Cereus Cultures Research Articles

Synthesis and characterization of bioplastics, Polyhydroxyalkanoates produced from sugarcane bagasse by using Bacillus cereus

Polyhydroxyalkanoates (PHA) are biodegradable bioplastics accumulated in microbial cells which have immense potential to replace the existing non-biodegradable plastics. This study focuses on optimizing Bacillus cereus growth conditions to enhance PHA formation, utilizing the sugarcane bagasse (SCB) as the primary feedstock. The SCB was subjected to enzymatic hydrolysis, resulting in 8.33 mg/g of total sugar production. Subsequently, Bacillus cereus was cultivated and the treated SCB was employed for PHA synthesis. An essential precursor to this synthesis involves an in-depth investigation into the characterization of the PHA produced using Bacillus cereus. A total of 20 experimental runs of different parameter conditions for Bacillus cereus culture to produce PHA were optimized by using the Response Surface Methodology (RSM). At an inoculum size of 8% (v/v), temperature of 25 °C, and incubation time of 48 h, the highest PHA was produced with 86.4 g/L or 62.1% (w/w). The optimal growth rate of Bacillus cereus was obtained at 7.71% (v/v), 35.1 °C for 48.3 h. The greatest PHA content and yield were found to be 57.9% (w/w) of PHA accumulation and 90.39 g/L, respectively. The synthesized PHA is characterized by using FTIR, XRD, FESEM-EDX, and soil burial test. From the analysis done, the properties of the produced sample were confirmed as the PHA. The soil burial test observed that the polymer was a PHA with a biodegradability rate of 41.73% in a week. The study successfully optimized the Bacillus cereus culture conditions for PHA production by using sugarcane bagasse hydrolysate at 8% (v/v) as the feedstock.

Design and testing of a novel cost-effective lethal ovitrap for the control of Aedes aegypti (Linnaeus 1762)

An indigenous, novel, cost-effective, non-electric lethal ovitrap was designed for the dengue vector. The bacteria Bacillus cereus VCRC 641 was grown in a cost-effective culture medium of chicken feather waste. The culture supernatant was used as ovitrap attraction of the dengue vector of Aedes aegypti (Linnaeus 1762). Twenty experiments with 80 ovitraps, three experiments with 15,000 ovitraps, and three experiments with 3,000 ovitraps in the laboratory, outdoor and households respectively conducted, to assess the efficacy, showed that the ovitrap with B. cereus culture supernatant (10%) significantly attracted the gravid A. aegypti to lay eggs in all experimental traps. More importantly, the first instar larvae from the eggs on the traps had the mortality immediately. The novelty of this trap is proved to exhibit a dual function for attracting gravid mosquitoes and killing the newly emerged larvae. This is the first report asserting the dual role of a cost-effective, non-electric ovitrap to control dengue vectors. The ovitrap contains only simple components of a plastic bowl, a cylinder, and a plastic plate. The total cost of these materials per trap was Rs15 only. The laboratory study showed that the experimental ovitrap attracted >10 times the control traps (experimental: 1465.90±251.48 and control: 140.00±23.95). This is the first report about the efficacy of an indigenously designed lethal ovitrap for attracting dengue vector.

Presence and function of Hbl B’, the fourth protein component encoded by the hbl operon in Bacillus cereus

ABSTRACT The genes hblC, hblD and hblA encode the components Hbl L2, L1 and B of the pore forming enterotoxin haemolysin BL of Bacillus cereus. Two variants of the operon existand the more common one additionally contains hblB downstream of hblCDA. Up to now, it was completely unclear whether the corresponding protein, Hbl B’, is widely expressed among B. cereus strains and if it has a distinct function. In the present study, it was shown that the hblB gene is indeed expressed and the Hbl B’ protein is secreted by nearly all analysed B. cereus strains. For the latter, a detection system was developed based on monoclonal antibody 11A5. Further, a distinct reduction of cytotoxic and haemolytic activity was observed when recombinant (r)Hbl B’ was applied simultaneously with L2, L1 and B. This effect was due to direct interaction of rHbl B’ with L1. D −6 B. cereusAltogether, we present the first simple tool for the detection of Hbl B’ in B. cereus culture supernatants. Moreover, an important regulatory function of Hbl B’ in the mechanism of Hbl was determined, which is best described as an additional control of complex formation, balancing the amounts of Hbl B-L1 complexes and the corresponding free subunits.

