Bacillus Thuringiensis Spores Research Articles

Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions.

Optimal Fast Integral Decontamination of Bacillus thuringiensis Aerosols and Fast Disinfection of Contaminated Surfaces.

Aerosolized anthrax (Bacillus anthracis) spores are of extreme health concern and can remain airborne for hours and contaminate all kinds of surfaces, constituting reservoirs from which resuspension is easily produced. The assessment of decontamination techniques must therefore consider both air and surfaces. In the present study, several kinds of disinfecting fogs were experimentally tested against Bacillus thuringiensis spores, which served as a surrogate for Bacillus anthracis, both as aerosols released into the air and spread on porous and non-porous surfaces with different positions and orientations. This technology removed Bacillus thuringiensis spores from the air in 20 min with just a 1 min application of fog. The dynamics and characteristics of the fog, related to aerosol and surface interactions, proved to be critical for optimal performance and decontamination. An optimal configuration could provide effective disinfection even on indirectly reached surfaces. In all cases, 8% hydrogen peroxide (H2O2) provided a higher disinfection rate than 2% glutaraldehyde.

Effect of treating the larval stage of the large waxworm Galleria mellonella with different concentrations of zinc oxide nanomaterials ZnO, titanium dioxide TiO2 and Bacillus thuringiensis spores on some aspects of the insect's life.

The present study was conducted to determine the impact of nanoparticles (TiO2 and ZnO) at concentrations (100,500,1000,5000) ppm and Bacillus thuringiensis spores at concentration (106 and109 cells/ml of D.W on some biological aspects of greater ,108 107 wax moth(Galleria mellonella) . The results showed that these materials have a significant effect on the development stages, larva and pupa of Galleria mellonella .The nanoparticles had a clear effect on the motility of the insect as high concentrations, 5000ppm of ZnO increased the killing rate in the larva and pupa to 80.10% and 34.64 respectively. Treatment with these materials also had a significant effect in some biological aspects of the first generation. The average number of eggs was 22.667 and 10.007 in the treatment with concentration 5000ppm of both TiO2 and ZnO respectively, while the average number of egg was 9.667 in the treatment with 106 of Bacillus thuringiensis spores . Treatment with concentration 5000ppm also caused a delay in the eggs laying of the greater wax moth to reach 87.720 and 95.163% of both, TiO2 and ZnO respectively, compared with Bacillus thuringiensis spores with showed 94.233% at106 concentration

Bacillus thuringiensis Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality.

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut – key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and ‘oxidation stress’ is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised.

Laser induced degradation of bacterial spores during micro-Raman spectroscopy

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores’ structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Differential side-effects of Bacillus thuringiensis bioinsecticide on non-target Drosophila flies

Bioinsecticides based on Bacillus thuringiensis (Bt) spores and toxins are increasingly popular alternative solutions to control insect pests, with potential impact of their accumulation in the environment on non-target organisms. Here, we tested the effects of chronic exposure to commercial Bt formulations (Bt var. kurstaki and israelensis) on eight non-target Drosophila species present in Bt-treated areas, including D. melanogaster (four strains). Doses up to those recommended for field application (~ 106 Colony Forming Unit (CFU)/g fly medium) did not impact fly development, while no fly emerged at ≥ 1000-fold this dose. Doses between 10- to 100-fold the recommended one increased developmental time and decreased adult emergence rates in a dose-dependent manner, with species-and strain-specific effect amplitudes. Focusing on D. melanogaster, development alterations were due to instar-dependent larval mortality, and the longevity and offspring number of adult flies exposed to bioinsecticide throughout their development were moderately influenced. Our data also suggest a synergy between the formulation compounds (spores, cleaved toxins, additives) might induce the bioinsecticide effects on larval development. Although recommended doses had no impact on non-target Drosophila species, misuse or local environmental accumulation of Bt bioinsecticides could have side-effects on fly populations with potential implications for their associated communities.

Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores.

A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 × 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.

New Biocide Foam Containing Hydrogen Peroxide for the Decontamination of Vertical Surface Contaminated With Bacillus thuringiensis Spores.

