Food-borne Pathogen Bacillus Cereus Research Articles

Diterpenoids with Antibacterial Activities from the Fungus Trichoderma harzianum.

A new fusicoccane diterpenoid, harziaderma A (1), two novel harziane diterpenoids, harzianones G and H (2 and 3), one revised harziane diterpenoid (4), and two known diterpenoids (5 and 6) were isolated from the fungus Trichoderma harzianum and established via NMR, HRESIMS, Mo2(OAc)4-induced circular dichroism (ICD) and electronic circular dichroism (ECD) calculations. It is worth noting that compound 1 represents the first instance of a fusicoccane-type diterpenoid derived from T. harzianum. The structure of furanharzianone B was revised to 4 via careful spectroscopic analyses. Additionally, compounds 2 and 5 could suppress the overall growth of the foodborne bacterial pathogen Bacillus cereus. Compound 4 showed a moderate suppressive impact on NO generation in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The discoveries from the current study not only expanded the structural variety of diterpenoids isolated from T. harzianum but also laid a robust foundation for the development of harziane diterpenoids as anti-foodborne pathogen agents.

Acid-tolerant Lactiplantibacillus plantarum ZDY2013 shows a colonization niche preference and interacts with enterotoxigenic Bacillus cereus in specific-pathogen-free mice.

Probiotics have long been utilized as functional food and modulate gut microbial homeostasis, but their colonization niche is mostly unclear and transient, which restrains the development of microbiome-targeted strategies. Lactiplantibacillus (L.) plantarum ZDY2013 is an allochthonous species of the human gastrointestinal tract with acid-tolerant properties. It serves as an antagonistic agent against the food-borne pathogen Bacillus (B.) cereus and a potent regulator of the gut microbiota. However, there is a knowledge gap regarding the colonization dynamics of L. plantarum ZDY2013 in the host intestine and the colonization niche of its interaction with pathogens. Here, we designed a pair of specific primers targeting L. plantarum ZDY2013 based on its whole genome sequence. We evaluated their accuracy and sensitivity with other host-derived strains and confirmed their availability with artificially spiked fecal samples from different mouse models. Additionally, the content of L. plantarum ZDY2013 was quantified by qPCR in fecal samples from BALB/c mice, followed by the analysis of its colonization niche preference. Moreover, the interactions between L. plantarum ZDY2013 and enterotoxigenic B. cereus HN001 were also elucidated. The results revealed that the newly designed primers could identify L. plantarum ZDY2013 with high specificity and were resistant to the influence of the complex fecal matrix and gut microbes from different hosts. Interestingly, the content of mixed L. plantarum ZDY2013 and B. cereus HN001 when orally administered remained higher when compared with the single strain group in BALB/c mice upon discontinuation of intragastric administration. In addition, L. plantarum ZDY2013 was mainly enriched in the large intestine during the ingestion period and maintained the highest content in the stomach after discontinuing supplementation on day 7. Moreover, L. plantarum ZDY2013 colonization neither damaged the intestine nor ameliorated the damage triggered by B. cereus in BALB/c mice. Overall, our study constructed two efficient specific primers targeting L. plantarum ZDY2013 and provided the potential to explore the underlying mechanism of competition between L. plantarum ZDY2013 and pathogens in host species.

Synthesis, characterization and molecular docking studies of highly functionalized and biologically active derivatives of 2-aminothiazole

Many derivatives of 2-aminothiazoles are found to have significant biological properties and used as potential antibacterial and antifungal drugs. Therefore new synthetic methodologies for deriving 2-aminothiazole scaffolds are enabled in order to develop enhanced drug activity and therapeutic properties. In this study, a new series of 2-aminothiazole derivatives are synthesized via the cyclocondensation reaction of thiourea with appropriate acid halides followed by condensation with highly functionalized amines to get the amides of biological interest. The products thus obtained are characterized by IR, 1H NMR, 13C NMR and mass spectral data. The synthesized 2-aminothiazole derivatives were screened for antibacterial activity against Bacillus cereus bacterial strains using serial broth dilution methods. In addition the 2-aminothiazole derivatives are screened for their in vitro anti-bacterial and anti-fungal activity through microbial incubatory concentration (MIC) and molecular docking. This in silico technique is used to identify the interaction modes of the derived products with the active site of foodborne pathogen Bacillus cereus and yeast form of fungal pathogen Candida albicans. The study shows good docking scores with acceptable binding interactions.

