Bacterial Endospores Research Articles

Mechanism of Sporicidal Activity for the Synergistic Combination of Peracetic Acid and Hydrogen Peroxide.

There is still great interest in controlling bacterial endospores. The use of chemical disinfectants and, notably, oxidizing agents to sterilize medical devices is increasing. With this in mind, hydrogen peroxide (H2O2) and peracetic acid (PAA) have been used in combination, but until now there has been no explanation for the observed increase in sporicidal activity. This study provides information on the mechanism of synergistic interaction of PAA and H2O2 against bacterial spores. We performed investigations of the efficacies of different combinations, including pretreatments with the two oxidizers, against wild-type spores and a range of spore mutants deficient in the spore coat or small acid-soluble spore proteins. The concentrations of the two biocides were also measured in the reaction vessels, enabling the assessment of any shift from H2O2 to PAA formation. This study confirmed the synergistic activity of the combination of H2O2 and PAA. However, we observed that the sporicidal activity of the combination is largely due to PAA and not H2O2. Furthermore, we observed that the synergistic combination was based on H2O2 compromising the spore coat, which was the main spore resistance factor, likely allowing better penetration of PAA and resulting in the increased sporicidal activity.

Improving Dispersion of Bacterial Endospores for Enumeration

Improving Dispersion of Bacterial Endospores for Enumeration

Natural occurrence of Pasteuria nishizawae in soybean areas

Soybean ( Glycine max (L.) Merrill) is a widely grown crop of economic prominence. Brazil is considered as the second largest worldwide producer and exporter. Soybean cyst nematode, Heterodera glycines Ichinohe, in one of the most serious threats for this crop, considered as its most destructive parasite. The first report of this disease in Brazil was recorded during the 1991/1992 harvest. Control of nematodes is more challenging if compared with other plant diseases control. Thus, there is a growing demand to search for alternative control practices that will not harm the environment nor the human being. Therefore, the highly specific bacteria Pasteuria spp. Metchnikoff, represents an auspicious biological control agent against nematodes. The biological control of the soybean cyst nematode with the bacterium Pasteuria nishizawae Sayre has proven an excellent choice and has been studied by different scientists. The objective of this work was to determine the natural occurrence of Pasteuria nishizawae in Brazilian soils. The experiment was performed under greenhouse conditions at the Research unit from Syngenta® in Uberlandia-MG, Brazil, with soil samples originated from soybean planted areas from the Brazilian states of Bahia, Goias, Mato Grosso and Parana. Fractions of 150 cm 3 of soil were withdrawn to be processed by the centrifugal flotation technique in sucrose solution. Aliquots of 1 mL from the obtained suspension were observed in Peters’ chamber with the aid of an inverted light microscope, in order to verify the absence or presence of bacterial endospores attached to the cuticle of the nematodes extracted from the soil samples. The frequency of Pasteuria nishizawae incidence was of 100% within the samples analyzed.

TENCEL$^{\mbox{\circledR}}$ with a Microbial Barrier for Medical Bras

The goal of the research was to determine the properties of the microbial barrier TENCEL knitted fabrics intended for medical bras. The samples were tested according to a new method developed in the earlier research by the same authors. The most resistant forms of microorganisms of a pathogenic bacterial endospore of the Bacilllus genus were used to investigate microbial barrier permeability. Based on the samples tested and their characteristics, medical bra construction was developed with the aim of reducing irritation for the body part that underwent surgery. The data on the forms and measures were obtained using a 3D body scanner.

Spore Germination.

Despite being resistant to a variety of environmental insults, the bacterial endospore can sense the presence of small molecules and respond by germinating, losing the specialized structures of the dormant spore, and resuming active metabolism, before outgrowing into vegetative cells. Our current level of understanding of the spore germination process in bacilli and clostridia is reviewed, with particular emphasis on the germinant receptors characterized in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. The recent evidence for a local clustering of receptors in a "germinosome" would begin to explain how signals from different receptors could be integrated. The SpoVA proteins, involved in the uptake of Ca2+-dipicolinic acid into the forespore during sporulation, are also responsible for its release during germination. Lytic enzymes SleB and CwlJ, found in bacilli and some clostridia, hydrolyze the spore cortex: other clostridia use SleC for this purpose. With genome sequencing has come the appreciation that there is considerable diversity in the setting for the germination machinery between bacilli and clostridia.

