Cereus Endospores Research Articles

Flower-Shaped PCR Scaffold-Based Lateral Flow Bioassay for Bacillus cereus Endospores Detection

Bacillus cereus, a foodborne pathogen, produces resilient endospores that are challenging to detect with conventional methods. This study presents a novel Flower-Shaped PCR Scaffold-based Lateral Flow Biosensor (FSPCRS-LFB), which employs an aptamer-integrated PCR scaffold as capture probes, replacing the traditional streptavidin-biotin (SA-Bio) approach. The FSPCRS-LFB demonstrates high sensitivity and cost-efficiency in detecting B. cereus endospores, with a limit of detection (LOD) of 4.57 endospores/mL a visual LOD of 102 endospores/mL, and a LOD of 6.78 CFU/mL for endospore-cell mixtures. In chicken and tea samples, the platform achieved LODs of 74.5 and 52.8 endospores/mL, respectively, with recovery rates of 82.19% to 97.88%. Compared to existing methods, the FSPCRS-LFB offers a 3.7-fold increase in sensitivity while reducing costs by 26% over the SA-Bio strategy and 87.5% over rolling circle amplification (RCA). This biosensor provides a rapid, sensitive and cost-effective solution for point-of-care testing (POCT) of B. cereus endospores, expanding detection capabilities and offering novel approaches for pathogen detection.

Simultaneous combination treatment with superheated steam and nebulized organic acid to inactivate Bacillus cereus endospores on stainless steel surfaces

Simultaneous combination treatment with superheated steam and nebulized organic acid to inactivate Bacillus cereus endospores on stainless steel surfaces

Correlation analysis of reactive oxygen and nitrogen species (RONS) components in plasma activated water (PAW) and its inactivation of Bacillus cereus endospore

Plasma activated water (PAW), a physicochemical approach that can efficiently enable the inactivation effect of Bacillus cereus endospores, is receiving numerous attentions in food and other areas. However, the potential RONS featuring PAW that inactivates bacteria is an open issue. Here, we for the first time thoroughly clarified the functioning chemicals on antimicrobial ability in PAW by adding each corresponding scavenger at an optimized dose, including superoxide anion (O2−), singlet oxygen (1O2), hydroxyl radical (OH), hydrogen peroxide (H2O2), nitrite ions (NO2−), nitric oxide (NO) and peroxynitrite (ONOOH). An overall reduction of 1.74 log CFU/mL is determined by the sporicidal effects of PAW; however, when adding their corresponding scavengers, the effect was attenuated to different extents. The internal correlations are constructed using Spearman analysis based on the inactivation of endospores for determining their specific contributions in such a complex PAW system and corresponding effective factors are calculated. The Spearman coefficients of NO2−, O2−, H2O2, and NO were 0.476, 0.405, 0.168, and 0.143, revealing that the log CFU/mL reduction of B. cereus endospore was positively proportional to the contents of NO2−, O2−, H2O2, and NO. This study addresses the fundamental issues related to the chemicals in PAW and may initiate wide interest in PAW in physiochemistry and biology.

Inactivation of Bacillus cereus endospores on black pepper by pulsed superheated steam system

In this study, a superheated steam (SHS) system was constructed to inactivate Bacillus cereus endospores on the surface of black pepper, and continuous and pulsed treatment was applied to compare sporicidal effects. Additionally, inactivation mechanisms were analyzed to investigate the differences between pulsed and continuous SHS treatments. SHS at 250 °C and 300 °C for 1 min achieved more than a 3 log reduction, whereas SHS at 200 °C for 1 min achieved less than 2 log reduction in the number of endospores. In addition, higher microbicidal effects were confirmed with pulsed SHS treatment with a shorter duty ratio. To elucidate the inactivation mechanisms, inner membrane damage (dipicolinic acid release), intracellular enzyme activities, and DNA integrity were measured after 300 °C SHS pulsed or continuous treatments. After pulsed SHS treatment for up to 20 s, intracellular enzymes were inactivated more rapidly than after continuous treatment, and more DPA was released after 40 s of treatment, indicating that enzyme inactivation occurred prior to inner membrane damage, and pulsed treatment accelerated this mode of action. DNA integrity was significantly lower after 60 s of pulsed or continuous treatment; however, there was no difference in between pulsed and continuous treatments. Our results provide fundamental insights for the sterilization of black pepper by SHS treatment in food industries.

