication; 15% were receiving 6–8 hour stimulant medication and 23% 12-hour stimulant medication. Compared with the normative population sample, parents reported that ADHD children consistently displayed more demanding, noisy, disruptive, disorganised and impulsive behaviour. Significantly more parents reported that ADHD children experienced challenges throughout the day, from morning until bedtime, compared with the normative population sample. Parents reported that children with ADHD receiving 12-hour stimulant medication experienced fewer challenges during early afternoon and late afternoon/early evening than children receiving 6–8 hour stimulant medication; by late evening and bedtime however, this difference was not apparent. ADHD was reported to impact most significantly on activities such as homework, family routines and playing with other children. All relationships between ADHD children and others were also negatively affected, especially those between parent and child (72% of respondents). Parents reported that more children with ADHD experienced a personal injury in the preceding 12 months, including those requiring the attention of healthcare professionals. Although 68% of parents were satisfied with their child's current treatment, 35–40% stated that their child's ADHD symptoms needed to be more effectively treated during the afternoon and evening. Conclusion This parent survey highlights the breadth of problems experienced by ADHD children and the impact throughout the day on both activities and relationships. Therefore, there is a need for treatment approaches that take into account the 24-hour impact of the disorder and include all-day coverage with effective medication.

Members of the B. cereus group are spore-forming organisms commonly associated with spoilage of milk and dairy products. We have determined the genetic identity and growth characteristics of 57 B. cereus isolates collected from a Norwegian ice cream production plant. Our findings revealed persistence of B. cereus spp. strains for up to 19 months, suggesting the plant's susceptibility to long-term colonization. One of the mesophilic isolates, NVH-YM303, carried a complete cereulide synthetase operon. To assess the potential food poisoning risk associated with the presence of cereulide-producing strains in the production line, we examined the production of cereulide in ice cream and milk at different temperatures by NVH-YM303 and by the emetic psychrotrophic B. weihenstephanensis strain BtB2-4. Our findings revealed that NVH-YM303 produced higher levels of cereulide in ice cream as compared to milk. Furthermore, it was observed that NVH-YM303 produced more cereulide in ice cream at 25 °C compared to 15 °C. Conversely, BtB2-4 produced more cereulide in ice cream at 15 °C than at 25 °C. The results obtained in this study contribute to knowledge important for risk assessment of the potential hazards posed by the presence of B. cereus within ice cream production facilities.

Despite the lack of scientific evidence, some consumers assert that raw milk is a natural food with nutritional and immunological properties superior to pasteurized milk. This has led to the increased popularity of unpasteurized cow milk (UPM) and disregard for the risks of being exposed to zoonotic infections. Dairy cattle are healthy carriers of Shiga toxin (Stx)-producing E. coli (STEC), and contaminated UPM has caused STEC outbreaks worldwide. The association between STEC, carrying the eae (E. coli attachment effacement) gene, and severe diseases is well-established. We have previously isolated four eae positive STEC isolates from two neighboring dairy farms in the Southeast of Norway. A whole genome analysis revealed that isolates from different farms exhibited nearly identical genetic profiles. To explore the risks associated with drinking UPM, we examined the ability of the isolates to produce Stx and their growth in UPM at different temperatures. All the isolates produced Stx and one of the isolates was able to propagate in UPM at 8 °C (p < 0.02). Altogether, these results highlight the risk for STEC infections associated with the consumption of UPM.

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., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Background: Enterohemorrhagic Escherichia coli (EHEC) is an emerging health challenge worldwide and outbreaks caused by this pathogen poses a serious public health concern. Shiga toxin (Stx) is the major virulence factor of 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 which 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., Results: In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and three already described Erus, ten novel Erus (Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 are 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., Conclusion: 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., (© 2022. The Author(s).)

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. IMPORTANCE Listeria monocytogenes is 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 that L. monocytogenes can 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 of L. monocytogenes from a wide range of environments, including natural, urban, agricultural, livestock, food production, and food processing environments, 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., (© 2021 The Authors.)