Spore Peptidoglycan.

Bacterial endospores possess multiple integument layers, one of which is the cortex peptidoglycan wall. The cortex is essential for the maintenance of spore core dehydration and dormancy and contains structural modifications that differentiate it from vegetative cell peptidoglycan and determine its fate during spore germination. Following the engulfment stage of sporulation, the cortex is synthesized within the intermembrane space surrounding the forespore. Proteins responsible for cortex synthesis are produced in both the forespore and mother cell compartments. While some of these proteins also contribute to vegetative cell wall synthesis, others are sporulation specific. In order for the bacterial endospore to germinate and resume metabolism, the cortex peptidoglycan must first be degraded through the action of germination-specific lytic enzymes. These enzymes are present, yet inactive, in the dormant spore and recognize the muramic-δ-lactam modification present in the cortex. Germination-specific lytic enzymes across Bacillaceae and Clostridiaceae share this specificity determinant, which ensures that the spore cortex is hydrolyzed while the vegetative cell wall remains unharmed. Bacillus species tend to possess two redundant enzymes, SleB and CwlJ, capable of sufficient cortex degradation, while the clostridia have only one, SleC. Additional enzymes are often present that cannot initiate the cortex degradation process, but which can increase the rate of release of small fragments into the medium. Between the two families, the enzymes also differ in the enzymatic activities they possess and the mechanisms acting to restrict their activation until germination has been initiated.

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption

In Western countries, the popularity of edible insects as an alternative animal protein source is increasing. Nevertheless, there is a lack of profound insight into the microbial safety and shelf life of living insects sold for human consumption. The purpose of this study was to characterise the microflora of fresh edible mealworm larvae and grasshoppers in a quantitative and qualitative way. Therefore, culture-dependent analyses (the total viable aerobic count, Enterobacteriaceae, lactic acid bacteria, yeasts and moulds, and bacterial endospores) and next-generation sequencing (454amplicon pyrosequencing) were performed. High microbial counts were obtained for both insect species. Different insect batches resulted in quite similar microbial numbers, except for bacterial endospores. However, the bacterial community composition differed between both insect species. The most abundant operational taxonomic unit in mealworm larvae was Propionibacterium. Also members of the genera Haemophilus, Staphylococcus and Clostridium were found. Grasshoppers were mainly dominated by Weissella, Lactococcus and Yersinia/Rahnella. Overall, a variety of potential spoilage bacteria and food pathogens were characterised. The results of this study suggest that a processing step with a microbiocidal effect is required to avoid or minimize risks involved with the consumption of edible insects.

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC.

Small acid-soluble proteins (SASPs) play an important role in protection of DNA in dormant bacterial endospores against damage by heat, UV radiation or enzymic degradation. In the genome of the strict anaerobe Clostridium acetobutylicum, five genes encoding SASPs have been annotated and here a further sixth candidate is suggested. The ssp genes are expressed in parallel dependent upon Spo0A, a master regulator of sporulation. Analysis of the transcription start points revealed a σG or a σF consensus promoter upstream of each ssp gene, confirming a forespore-specific gene expression. SASPs were termed SspA (Cac2365), SspB (Cac1522), SspD (Cac1620), SspF (Cac2372), SspH (Cac1663) and Tlp (Cac1487). Here it is shown that with the exception of Tlp, every purified recombinant SASP is able to bind DNA in vitro thereby protecting it against enzymic degradation by DNase I. Moreover, SspB and SspD were specifically cleaved by the two germination-specific proteases GPR (Cac1275) and YyaC (Cac2857), which were overexpressed in Escherichia coli and activated by an autocleavage reaction. Thus, for the first time to our knowledge, GPR-like activity and SASP specificity could be demonstrated for a YyaC-like protein. Collectively, the results assign SspA, SspB, SspD, SspF and SspH of C. acetobutylicum as members of α/β-type SASPs, whereas Tlp seems to be a non-DNA-binding spore protein of unknown function. In acetic acid-extracted proteins of dormant spores of C. acetobutylicum, SspA was identified almost exclusively, indicating its dominant biological role as a major α/β-type SASP in vivo.