Design and efficiency evaluation of a mid-size serpentine Dean flow UV-C system for the processing of whole milk using computational fluid dynamics and biodosimetry

UV-C processing of whole milk (WM) using a designed novel serpentine coil Dean flow system was demonstrated at flow rates of 11.88, 23.77, and 47.55 gph. Computational fluid dynamics (CFD) and biodosimetry studies were conducted to assess the flow field and fluence delivered at selected experimental flow rates. The CFD results revealed that as the flow rate increases, the ratio between average velocity to maximum velocity ( V avg /V max ) increases from 0.58 to 0.72 and enhanced Dean effects of the fluid domain at serpentine bend locations. Delivered UV fluence rate (REF rate) was 1.54 times higher at 47.88 gph than 11.88 gph. Microbial inactivation studies showed 2.75 and 4.29 log reduction (CFU/mL) of B. cereus endospores and T1UV phage after three and two passes of WM through the system at a flow rate of 47.88 gph. UV-C irradiated WM quality analysis revealed no significant effect on lipid peroxidation and volatiles profile at REF of 60 mJ/cm 2 . Lower electrical energy per order (E EO ) value also signifies the higher electrical efficiency of the system at a flow rate of 47.88 gph. • Designed and tested serpentine coiled Dean flow UV system for whole milk processing. • Demonstrated flow field at various flow rates using computational fluid dynamics. • Validated the fluence delivery and demonstrated the effect of flow rate on system's performance. • Verified microbial inactivation against bacterial endospores and virus particles. • Assessed product's quality (lipid peroxidation and volatiles profile) at different fluence levels.

Evaluation of UV-C Irradiation Treatments on Microbial Safety, Ascorbic Acid, and Volatile Aromatics Content of Watermelon Beverage

UV-C treatment technology is proposed as a potential alternative to thermal treatments for decontamination of beverages because heat may adversely affect the nutritive value and quality. Effects of UV-C irradiation processing on microbial inactivation, ascorbic acid, and volatile aromatics of watermelon beverage were evaluated using a flow-through UV system. Selected microbial agents Escherichia coli O157:H7 and Bacillus cereus endospores were inactivated by 5.31 ± 0.01 and 6.1 ± 0.01 log CFU mL−1 at UV equivalent fluence levels of 12 and 60 mJ cm−2, respectively. At UV pasteurization equivalent exposure (40 mJ cm−2), 93% ascorbic acid was retained. No significant loss of volatile aromatic aldehydes was noticed at fluence 40 mJ cm−2. Overall, the results support the UV-C processing as an alternative to traditional thermal processing to preserve quality in conjunction with microbial safety aspects of watermelon beverage.

Purification and characterization of an antimicrobial compound produced by Lactobacillus plantarum EM showing both antifungal and antibacterial activities

Lactobacillus plantarum EM exhibited antimicrobial activities against different food-borne pathogenic bacteria and food spoilage fungi. The compounds showing antibacterial and antifungal activities were identified as organic acids, lactic and acetic acids, and unidentified antimicrobial compounds. Thus the unidentified antimicrobial compounds were purified using solid phase extraction and recycling preparative HPLC and its structure was elucidated using NMR and LC/MS. The purified active compound of L. plantarum EM was identified as 3-hydroxy-5-dodecenoic acid (MW 214). Furthermore, the antimicrobial effects of the active compounds, 3-hydroxy-5-dodecenoic acid, lactic and acetic acids were greater with combined usage. 3-Hydroxy-5-dodecenoic acid caused severe damage to the surface of vegetative cells of B. cereus and led to shrunken B. cereus endospores and A. fumigatus conidium cells along with aggregation of damaged cells, resulting in bactericidal and fungicidal effects against B. cereus and A. fumigatus. The MICs of 3-hydroxy-5-dodecenoic acid against A. fumigatus and B. cereus were 0.21 g/L and 0.25 g/L, respectively, which were lower than those of the commonly used chemical preservative, sodium benzoate. Overall, these results suggest that the antimicrobial activity of L. plantarum EM may lead to the development of promising biopreservatives that prevent fungal spoilage and food-borne pathogens.