Influence of culture age on exopolymeric substances from common laboratory bacterial strains: a study on yield, profile and Cu(II) biosorption

Extracellular polymeric substances (EPS) produced by laboratory strains Bacillus cereus and Pseudomonas aeruginosa were extracted from cultures incubated at various incubation periods (24, 48, 72, 96 and 120 h). At each sampling time, the EPS were analysed for yield, quality, functional groups present, and their efficacies in copper (Cu(II)) biosorption (using 30 and 50 ppm EPS). Results revealed that EPS yield was influenced by incubation period, with 48-h culture of B. cereus and 96-h culture of P. aeruginosa producing the highest yield of EPS at 8.30 mg and 6.95 mg, respectively. The EPS produced at various incubation periods have similar characteristics in solubility, quality and major functional groups (C-O, CH3, C=C, O-H) present. Efficacy of Cu(II) biosorption was influenced by the amount of EPS used and the EPS-metal incubation time. Although Cu(II) removal was higher for EPS from 24-h B. cereus (18.96%) and 48-h P. aeruginosa (19.19%) when 30 ppm was used, application of 50 ppm EPS demonstrated no distinct differences in amount of Cu(II) removed. This suggested that higher biomass of EPS used and longer EPS-metal incubation period, superseded the efficacy of EPS from various incubation periods.

Influence of culture age on exopolymeric substances from common laboratory bacterial strains: a study on yield, profile and Cu(II) biosorption

Extracellular polymeric substances (EPS) produced by laboratory strains Bacillus cereus and Pseudomonas aeruginosa were extracted from cultures incubated at various incubation periods (24, 48, 72, 96 and 120 h). At each sampling time, the EPS were analysed for yield, quality, functional groups present, and their efficacies in copper (Cu(II)) biosorption (using 30 and 50 ppm EPS). Results revealed that EPS yield was influenced by incubation period, with 48-h culture of B. cereus and 96-h culture of P. aeruginosa producing the highest yield of EPS at 8.30 mg and 6.95 mg, respectively. The EPS produced at various incubation periods have similar characteristics in solubility, quality and major functional groups (C-O, CH3, C=C, O-H) present. Efficacy of Cu(II) biosorption was influenced by the amount of EPS used and the EPS-metal incubation time. Although Cu(II) removal was higher for EPS from 24-h B. cereus (18.96%) and 48-h P. aeruginosa (19.19%) when 30 ppm was used, application of 50 ppm EPS demonstrated no distinct differences in amount of Cu(II) removed. This suggested that higher biomass of EPS used and longer EPS-metal incubation period, superseded the efficacy of EPS from various incubation periods.

Inhibition of Collagenase Q1 of Bacillus cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections.

Despite the progress in surgical techniques and antibiotic prophylaxis, opportunistic wound infections with Bacillus cereus remain a public health problem. Secreted toxins are one of the main factors contributing to B. cereus pathogenicity. A promising strategy to treat such infections is to target these toxins and not the bacteria. Although the exoenzymes produced by B. cereus are thoroughly investigated, little is known about the role of B. cereus collagenases in wound infections.In this report, the collagenolytic activity of secreted collagenases (Col) is characterized in the B. cereus culture supernatant (csn) and its isolated recombinantly produced ColQ1 is characterized. The data reveals that ColQ1 causes damage on dermal collagen (COL). This results in gaps in the tissue, which might facilitate the spread of bacteria. The importance of B. cereus collagenases is also demonstrated in disease promotion using two inhibitors. Compound 2 shows high efficacy in peptidolytic, gelatinolytic, and COL degradation assays. It also preserves the fibrillar COLs in skin tissue challenged with ColQ1, as well as the viability of skin cells treated with B. cereus csn. A Galleria mellonella model highlights the significance of collagenase inhibition in vivo.

