From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

Jeßberger, Nadja; Krey, Viktoria M.; Rademacher, Corinna; Böhm, Maria-Elisabeth; Mohr, Ann-Katrin; Ehling‐Schulz, Monika; Scherer, Siegfried; Märtlbauer, Erwin

doi:10.3389/fmicb.2015.00560

Cited by 74 publications

(149 citation statements)

References 79 publications

(113 reference statements)

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“…nheB (A) and hblD (C) transcription was determined by qRT-PCR, normalized to 16S rrn levels of the same sample and relative to the transcript level of an external calibrator. hblD expression of reference strain F837/76 at 6 h growth in CGY medium (Jeßberger et al, 2015) was used as calibrator and set to 100% (log-2 = 0). With the 2 −ΔΔ C T method, transcription of all other samples was compared to this calibrator.…”

Section: Resultsmentioning

confidence: 99%

“…While in CGY medium strains grew to maximum optical densities of 20 (Jeßberger et al, 2015), the average maximum OD 600 in cRPMI was 4–5. Log phases were shortened (2–4 h in cRPMI compared to 2–6 h in CGY).…”

Section: Resultsmentioning

confidence: 99%

“…* Strain showed high toxicity but particularly low NheB titers in CGY due to binding failure of mAb 2B11 in sandwich EIA (Didier et al, 2015) .** Sequence analysis revealed a truncated hbl operon; as strain is not able to produce Hbl L2 and Hbl B protein (negative in EIAs), it was allocated to profile F (Jeßberger et al, 2015) . Genotyping of the strains had been conducted by sequence analyses of the genetic markers spoIIIAB and panC. PCR analyses with specific primers for ces and enterotoxin genes had been used for toxin profiling.…”

Section: Methodsmentioning

confidence: 99%

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Simulating Intestinal Growth Conditions Enhances Toxin Production of Enteropathogenic Bacillus cereus

et al. 2017

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Bacillus cereus is a ubiquitous bacterial pathogen increasingly reported to be the causative agent of foodborne infections and intoxications. Since the enterotoxins linked to the diarrheal form of food poising are foremost produced in the human intestine, the toxic potential of enteropathogenic B. cereus strains is difficult to predict from studies carried out under routine cultivation procedures. In this study, toxigenic properties of a panel of strains (n = 19) of diverse origin were compared using cell culture medium pre-incubated with CaCo-2 cells to mimic intestinal growth conditions. Shortly after contact of the bacteria with the simulated host environment, enterotoxin gene expression was activated and total protein secretion of all strains was accelerated. Although the signal stimulating enterotoxin production still needs to be elucidated, it could be shown that it originated from the CaCo-2 cells. Overall, our study demonstrates that the currently used methods in B. cereus diagnostics, based on standard culture medium, are not allowing a conclusive prediction of the potential health risk related to a certain strain. Thus, these methods should be complemented by cultivation procedures that are simulating intestinal host conditions.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Simulating Intestinal Growth Conditions Enhances Toxin Production of Enteropathogenic Bacillus cereus

et al. 2017

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“…However, numerous studies have already demonstrated that Nhe, Hbl and CytK (separately or in combination) do not seem to fully explain the diarrhoeal syndrome related to B. cereus [46,47,48,49]. Indeed, although nhe and hbl genes detection in B. cereus is generally applied to assess the enterotoxic potential of strains, it has been shown that the gene presence does not guaranty the cytotoxicity of the bacteria.…”

Section: Discussionmentioning

confidence: 99%

Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells

Castiaux

Laloux

Schneider

et al. 2016

Toxins

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B. cereus is an opportunistic foodborne pathogen able to cause diarrhoea. However, the diarrhoeal potential of a B. cereus strain remains difficult to predict, because no simple correlation has yet been identified between the symptoms and a unique or a specific combination of virulence factors. In this study, 70 B. cereus strains with different origins (food poisonings, foods and environment) have been selected to assess their enterotoxicity. The B. cereus cell-free supernatants have been tested for their toxicity in vitro, on differentiated (21 day-old) Caco-2 cells, using their ATP content, LDH release and NR accumulation. The genetic determinants of the main potential enterotoxins and virulence factors (ces, cytK, entFM, entS, hbl, nhe, nprA, piplC and sph) have also been screened by PCR. This analysis showed that none of these genes was able to fully explain the enterotoxicity of B. cereus strains. Additionally, in order to assess a possible effect of the mucus layer in vitro, a cytotoxicity comparison between a monoculture (Caco-2 cells) and a co-culture (Caco-2 and HT29-MTX mucus-secreting cells) model has been performed with selected B. cereus supernatants. It appeared that, in these conditions, the mucus layer had no notable influence on the cytotoxicity of B. cereus supernatants.

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“…Probiotics may interfere with pathogen invasion by reducing or inhibiting pathogen adherence, producing antimicrobials, or interfering with toxins (41). Indeed, some pathogens, such as C. difficile, B. cereus, Vibrio cholerae, and Escherichia coli, may secrete toxins that are implicated in bacterial virulence (11,(42)(43)(44). Proteins secreted and released into the environment might mediate some of the positive effects observed in probiotics (45,46).…”

Section: Discussionmentioning

confidence: 99%

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins

Ripert

Racedo

Elie

et al. 2016

Antimicrob Agents Chemother

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Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.T he gut, one of the biggest surfaces of exchange between the body's internal and external environment, is in permanent contact with antigens. Indeed, the intestinal barrier constitutes an important playground for numerous intestinal pathogen bacteria and toxins.Among them, Clostridium difficile, an anaerobic Gram-positive bacterium, is a major cause of diarrhea, pseudomembranous colitis, and septicemia, and it can lead to death. Hospital stay and use of antibiotics are major risk factors for contracting this disease (1). Over the past decade, the frequency and severity of C. difficile infections have increased markedly due to the emergence of socalled hypervirulent strains that overproduce toxins (2-4). The C. difficile toxins modify target cell proteins to cause disassembly of the actin cytoskeleton and induce severe inflammation (5).Expression of several host genes, mainly those involved in cellular processes, cell-cell interactions, apoptosis, and inflammation, is modified during C. difficile infection (6). These include host genes encoding RhoB (regulating actin cytoskeleton and interactions between cells), ZO-1 (tight-junction protein), and ROCK2 (actin cytoskeleton and signaling protein regulator) (7-9).Equally, many other pathogens, such as Salmonella species, E...

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From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

Cited by 74 publications

References 79 publications

Simulating Intestinal Growth Conditions Enhances Toxin Production of Enteropathogenic Bacillus cereus

Simulating Intestinal Growth Conditions Enhances Toxin Production of Enteropathogenic Bacillus cereus

Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins

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