Molecular Pathogenesis of Bacillus spp., with Emphasis on the Dairy Industry

The bacterial species Bacillus cereus accounts for 1.4-12% of foodborne illness outbreaks worldwide, a statistic that is certainly an underestimate. This bacterial genus is capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. B. cereus endospores are partially resistant to pasteurization, dehydration, gamma radiation, and other physical stresses used in food processing, and their adhesive characteristics promote biofilm-forming capability on a variety of substrates in dairy operations. B. cereus and other closely-related species produce several types of exotoxins, including at least four hemolysins, three phospholipases, a heat/acid stable emetic toxin called cereulide, and three well-studied heat-labile enterotoxins that all cause gastroenteritis following ingestion. While a great deal of information on virulence gene presence and expression is known in B. cereus, very little has been done to explore the virulence potential of thermoduric spore-formers that may be found in ultrahigh temperature (UHT) pasteurized milk, and their ability to produce biofilms. Biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants. This chapter describes the current status of knowledge with Bacillus spp. relevant to the dairy industry, virulence potential, and biofilm production from the perspective of food safety.

Effects of Bacillus cereus Endospores on Free-Living Protist Growth.

We studied the predator-prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.

Internal Hydration Properties of Single Bacterial Endospores Probed by Electrostatic Force Microscopy.

We show that the internal hydration properties of single Bacillus cereus endospores in air under different relative humidity (RH) conditions can be determined through the measurement of its electric permittivity by means of quantitative electrostatic force microscopy (EFM). We show that an increase in the RH from 0% to 80% induces a large increase in the equivalent homogeneous relative electric permittivity of the bacterial endospores, from ∼4 up to ∼17, accompanied only by a small increase in the endospore height, of just a few nanometers. These results correlate the increase of the moisture content of the endospore with the corresponding increase of environmental RH. Three-dimensional finite element numerical calculations, which include the internal structure of the endospores, indicate that the moisture is mainly accumulated in the external layers of the endospore, hence preserving the core of the endospore at low hydration levels. This mechanism is different from what we observe for vegetative bacterial cells of the same species, in which the cell wall at high humid atmospheric conditions is not able to preserve the cytoplasmic region at low hydration levels. These results show the potential of quantitative EFM under environmental humidity control to study the hygroscopic properties of small-scale biological (and nonbiological) entities and to determine its internal hydration state. A better understanding of nanohygroscopic properties can be of relevance in the study of essential biological processes and in the design of bionanotechnological applications.

Sporicidal Effects of High‐Intensity 405 nm Visible Light on Endospore‐Forming Bacteria

Resistance of bacterial endospores to treatments, including biocides, heat and radiation is a persistent problem. This study investigates the susceptibility of Bacillus and Clostridium endospores to 405 nm visible light, wavelengths which have been shown to induce inactivation of vegetative bacterial cells. Suspensions of B. cereus endospores were exposed to high-intensity 405 nm light generated from a light-emitting diode array and results demonstrate the induction of a sporicidal effect. Up to a 4-log(10) CFU mL(-1) reduction in spore population was achieved after exposure to a dose of 1.73 kJ cm(-2). Similar inactivation kinetics were demonstrated with B. subtilis, B. megaterium and C. difficile endospores. The doses required for inactivation of endospores were significantly higher than those required for inactivation of B. cereus and C. difficile vegetative cells, where ca 4-log(10) CFU mL(-1) reductions were achieved after exposure to doses of 108 and 48 J cm(-2), respectively. The significant increase in dose required for inactivation of endospores compared with vegetative cells is unsurprising due to the notorious resilience of these microbial structures. However, the demonstration that visible light of 405 nm can induce a bactericidal effect against endospores is significant, and could have potential for incorporation into decontamination methods for the removal of bacterial contamination including endospores.

Inactivation of recalcitrant protozoan oocysts and bacterial endospores in drinking water using high-intensity pulsed UV light irradiation

This constitutes the first study to compare the use of high-intensity pulsed UV light (PUV) irradiation for the novel destruction of harmful protozoan (Cryptosporidium parvum Iowa isolate) oocysts and bacterial (Clostridium perfringens ATCC 13124 and Bacillus cereus ATCC 11178) endospores in artificially-spiked water where these organisms are resistant to conventional chlorination. Experimental results revealed that all three test organisms in their dormant recalcitrant state required extended levels of pulsing to achieve significant reductions in numbers compared to other similarly PUV-treated Escherichia coli ATCC 25922 that is a non-spore forming indicator of faecal pollution in water. 120 pulses at 900 V or 16.2 J per pulse (equivalent to a UV dose of 8.39 μJ cm−2) were required to achieve ca. 2 log C. perfringens spore numbers, whereas a similar level of PUV irradiation reduced both C. parvum oocysts and B. cereus endospores by ca. 5 log orders. A comparative ca. 5 log reduction of E. coli cell numbers was achieved after only 25 pulses at 900 V (equivalent to a UV dose of 1.74 μJ cm−2). A clear trend emerged where the order of resistance to PUV-irradiation observed was C. perfringens endospores > C. parvum oocysts, B. cereus endospores > E. coli cells. This study suggests disinfection kinetic data for the more resistant C. perfringens endospores can be used as a measure of estimating disinfection efficacy of PUV treatments for C. parvum oocysts in water, avoiding the need to use complex animal or cell culture infectivity models that are only available in specialised laboratories with highly trained technicians. This study will inform future studies exploring scale-up of PUV at waste-water treatment plants.