Chemical Composition and Antibacterial Activity of Extracts of Cymbopogon Citratus (Lemon Grass) and Phyllantus Amarus (Stone Breaker) Leaves

This study described the chemical composition, antioxidant and antibacterial activities of the ethanol and aqueous extracts of fresh and dried leaves of Phyllantus amarus (PA) (Stone breaker), Cymbopogon citratus (CC) (Lemon grass) and combination of stone breaker and lemon grass (PA/CC and DL/DP) using standard methods. The proximate and mineral results showed that both fresh and dried samples of Phyllantus amarus and Cymbopogon citratus were rich in crude fibre (18.53%, 14.65%), ash (16.27%, 2.29%), potassium (95.56 mg/100g, 54.04 mg/100g), calcium (58.57 mg/100g, 7.05 mg/100g), and sodium (15.23 mg/100g, 9.84 mg/100g) respectively. The ability of the extracts to scavenge 2, 2 diphenyl -2- picryl-hydrazyl (DPPH) radical showed that the fresh PA had the highest scavenging activity of 91.12 mg/g, followed (PA/CC) 90.93 mg/g and dried PA (85.61mg/g). The phytochemical composition of the extracts also revealed that they contain high content of cardiac glycoside (11.62 mg/g - 64.81 mg/g), terpenoid (14.56 mg/g - 69.66 mg/g), phenol (14.73 mg/g - 53.93 mg/g), saponin (22.61 mg/g - 30.47mg/g). Dried lemon and dried Phyllantus amarus (DL/DP) was found to be rich in Vitamin A (22.22 mg/g), while the lowest value of Vitamin E (0.36 mg/g) was obtained from fresh lemon grass (0.36mg/g). Antibacterial assay carried out on the two leaves extracts showed that they produced mild antibacterial activity against some foodborne pathogen (Bacillus cereus, Shigella dysenteriae, Escherichia coli, Salmonella spp, Staphylococcus aureus, Pseudomonas spp). with zones of inhibition of 4.3 mm -9.1mm. Further studies should be carried out to investigate the employment of the leaves in production of functional food

The effect of natural feed additive on productive performance of broiler chickens

The effect of natural feed additive on productive performance of broiler chickens

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6–7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Toxins of Foodborne Pathogen Bacillus cereus and the Regulatory Factors Controlling the Biosynthesis of Its Toxins

Bacillus cereus is a versatile organism which causes two distinct types of food poisoning by producing toxins. Toxin formation in B. cereus is very much a complex process involving co-regulation of multiple genes exerting control at transcriptional, translational and post-translational level and such regulations too are often influenced by extrinsic factors. A comprehensive understanding of such factors is very crucial for holistic approaches and strategies in order to minimise food poisoning risk. Hence, this review will focus on the intrinsic and extrinsic factors controlling toxin biosynthesis in B. cereus.

Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress.

Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Short-term intake of Lactiplantibacillus plantarum ZDY2013 fermented milk promotes homoeostasis of gut microbiota under enterotoxigenic Bacillus cereus challenge.

Probiotics have long been used as functional starter cultures for fermented foods and are associated with numerous health benefits. Lactiplantibacillus (L.) plantarum ZDY2013 is an acid-tolerant probiotic candidate owning antagonistic properties against the food-borne pathogen Bacillus (B.) cereus and serves as a potent regulator of the gut microbiota. However, whether it retains these properties when used as dietary supplements in functional foods remains unknown. Accordingly, we investigated the ameliorating effects of L. plantarum fermented milk on disease phenotypes triggered by enterotoxigenic B. cereus in mice. The results revealed that administration of 3.0 × 108 cfu pathogenic B. cereus for one week induced damage to intestinal structures and bowel function, accompanied by an imbalance of gut microbiota. However, before or after B. cereus infection, oral administration of L. plantarum fermented milk mitigated losses of body weight and damage in the histological structure of the gastrointestinal tract, restored serum levels of IL-1β and IL-10, and contributed to significant decreases in platelet counts and uric acid levels. Most importantly, it restored the dissimilarity of gut microbiota and the abundance of bacterial taxa (i.e., reduced the abundance of Deferribacteres and Bacilli and increased the abundance of Lactobacillus and Bifidobacterium) without impacting the taxonomic composition. Combining these results, we speculate that enterotoxigenic B. cereus damages the intestinal epithelium and weakens its adherence capacity for the microbe, which is rescued by the supplementation of L. plantarum fermented milk. Overall, our findings revealed that L. plantarum ZDY2013 has the potential to be a fermented starter in functional foods and retains its antagonism against B. cereus pathogenesis.