Despite scientific advances, bacterial spores remain a major preoccupation in many different fields, such as the hospital, food, and CBRN-E Defense sector. Although many disinfectant technologies exist, there is a lack for the decontamination of difficult to access areas, outdoor sites, or large interior volumes. This study evaluates the decontamination efficiency of an aqueous foam containing hydrogen peroxide, with the efficiency of disinfectant in the liquid form on vertical surfaces contaminated by Bacillus thurengiensis spores. The decontamination efficiency impact of the surfactant and stabilizer agents in the foam and liquid forms was evaluated. No interferences were observed with these two chemical additives. Our results indicate that the decontamination kinetics of both foam and liquid forms are similar. In addition, while the foam form was as efficient as the liquid solution at 4°C, it was even more so at 30°C. The foam decontamination reaction follows the Arrhenius law, which enables the decontamination kinetic to be predicted with the temperature. Moreover, the foam process used via spraying or filling is more attractive due to the generation of lower quantity of liquid effluents. Our findings highlight the greater suitability of foam to decontaminate difficult to access and high volume facilities compared to liquid solutions.

Effect of Humidity on Sporicidal Activity of Iodine Vapor on Bacillus thuringiensis.

The emergence of Bacillus anthracis as a potential bioterrorism and biological warfare agent points to the need for safe, effective, and economical sporicides for infection prevention and control. This work examined the efficacy of iodine vapor decontamination technologies to inactivate a surrogate for B. anthracis, Bacillus thuringiensis spores on glass materials. 106-107 colony-forming units of spores inoculated onto circular glass cover slips were treated with different concentrations of iodine vapor under various temperature and relative humidity. Only minimal spore killing activity was observed at low humidity. Higher humidity levels, as well as pre-hydration or post-hydration of the spores, increased the rate of inactivation as long as the contact between spores and iodine was maintained in a hydrated environment. Significant sporicidal activity of 3-log and 6-log spore reduction has been observed with 2.1mgL-1 iodine vapor concentration at 90% relative humidity and 22°C, with 1 and 24h of exposure, respectively. The results showed that the relative humidity of the environment is of major importance in regulating the rate at which the spores are inactivated by iodine. The results of this study may provide insight into the parameters of effective decontamination procedures for Bacillus spores using gaseous iodine.

Probing the germination kinetics of ethanol-treated Bacillus thuringiensis spores.

Bacillus thuringiensis (Bt) is the most widely used microbial insecticide. To clarify the mechanism of bacterial resistance to ethanol toxicity, the present study investigated the effects of 70% (v/v) ethanol at a moderate temperature (65°C) on Bt spore germination by single-cell Raman spectroscopy and differential interference contrast microscopy. We found that over 80% of Bt spores were inviable after 30 min of treatment. Moreover, ethanol treatment affected spore germination; the time for initiation of rapid calcium dipicolinate (CaDPA) release (i.e., lag time, Tlag), time taken for rapid CaDPA release (i.e., ΔTrelease), and time required for complete hydrolysis of the peptidoglycan cortex of spores (i.e., ΔTlys) were increased with longer treatment times. Alanine-initiated germination upon ethanol treatment for 30-90 min showed a 2- to 4-fold longer Tlag, 2- to 3.5-fold longer ΔTrelease, and ∼2-fold longer ΔTlys relative to the control. Dodecylamine-initiated germination treated for 15-30 min had 3- to 5-fold longer Tlag and 1.4- to 1.7-fold longer ΔTrelease than the control. Germination induced by exogenous CaDPA was observed only in a small fraction of spores treated with ethanol for 5 min. Single-cell Raman spectroscopy revealed that more than 52% of spores lost CaDPA after 30 min of ethanol treatment; these showed reductions in the intensity of 1280 and 1652 cm-1 bands (corresponding to protein α-helical structure) and increases in that of 1245 and 1665 cm-1 bands (attributed to irregularities in protein structure). These results indicate that CaDPA in the core of Bt spores confers resistance to ethanol, and that damage to the spore inner membrane by ethanol treatment results in CaDPA leakage. Additionally, moderate-temperature ethanol treatment and consequent denaturation of germination-related proteins affected spore germination, specifically by inactivating the cortex-lytic enzyme CwlJ. Our findings provide a theoretical basis for the development of more effective methods for killing spore-forming bacteria; microscopy imaging and Raman spectroscopy can provide novel insight into the effects of chemical agents on microbial cells.

Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples

Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×101CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×103CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined

In the agricultural environment, honey bees may be exposed to combinations of pesticides. Until now, the effects of these combinations on honey bee health have been poorly investigated. In this study, we assessed the impacts of biological and chemical insecticides, combining low dietary concentrations of Bacillus thuringiensis (Bt) spores (100 and 1000µg/L) with the chemical insecticide fipronil (1µg/L). In order to assess the possible effects of Cry toxins, the Bt kurstaki strain (Btk) was compared with a Bt strain devoid of toxin-encoding plasmids (Bt Cry−). The oral exposure to fipronil and Bt spores from both strains for 10 days did not elicit significant effects on the feeding behavior and survival after 25 days. Local and systemic physiological effects were investigated by measuring the activities of enzymes involved in the intermediary and detoxication metabolisms at two sampling dates (day 10 and day 20). Attention was focused on head and midgut glutathione-S-transferase (GST), midgut alkaline phosphatase (ALP), abdomen glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD). We found that Bt Cry− and Btk spores induced physiological modifications by differentially modulating enzyme activities. Fipronil influenced the enzyme activities differently at days 10 and 20 and, when combined with Bt spores, elicited modulations of some spore-induced physiological responses. These results show that an apparent absence of toxicity may hide physiological disruptions that could be potentially damaging for the bees, especially in the case of combined exposures to other environmental stressors.

Fighting Ebola with novel spore decontamination technologies for the military.

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.

Bio-insecticide Bacillus thuringiensis spores encapsulated with amaranth derivatized starches: studies on the propagation "in vitro".

Bacillus thuringiensis (Bt) is one of the bioinsecticides used worldwide due to its specific toxicity against target pests in their larval stage. Despite this advantage, its use is limited because of their short persistence in field when exposed to ultra violet light and changing environmental conditions. In this work, microencapsulation has been evaluated as a promising method to improve Bt activity. The objective of this study was to develop and characterize native and modified amaranth starch granules and evaluate their potential application as wall materials in the microcapsulation of B thuringiensis serovar kurstaki HD-1 (Bt- HD1), produced by spray drying. Native amaranth starch granules were treated by hydrolyzation, high energy milling (HEM) and were chemically modified by phosphorylation and succinylation. The size of the Bt microcapsules varied from 12.99 to 17.14 μm adequate to protect the spores of Bt from ultraviolet radiation. The aw coefficient of the microcapsules produced by the modified starches after drying was low (0.14-1.88), which prevent microbial growth. Microcapsules prepared with phosphorylated amaranth starch presented the highest bacterial count and active material yield. Different concentrations of the encapsulated Bt formulation in phosphorylated amaranth starch showed a high level of insecticidal activity when tested on M. sexta larvae and has great potential to be developed as a bioinsecticide formulation, also, the level of toxicity is much higher than that found in some of the products commercially available.

Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment

Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in‐line advanced oxidation to inactivate microbes, and document the inactivation via an in‐line, real‐time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry® was evaluated for its potential to monitor inactivation in real‐time. B. thuringiensis was selected as a non‐pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in‐line sensor was not successful in documenting destruction of the microbial cells in real‐time.

Bacillus cereus and Bacillus thuringiensis spores in Korean rice: Prevalence and toxin production as affected by production area and degree of milling

We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

Evaluation of a Disinfectant Wipe Intervention on Fomite-to-Finger Microbial Transfer