UV-C treatment of soymilk in coiled tube UV reactors for inactivation of Escherichia coli W1485 and Bacillus cereus endospores

Coiled tube UV reactors were used to investigate the influence of tube diameter (1.6mm ID, and 3.2mm ID) and Reynolds number (Re) to inactivate Escherichia coli W1485 and Bacillus cereus spores in raw soymilk (RSM). Four levels of Re (343, 686, 1029 and 1372) were tested in RSM inoculated separately with each bacterium and treated in the UV reactors at a constant residence time of 11.3s with UV-C dose of 11.187mJ/cm2 at 253.7nm. Inactivation efficiency of both microorganisms increased with Re. Maximum reductions of 5.6log10CFU/ml of E. coli and 3.29log10CFU/ml of B. cereus spores were achieved in the 1.6mm ID UV reactor. Inactivation efficiency was higher in the 1.6mm ID UV reactor than the 3.2mm ID UV reactor for both the organisms. Effect of UV-C light on lipid oxidation of untreated RSM, measured as malondialdehyde and other reactive substances (MORS) content, was much higher (95nmol/ml) than the UV-treated (58nmol/ml) and thermally pasteurized (55nmol/ml) RSM during the storage period of 7 days. The UV-C treatment can be effectively used for reducing E. coli cells and B. cereus spores in soymilk without compromising its quality.

Antimicrobial activities of hydrogen peroxide and its activation by a novel heterogeneous Fenton’s-like modified PAN catalyst

To investigate the potential activation of hydrogen peroxide by a novel catalyst, reducing the concentration of hydrogen peroxide required and the time taken for microbial inactivation. The antimicrobial properties of an iron-based novel heterogeneous polyacrylonitrile catalyst in combination with hydrogen peroxide were examined against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using a modified version of the European suspension test. Antimicrobial activity against Bacillus cereus and Bacillus subtilis endospores was also investigated. Bactericidal activity was significantly increased when the polyacrylonitrile catalyst was combined with hydrogen peroxide. 0.2, 0.5 and 1% w/v hydrogen peroxide resulted in average log reductions of 4.76, 5.59 and 5.37 for E. coli, Ps. aeruginosa and Staph. aureus, respectively, after 60 min exposure at room temperature. The catalyst also significantly increased the activity of hydrogen peroxide against B. subtilis and B. cereus endospores. These studies have demonstrated the potential biocidal use of the novel polyacrylonitrile catalyst when combined with hydrogen peroxide. This is the first publication to demonstrate the enhanced activity gained using the novel heterogeneous catalyst to potentiate the activity of hydrogen peroxide as a biocide.

Performance of coiled tube ultraviolet reactors to inactivate Escherichia coli W1485 and Bacillus cereus endospores in raw cow milk and commercially processed skimmed cow milk

Two coiled tube reactors were designed to investigate the influence of Reynolds number (Re) and diameter of fluid carrying tube on UV-C inactivation of Escherichia coli W1485 and Bacillus cereus endospores in raw cow milk (RCM) and skimmed cow milk (SCM) at room temperature. UV reactors were constructed using perfluoroalkoxy (PFA) tubing having internal diameters of 1.6 and 3.2mm and each had a residence time of 11.3s. Four levels of Re were tested for each milk type, each tube size and each bacteria type. Inactivation efficiency increased as the Re increased in both the reactors for both types of milk. The inactivation of both bacteria was higher in the 1.6mm UV reactor than the 3.2mm UV reactor. Maximum reduction of 7.8log10CFU/ml of E. coli was achieved in SCM in the 1.6mm UV reactor corresponding to the Re of 532 and higher, whereas the maximum reduction of E. coli in RCM was 4.1log10CFU/ml at the highest level of Re (713) tested. For B. cereus, the maximum reduction was 2.72log10CFU/ml in 1.6 UV reactor, in SCM at Re of 1024; whereas the maximum reduction of B. cereus in RCM was 2.65log10CFU/ml at Re value of 713. Inactivation efficiency of both bacteria was more in SCM than RCM. The coiled tube reactor design provided adequate mixing and UV-C dosage for efficient disinfection of E. coli cells in milk.