Establishing a MALDI-TOF-TOF-MS method for rapid identification of three common Gram-positive bacteria (Bacillus cereus, Listeria monocytogenes, and Micrococcus luteus) associated with foodborne diseases

We aimed to establish a method for the rapid identification of three common Gram-positive bacteria (Bacillus cereus, Listeria monocytogenes, and Micrococcus luteus) associated with foodborne diseases. MALDI-TOF-MS was used to determine the effects of sample pretreatment, culture medium, and culture time on the identification results. Then, MALDI-TOF-TOF-MS was used to establish an optimized method and further explore the effects of culture time on secondary proteins. Among the three sample pretreatment methods, formic acid extraction outperformed direct transfer and extended direct transfer, resulting in more protein peaks and higher peak intensity in B. cereus culture samples. However, with different culture times (1–7 d), ion peaks of all bacterial proteins were relatively stable according to MALDI-TOF-TOF-MS data. The protein peaks of B. cereus (1430 m/z), L. monocytogenes (2100 m/z), and M. luteus (2140 m/z) were split into small ion peaks at 1162, 1465, and 1625 m/z, respectively. After 7 d of culture, the secondary spectra, peak intensities, and peak values of formic acid-treated samples were relatively stable, indicating that secondary protein peaks were less affected by culture time. It provides a new approach for the routine identification and market supervision of food safety in China.

Toxigenic and pathogenic potential of enteric bacterial pathogens prevalent in the traditional fermented foods marketed in the Northeast region of India

The microbial risk involved with natural food fermentation is largely unknown. Here, we report the prevalence of enteric bacterial pathogens in the traditional fermented foods marketed in Northeast region of India. A total of 682 samples of 39 food types (broadly categorized into fermented soybean, bamboo shoot, fish, milk and pork products) collected over four different seasons from seven states of India were analyzed in this study. Cultivation-independent analysis by MiSeq amplicon sequencing of V4-V5 region of the 16S rRNA gene showed the bacterial community structure in the foods. Among the WHO prioritized foodborne bacterial pathogens, we detected the prevalence of phylotypes related to Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, and Escherichia coli in these ethnic foods. We also observed the occurrence of other well known human enteric pathogens like Proteus mirabilis, Clostridium difficile, and Yersinia enterocolitica. Further pathogen-specific qPCR assays confirmed a higher population (>107 cells/g) of B. cereus, P. mirabilis, and a C. botulinum related phylotype in the fermented soybean, fish, and pork products. We noticed a general trend of higher pathogen occurrence during the colder months without any seasonal variation of total bacterial load in the fermented foods. Further qPCR analysis on toxigenic and pathogenic potential, and toxins production by immunoassays showed that all the soybean samples and the isolated B. cereus cultures were positive for diarrheal toxins (Nhe and Hb1), and nearly half of the samples were positive for emetic toxin (cereulide). Similarly, the food samples and associated swarming P. mirabilis cultures were positive with the pathogenic factors like hemolysin (hpm), urease (ure) and multidrug resistance. However, we could not confirm the presence of botulinum neurotoxin (toxins A, B, E, and F) in the C. botulinum positive food samples. This is the first baseline data of the enteric bacterial pathogens prevalent in the traditional fermented foods of India, which will support the sustained effort of WHO to estimate the global foodborne disease burden. The unusual presence of P. mirabilis in the fermented foods marketed in the Indian region with high incidence of urolithiasis cases is a concern. Our study emphasizes the need of the hour to have a coordinated action to control and prevent the spread of enteric bacterial pathogens through fermented foods marketed in India. Moreover, replacing the indigenous process with a defined starter culture based controlled fermentation will enhance the safety of Indian fermented foods.

Survival and detection of Bacillus cereus in the presence of Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans after rechallenge in make-up removers.