Inanimate surfaces, or fomites, can serve as routes of transmission of enteric and respiratory pathogens. No previous studies have evaluated the impact of surface disinfection on the level of pathogen transfer from fomites to fingers. Thus, the present study investigated the change in microbial transfer from contaminated fomites to fingers following disinfecting wipe use. Escherichia coli (10(8) to 10(9) CFU/ml), Staphylococcus aureus (10(9) CFU/ml), Bacillus thuringiensis spores (10(7) to 10(8) CFU/ml), and poliovirus 1 (10(8) PFU/ml) were seeded on ceramic tile, laminate, and granite in 10-μl drops and allowed to dry for 30 min at a relative humidity of 15 to 32%. The seeded fomites were treated with a disinfectant wipe and allowed to dry for an additional 10 min. Fomite-to-finger transfer trials were conducted to measure concentrations of transferred microorganisms on the fingers after the disinfectant wipe intervention. The mean log10 reduction of the test microorganisms on fomites by the disinfectant wipe treatment varied from 1.9 to 5.0, depending on the microorganism and the fomite. Microbial transfer from disinfectant-wipe-treated fomites was lower (up to <0.1% on average) than from nontreated surfaces (up to 36.3% on average, reported in our previous study) for all types of microorganisms and fomites. This is the first study quantifying microbial transfer from contaminated fomites to fingers after the use of disinfectant wipe intervention. The data generated in the present study can be used in quantitative microbial risk assessment models to predict the effect of disinfectant wipes in reducing microbial exposure.

D-cycloserine or similar physiochemical compounds may be uniquely suited for use inBacillus anthracisspore decontamination strategies

As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of d-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting l-alanine to d-alanine, as a spore germination enhancer and antimicrobial agent. We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B.anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by l-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B.anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B.anthracis spores when exposed to DCS. These results further support that DCS augments the germination response of spores in the presence of l-alanine but also reveal that DCS is bactericidal towards germinating spores. DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

Evaluation of Real-Time Water Quality Sensors for the Detection of Intentional Bacterial Spore Contamination of Potable Water

Water utility treatment failure, as well as intentional or accidental water intrusions can introduce biological and/ or chemical contaminants into public drinking water distribution systems. However, recently developed real-time water quality sensors can be implemented to detect such contamination events. The overall objective of this study was to evaluate the potential for real-time monitoring of bacterial spore contamination of potable water using several different water quality sensors including: the HACH Monitoring Platform; the JMAR BioSentry unit; and the S::CAN spectro::lyser technology. For this, Bacillus thuringiensis spores were used as a surrogate for Bacillus anthracis. The minimum threshold response of sensors to the microbial contaminant was determined by injecting B. thuringiensis spores into Deionized (DI), filtered or unfiltered tap water. Out of these three evaluated sensors, the BioSentry sensor was capable of detecting introduced spores and responded to B. thuringiensis spores over a concentration range of 10 2 -10 5 spores/ml. In contrast, The HACH and S::CAN units were not capable of direct detection of spores. However, these two sensors can detect changes in water quality parameters such as turbidity, pH, temperature, total organic carbon and conductivity, due to media that may be associated with spores. Thus, these sensors can be integrated into a contaminant warning system for monitoring intrusion events in water distribution systems.

Synthesis of Bioactive Microcapsules Using a Microfluidic Device

Bioactive microcapsules containing Bacillus thuringiensis (BT) spores were generated by a combination of a hydro gel, microfluidic device and chemical polymerization method. As a proof-of-principle, we used BT spores displaying enhanced green fluorescent protein (EGFP) on the spore surface to spatially direct the EGFP-presenting spores within microcapsules. BT spore-encapsulated microdroplets of uniform size and shape are prepared through a flow-focusing method in a microfluidic device and converted into microcapsules through hydrogel polymerization. The size of microdroplets can be controlled by changing both the dispersion and continuous flow rate. Poly(N-isoproplyacrylamide) (PNIPAM), known as a hydrogel material, was employed as a biocompatible material for the encapsulation of BT spores and long-term storage and outstanding stability. Due to these unique properties of PNIPAM, the nutrients from Luria-Bertani complex medium diffused into the microcapsules and the microencapsulated spores germinated into vegetative cells under adequate environmental conditions. These results suggest that there is no limitation of transferring low-molecular-weight-substrates through the PNIPAM structures, and the viability of microencapsulated spores was confirmed by the culture of vegetative cells after the germinations. This microfluidic-based microencapsulation methodology provides a unique way of synthesizing bioactive microcapsules in a one-step process. This microfluidic-based strategy would be potentially suitable to produce microcapsules of various microbial spores for on-site biosensor analysis.