Morphological Changes Induced by Wet-heat in Bacillus cereus Endospores

Morphological Changes Induced by Wet-heat in Bacillus cereus Endospores

Differentiation of Bacillus endospore species from fatty acid methyl ester biomarkers

A simple method to detect and differentiate Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) endospores using biomarker compounds, including dipicolinic acid methyl ester (DPAME) and fatty acid methyl esters (FAMEs), has been developed. The method is based on thermochemolysis methylation (TCM) of the endospores and gas chromatography-mass spectrometry (GC-MS) of the reaction products. A suspension of the sample mixed with sulfuric acid (H2SO4) and tetramethylammonium hydroxide (TMAH) in methanol (MeOH) at room temperature is sampled using a coiled wire filament (CWF) device, which consists of a tiny platinum helical wire coil attached to a retractable plunger that moves the coil in and out of a syringe needle housing. Sampling is accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port. While DPAME can be used for the general detection of endospores, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME could be detected in samples containing as few as 6000 endospores, and the GC-MS peak area percent reproducibility for FAMEs varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples.

Bacillus cereus endospores exhibit a heterogeneous response to heat treatment and low-temperature storage

Bacillus cereus endospores were challenged by heat treatments simulating typical domestic/industrial cooking regimes and the resulting effects on germination, viability and sub-lethal heat damage determined using differential plate counting on a rich versus selective medium, flow cytometry (FCM), β- d-glucuronidase (GUD) activity and OD 600 measurement. Additionally, these techniques were used to investigate the effect on endospores of storage in a non-nutrient medium at 4 °C for 1 month. Plate counting revealed that heating generated sub-populations of sub-lethally damaged endospores, with the more severe heat treatments generating larger proportions of sub-lethally damaged endospores. These findings were also reflected in FCM analyses, which detected large amounts of heterogeneity among the populations of heat-treated endospores and uncovered differences in the proportions of membrane-damaged endospores and those displaying esterase activity pre- and post-treatment. Plate count data suggested that both the control and heat-treated endospores lost viability during storage, with FCM data indicating that the proportion of membrane-damaged endospores increased and those displaying the esterase activity decreased. The FCM, GUD and OD 600 data suggested that germination rates decreased with the increasing severity of heat treatment. This study demonstrates that a combination of plate counting and FCM can be used to detect heterogeneity in the response of endospores to insults.

Evaluation of endospore purification methods applied to Bacillus cereus

Research on spore inactivation techniques requires highly pure spore suspensions, but the efficacy of commonly used purification methods has not been systematically compared. By providing this comparison, the present study will help researchers improve the consistency and validity of their experiments. The current study evaluated alternative purification methodologies applied to Bacillus cereus endospores on the basis of criteria of purity, yield, time investment and effect on integrity of spores. The ASTM E2111-00, ethanol, heat shock, daily water washes and lysozyme treatment methods were applied to B. cereus cultures incubated for 3 and 10 days. Purities achieved ranged from 67 to 99% spores as a proportion of total colony forming units in the purified suspension, whereas yields ranged from less than 1 to 39% spores in proportion to total colony forming units of B. cereus culture. TEM images showed that only lysozyme treatment substantially altered spore morphology. Ethanol treatment is optimum for 3-day B. cereus cultures, whereas the ASTM method is most favorable for 10-day-old cultures. Lysozyme treatment is the least attractive purification method because of its large time investment and damage to spores.

Kinetics of deactivation of Bacillus cereus spores

The deactivation process for Bacillus cereus endospores was studied. Results indicated that, after being activated, endospores deactivate as a function of storage time. The ageing of endospores affects the deactivation process, with older endospores showing a lower reduction percentage than fresh ones in counts. The presence of germinant appears to affect the reduction percentage, but only at storage temperatures of 12°C and 20°C. The deactivation process showed a large tail with activated endospores for germination and growth, and it can be described by using the Weibull probability distribution function as a model.