Pathogenic contamination of cosmetics intended to be applied on or around the eye area, including make-up removers, may lead to severe eye infections. To assess the efficacy of antimicrobial preservatives in these products, we investigated the survival and detection of Bacillus cereus F 4227A spiked into make-up removers, alone and in the presence of other relevant micro-organisms. Four brands of make-up removers, A, B, C and D, were challenged three times (day 0, day 7 and day 14) using B. cereus, in pure and mixed cultures, at a final concentration of 5 log CFU per mL of Bacillus cereus or 6 log CFU per mL for other micro-organisms. Inoculated samples were diluted and spiral-plated after 30 min and 24 h of each challenge onto selective media for recovery of surviving micro-organisms: BACARA (B. cereus), MacConkey (E. coli), ChromID (P. aeruginosa), XLT4 (S. enteritidis), Baird Parker agar (Staph. aureus) and PDA+chlortetracycline HCL (C. albicans). The population of B. cereus spiked as a pure culture increased significantly from the first to the third challenge after 30-min exposure time, going from 0.73 to 2.59 in A, from 0.80 to 2.69 in B and from 0.80 to 1.67 log CFU per mL in C (P < 0.05). Likewise, the B. cereus population from the mixed cultures had a significantly higher survival count at the third challenge: from 0.12 log MPN per mL to 2.16 log CFU per mL in A, 0.57 to 2.27 log CFU per mL in B and from undetected (LOD = 0.48 log MPN) to 0.98 log CFU per mL in C, respectively. After challenges, Staph. aureus, C. albicans and P. aeruginosa increased in B; Staph. aureus and C. albicans in C; and E. coli and Staph. aureus in D. The growth of other bacteria types was unaffected by the number of challenges, but B. cereus population was detected with the third challenge. It is appropriate to assess the antimicrobial efficacy of preservatives using at least three challenges, especially for cosmetics that are subjected to repetitive contamination by users.

Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution.

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L2, L1 and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L2, L1 and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L1 and B, as well as L1 and L2 in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with KD values of 4.7 × 10−7 M and 1.5 × 10−7 M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin.

Biodegradation of endosulfan and pendimethalin by three strains of bacteria isolated from pesticides-polluted soils in the Sudan

Biodegradation of endosulfan (α and β) and pendimethalin by Bacillus safensis strain FO-36bT, Bacillus subtilis subsp. inaquosorum strain KCTC 13429T and Bacillus cereus strain ATCC14579T isolated from pesticides-polluted soil was studied in mineral salt medium. Endosulfan and pendimethalin were incubated with the three bacterial strains with samples drawn at various intervals for GC analysis. Representative samples were subject to GC–MS analysis. The loss in the initial concentrations, 0.663 mM (α endosulfan), 0.319 mM (β endosulfan) and 1.423 mM (pendimethalin), was monitored and used to compute the half-lives following biphasic model. Removal percentage of endosulfan and pendimethalin in the media inoculated with the bacterial strains ranged from 24 to 95% (α endosulfan), 21–91% (β endosulfan) and 51–97% (pendimethalin), respectively. Despite the significant decrease in starting material in B. safensis cultures, no metabolites were detected, whereas two major metabolites of endosulfan, 1,2,3,4,7,7-hexachloro-5,6-dihydroxybicyclo{2.2.1}-2-heptene and 1,2,3,4,7,7-hexachloro-formaldehyde-6-methylbicyclo{2.2.1}-2-heptene, were detected in the B. subtilis cultures, and one metabolite of pendimethalin metabolite; N-(1-ethylpropyl)-3-methyl-2, 6-diaminobenzine, was detected in the B. cereus culture. Generally, the result indicates the potential capability of these microorganisms in complete mineralization of endosulfan and pendimethalin. Based on half-lives, the efficiency of bacterial strains can be ordered as follows: B. subtilis > B. cereus > B. safensis for endosulfan and B. cereus > B. safensis > B. subtilis for pendimethalin.

Bacterial metabolites from intra- and inter-species influencing thermotolerance: the case of Bacillus cereus and Geobacillus stearothermophilus.

Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65°C respectively for 30min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50°C for 60min and viable cells determined. For spores, D values (85 and 95°C) were evaluated after 120h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.

Cloning and expression of vgb gene in Bacillus cereus, improve phenol and p-nitrophenol biodegradation