Non-haemolytic enterotoxin induces pore-formation and activation of the inflammasome and pyroptosis

Abstract Inflammasomes are central to innate immunity mediating host defence against pathogens and maintaining homeostasis with commensal microbes. The sensing of pathogens, danger, or homeostasis-altering signals by inflammasome sensor proteins is the molecular basis driving assembly of the inflammasome complex. Here, we identify non-haemolytic enterotoxin (NHE) as an activator of the NLRP3 inflammasome. We show that NHE, found in the neglected human foodborne pathogen Bacillus cereus, is a non-redundant toxin to haemolysin BL (HBL) despite exhibiting a similar mechanism of action. NHE-induced activation of NLRP3 has a delayed kinetic to that induced by HBL. Subunit C of NHE, via its putative transmembrane region, initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, forming a tripartite lytic pore permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting its versatile functional repertoire in different host species. Moreover, our data suggest that both NHE and HBL operate synergistically to induce inflammation in the host. Overall, our results highlight that multiple virulence factors from the same pathogen exhibiting conserved function and mechanism of action can be exploited for sensing by a single inflammasome, ultimately leading to increased capacity of the host to detect and defend against naturally-occurring genetic variants.

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome.

Molecular Detection of Food Borne Pathogen Bacillus cereus in Ready to Eat Meat Products

Molecular Detection of Food Borne Pathogen Bacillus cereus in Ready to Eat Meat Products

Reliable Identification of Bacillus cereus Group Species Using Low Mass Biomarkers by MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based pathogen identification relies on the ribosomal protein spectra provided in the proprietary database. Although these mass spectra can discern various pathogens at species level, the spectra-based method still has limitations in identifying closely-related microbial species. In this study, to overcome the limits of the current MALDI-TOF MS identification method using ribosomal protein spectra, we applied MALDI-TOF MS of low-mass profiling to the identification of two genetically related Bacillus species, the food-borne pathogen Bacillus cereus, and the insect pathogen Bacillus thuringiensis. The mass spectra of small molecules from 17 type strains of two bacilli were compared to the morphological, biochemical, and genetic identification methods of pathogens. The specific mass peaks in the low-mass range (m/z 500- 3,000) successfully identified various closely-related strains belonging to these two reference species. The intensity profiles of the MALDI-TOF mass spectra clearly revealed the differences between the two genetically-related species at strain level. We suggest that small molecules with low molecular weight, 714.2 and 906.5 m/z can be potential mass biomarkers used for reliable identification of B. cereus and B. thuringiensis.

WITHDRAWN: The complete genome sequence and comparative genome analysis of the multi-drug resistant food-borne pathogen Bacillus cereus

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Heterologous expression and purification of BtCspB, a novel cold-shock protein-like bacteriocin from Bacillus thuringiensis BRC-ZYR2.

A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20mg/L. The purified BtCspB showed a MIC value of 12.5µg/mL and a MBC value of 50.0µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

Heat-resistance of psychrotolerant Bacillus cereus vegetative cells

Spores of psychrotolerant strains of the foodborne pathogen Bacillus cereus can multiply during storage of cooked or pasteurized, refrigerated foods and can represent a risk if these cells are not eliminated during reheating of food product before consumption. We determined the heat-resistance of psychrotolerant B. cereus vegetative cells at different heating temperatures in laboratory medium and compared it with that of thermotolerant B. cereus vegetative cells. The z values, based on times for a 3 log10 reduction, of the vegetative cells of the three psychrotolerant phylogenetic groups of B. cereus varied between 3.02 °C and 4.84 °C. The temperature at which a 3 log10 reduction was achieved in 10 min varied between 47.6 °C and 49.2 °C for psychrotolerant vegetative cells and it was around 54.8 °C for thermotolerant vegetative cells. Moreover, 0.4 min at 60 °C would be sufficient for a 6 log10 CFU/ml reduction of the most heat resistant psychrotolerant B. cereus vegetative cells. These data clearly showed that psychrotolerant B. cereus vegetative cells can be rapidly eliminated by a mild heat treatment such as food reheating.

Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.