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 <0.005-0.110 hr -1 . A linear regression model was developed based on 528 observations and had an adjusted R-square of 0.80. The significant predictors were storage temperature, sodium lactate, interactions between sodium acetate and temperature, and MAP packaging and temperature. The model was validated in four laboratories in three countries. For conditions where the model predicted up to + log 2 cfu/g Listeria concentration, the observed concentrations were true or below the predicted concentration in 90% of the cases. For the remaining 10%, the roast beef was coated with spices and therefore different from the others. The model will be implemented in ListWare web-application for calculation of "Listeria shelf life"., (Copyright © 2021. Published by Elsevier Ltd.)

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia 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 the O and P genes 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 as eru genes (after EHEC phage replication unit 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Llarena, Aspholm, O’Sullivan, Wêgrzyn and Lindbäck.)

Flagellar motility is considered an important virulence factor in different pathogenic bacteria. In Listeria monocytogenes the transcriptional repressor MogR regulates motility in a temperature-dependent manner, directly repressing flagellar- and chemotaxis genes. The only other bacteria known to carry a mogR homolog are members of the Bacillus cereus group, which includes motile species such as B. cereus and Bacillus thuringiensis as well as the non-motile species Bacillus anthracis , Bacillus mycoides and Bacillus pseudomycoides. Furthermore, the main motility locus in B. cereus group bacteria, carrying the genes for flagellar synthesis, appears to be more closely related to L. monocytogenes than to Bacillus subtilis , which belongs to a separate phylogenetic group of Bacilli and does not carry a mogR ortholog. Here, we show that in B. thuringiensis , MogR overexpression results in non-motile cells devoid of flagella. Global gene expression profiling showed that 110 genes were differentially regulated by MogR overexpression, including flagellar motility genes, but also genes associated with virulence, stress response and biofilm lifestyle. Accordingly, phenotypic assays showed that MogR also affects cytotoxicity and biofilm formation in B. thuringiensis . Overexpression of a MogR variant mutated in two amino acids within the putative DNA binding domain restored phenotypes to those of an empty vector control. In accordance, introduction of these mutations resulted in complete loss in MogR binding to its candidate flagellar locus target site in vitro . In contrast to L. monocytogenes , MogR appears to be regulated in a growth-phase dependent and temperature-independent manner in B. thuringiensis 407. Interestingly, mogR was found to be conserved also in non-motile B. cereus group species such as B. mycoides and B. pseudomycoides , which both carry major gene deletions in the flagellar motility locus and where in B. pseudomycoides mogR is the only gene retained. Furthermore, mogR is expressed in non-motile B. anthracis. Altogether this provides indications of an expanded set of functions for MogR in B. cereus group species, beyond motility regulation. In conclusion, MogR constitutes a novel B. thuringiensis pleiotropic transcriptional regulator, acting as a repressor of motility genes, and affecting the expression of a variety of additional genes involved in biofilm formation and virulence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Smith, Josefsen, Lindbäck, Hegna, Finke, Tourasse, Nielsen-LeRoux, Økstad and Fagerlund.)

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., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Listeria monocytogenes , the causative agent of the serious foodborne 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 semisolid agar 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 2,409 genes belonging to 18 metabolic pathways 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 semisolid 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 cause 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 for 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 environment 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 and 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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 triggers spore germination by activating germination receptors (GRs), while 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., (Copyright © 2017 American Society for Microbiology.)

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., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Enterohemorrhagic E. coli (EHEC) is associated with severe gastrointestinal disease. Upon entering the gastrointestinal tract, EHEC is exposed to a fluctuating environment and a myriad of other bacterial species. To establish an infection, EHEC strains have to modulate their gene expression according to the GI tract environment. In order to explore the interspecies interactions between EHEC and an human intestinal commensal, the global gene expression profile was determined of EHEC O103:H25 (EHEC NIPH-11060424) co-cultured with B. thetaiotaomicron (CCUG 10774) or grown in the presence of spent medium from B. thetaiotaomicron. Microarray analysis revealed that approximately 1% of the EHEC NIPH-11060424 genes were significantly up-regulated both in co-culture (30 genes) and in the presence of spent medium (44 genes), and that the affected genes differed between the two conditions. In co-culture, genes encoding structural components of the type three secretion system were among the most affected genes with an almost 4-fold up-regulation, while the most affected genes in spent medium were involved in chemotaxis and were more than 3-fold up-regulated. The operons for type three secretion system (TTSS) are located on the Locus of enterocyte effacement (LEE) pathogenicity island, and qPCR showed that genes of all five operons (LEE1-LEE5) were up-regulated. Moreover, an increased adherence to HeLa cells was observed in EHEC NIPH-11060424 exposed to B. thetaiotaomicron. Expression of stx2 genes, encoding the main virulence factor of EHEC, was down-regulated in both conditions (co-culture/spent medium). These results show that expression of EHEC genes involved in colonization and virulence is modulated in response to direct interspecies contact between cells, or to diffusible factors released from B. thetaiotaomicron. Such interspecies interactions could allow the pathogen to recognize its predilection site and modulate its behaviour accordingly, thus increasing the efficiency of colonization of the colon mucosa, facilitating its persistence and increasing its virulence potential.

Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.

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.

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 structure of NheA, a component of the Bacillus cereus Nhe tripartite toxin, has been solved at 2.05 Å 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.

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., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.

A nonhemolytic Listeria monocytogenes strain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 10⁹ CFU were intraperitoneally injected into mice. Hemolytic L. monocytogenes bacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing of prfA from the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation of prfA and that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. Nonhemolytic L. monocytogenes strains with identical duplications in prfA were isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.

The antibacterial activity of the polysaccharide chitosan towards different bacterial species has been extensively documented. The response mechanisms of bacteria exposed to this biopolymer and the exact molecular mechanism of action, however, have hardly been investigated. This paper reports the transcriptome profiling using DNA microarrays of the type-strain of Bacillus cereus (ATCC 14579) exposed to subinhibitory concentrations of two water-soluble chitosan preparations with defined chemical characteristics (molecular weight and degree of acetylation (F(A))). The expression of 104 genes was significantly altered upon chitosan A (weight average molecular weight (M(w)) 36.0 kDa, F(A) = 0.01) exposure and 55 genes when treated with chitosan B (M(w) 28.4 kDa, F(A) = 0.16). Several of these genes are involved in ion transport, especially potassium influx (BC0753-BC0756). Upregulation of a potassium transporting system coincides with previous studies showing a permeabilizing effect on bacterial cells of this polymer with subsequent loss of potassium. Quantitative PCR confirmed the upregulation of the BC0753 gene encoding the K(+)-transporting ATPase subunit A. A markerless gene replacement method was used to construct a mutant strain deficient of genes encoding an ATP-driven K(+) transport system (Kdp) and the KdpD sensor protein. Growth of this mutant strain in potassium limiting conditions and under salt stress did not affect the growth pattern or growth yield compared to the wild-type strain. The necessity of the Kdp system for potassium acquisition in B. cereus is therefore questionable. Genes involved in the metabolism of arginine, proline and other cellular constituents, in addition to genes involved in the gluconeogenesis, were also significantly affected. BC2798 encoding a chitin binding protein was significantly downregulated due to chitosan exposure. This study provides insight into the response mechanisms of B. cereus to chitosan treatment and the significance of the Kdp system in potassium influx under challenging conditions.

Background: Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated., Results: Sec-type signal peptides were identified in all toxin components, and secretion of Hbl component B was shown to be dependent on an intact Sec-type signal peptide sequence. Further indication that secretion of Hbl, Nhe and CytK is dependent on the Sec translocation pathway, the main pathway on which bacterial secretion relies, was suggested by the observed intracellular accumulation and reduced secretion of the toxins in cultures supplemented with the SecA inhibitor sodium azide. Although a FEA deficient strain (a flhA mutant) showed reduced toxin expression and reduced cytotoxicity, it readily secreted overexpressed Hbl B, showing that the FEA is not required for Hbl secretion. Thus, the concurrent lack of flagella and reduced toxin secretion in the FEA deficient strain may point towards the presence of a regulatory link between motility and virulence genes, rather than FEA-dependent toxin secretion., Conclusions: The Hbl, Nhe and CytK toxins appear to be secreted using the Sec pathway, and the reduced Hbl expression of a FEA deficient strain was shown not to be due to a secretion defect.

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 beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-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.