In this work, the vgb gene from Vitrocilla stercoraria was used to genetically modify a Bacillus cereus strain isolated from pulp and paper wastewater effluent. The gene was cloned in a multicopy plasmid (pUB110) or uni-copy gene using a chromosome integrative vector (pTrpBG1). B. cereus and its recombinant strains were used for phenol and p-nitrophenol biodegradation using aerobic or micro-aerobic conditions and two different temperatures (i.e. 37 and 25 °C). Complete (100%) phenol degradation was obtained for the strain where the multicopy of vgb gene was present, 98% for the strain where uni-copy gene was present and 45% for wild type strain for the same experimental conditions (i.e. 37 °C and aerobic condition). For p-nitrophenol degradation at the same conditions, the strain with the multi-copy vgb gene was capable to achieve 50% of biodegradation, ∼100% biodegradation was obtained using the uni-copy strain and ∼24% for wild type strain. When the micro-aerobic condition was tested, the biodegradation yield showed a significant decreased. The biodegradation trend observed for aerobic was similar for micro-aerobic assessments: the modified strains showed higher degradation rates when compared with wild type strain. For all experimental conditions, the highest p-nitrophenol degradation was observed using the strain with uni-copy of vgb gene. Besides the increase of biodegradative capability of the strain, insertion of the vgb gene was observed able to modify other morphological characteristics such as avoiding the typical flake formation in the B. cereus culture. In both cases, the modification seems to be related with the enhancement of oxygen supply to the cells generated by the vgb gene insertion. The application of the genetically modified microorganism (GMM) to the biodegradation of pollutants in contaminated water possesses high potential as an environmentally friendly technology to facing this emergent problem.

Metal nanoparticles in DBS card materials modification

In the recent years the method of collecting and storing Dried Blood Spots (DBS) on special cellulose membrane (paper) has gained wide popularity. But possible damage of biosamples caused by microorganisms in case of their incomplete drying is a disadvantage of the method. It can be overcome by treating sample-collection membranes with colloidal solutions of metal nanoparticles, having antibacterial effect. The team studied antibacterial properties of nonwoven material samples with various coatings (alcohol sols of copper, aluminium, iron, titanium, silver and vanadium nanoparticles). Colloidal solutions of nanoparticles were obtained by means of electroerosion method with further low-temperature plasma condensation. Antibacterial activity of fiberglass and cellulose membrane samples with nanoparticle coatings was studied using B. cereus and plaque bacteria cultures. It was revealed that nanostructured coatings can suppress bacterial activity; in addition they can diffuse from the membrane surface into medium which leads to widening the areas of inhibiting testing cultures’ growth. Thus, membrane materials treatment with alcohol-sols of metal nanoparticles can be seen as promising for conferring antibacterial properties to DBS carriers.

Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells.

BackgroundSilver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs).MethodsHere, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins.ResultsAgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs.ConclusionOur results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required.

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC-TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC-electrospray ionization (ESI)-TOF MS and the HPLC-ESI-MS/MS-SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group

Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5 Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.

Bacteriostatic Mode of Action of Trypsin-Hydrolyzed Palm Kernel Expeller Peptide Against Bacillus cereus.

Palm kernel expeller (PKE), the by-product derived from the palm kernel oil milling industry, is commonly added to ruminant feed as a source of protein. Recent research has demonstrated that the enzymatically hydrolyzed protein is inhibitory to spore-forming bacteria including Bacillus cereus. The trypsin-hydrolyzed PKE peptide appears to disrupt the membrane integrity and inhibit the intracellular macromolecule metabolism of B. cereus. The addition of the PKE peptide (350 and 700μg/ml) to B. cereus cultures triggered the efflux of K(+) and caused the depletion of the intracellular ATP. However, no proportional increase in cell's extracellular ATP was observed. Analysis of the biosynthesis of macromolecules demonstrated that RNA was affected by the PKE peptide. Results of this study suggest that the PKE peptide is bacteriostatic interfering with membrane integrity and forming membrane pores permitting the efflux of K(+) and interferes with intracellular biopolymer synthesis.

Combined action of low temperature and magnetic field of different intensities on growth of some bacterial species &amp;lt;i&amp;gt;in vitro&amp;lt;/i&amp;gt;

Growth dynamic of bacterial population after influence of magnetic field and cryoaction has been studied. In vitro experiments were performed on Bacillus cereus, Staphylococcus aureus, and Lactobacillus delbrueckii cultures. The most significant result was obtained in experiments with Bacillus cereus. When applied separately, cryoaction and alternating magnetic field were not able to kill this microorganism, whereas combined action of these factors led to the complete devitalization of bacterial culture.

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R 2 values of 0.9969 and 0.9958 respectively. Linear correlations between the log 10 input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10 1 CFU/mL to 1.65 × 10 6 CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.