Plasma inactivation of bacterial endospores on wheat grains and polymeric model substrates in a dielectric barrier discharge

Motivated by the frequent microbial contamination of granular food products like cereal grains or sprout seeds, an atmospheric pressure dielectric barrier discharge (DBD) was developed to study plasma inactivation of microorganisms on granular materials. Wheat grains as well as polypropylene model substrates were artificially contaminated with endospores of Geobacillus stearothermophilus as a model organism and treated in a pulsed argon plasma discharge applying different combinations of treatment time, pulse voltage and frequency. While the treatment of polypropylene substrates resulted in an efficient reduction of microbes, wheat grains, having a rough surface and a deep ventral furrow, turned out to be more challenging to decontaminate. However, an improvement in treatment efficiency could be achieved by applying longer treatment, faster pulse frequency or higher pulse voltage. Furthermore, experiments demonstrated that endospore reduction was not caused by thermal, mechanical or electrical stress factors, but a direct effect of plasma-generated species, and chemical sputtering is supposed to be the predominant inactivation mechanism. Finally, it could be shown that functional wheat grain properties (Falling number, gluten content) are not negatively affected by our plasma treatment.

Inactivation of Bacterial Endospores in an Artificial Tissue for Electron Microscopy Analysis

Inactivation of Bacterial Endospores in an Artificial Tissue for Electron Microscopy Analysis

High pressure thermal inactivation of Clostridium botulinum type E endospores - kinetic modeling and mechanistic insights.

Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety.

Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress.

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores.

Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk

Bacterial endospores (also referred to as spores) present in raw milk are capable of surviving pasteurization and other adverse conditions encountered during dairy powder production. Therefore, requiring low spore levels in raw ingredients (e.g., raw milk) may be necessary for producing dairy powders with low spore counts. To identify potential associations between management practices and spore levels in raw milk, we sampled bulk tank raw milk from 33 farms throughout New York State every other month for 1yr. Following spore pasteurization (80°C for 12min), samples were incubated at 3 different temperatures to enumerate psychrotolerant (6°C for 10 d), mesophilic (32°C for 48h), and thermophilic (55°C for 48h) spores. An additional enrichment procedure was used to detect spores present at low levels (<10 spores/mL). Overall, psychrotolerant, mesophilic, and thermophilic spores were detected (at levels ≥10 spores/mL) in 1, 74, and 58% of bulk tank raw milk samples, respectively. Although thermophilic spore levels could not be quantified (due to bacterial swarming), mesophilic spore levels ranged from below detection (<10 spores/mL) to 680 spores/mL. Data collected through surveys were used to identify management practices associated with either mesophilic or thermophilic spore levels. We found that different management practices are associated with mesophilic and thermophilic spore levels. Low mesophilic spore levels in bulk tank raw milk samples were associated with (1) large herd size, (2) use of sawdust or sand bedding, and (3) not fore stripping during the premilking routine. Management practices that were associated with lower odds of having a thermophilic spore level ≥10 spores/mL are (1) large herd size, (2) spray-based application of the postmilking disinfectant, (3) dry massaging the udder during the premilking routine, and (4) the use of straw bedding. Collectively, these results suggest that different management practices may influence mesophilic and thermophilic spore levels in raw milk.

Forensic differentiation of Bacillus cereus spores grown using different culture media using Raman spectroscopy.

Some microorganisms have been shown to retain a chemical signature indicative of the medium used for culturing. However, the repeatability of medium-specific chemical signatures has not been demonstrated from samples of microorganisms produced in the same batch or in different batches by the same sporulation protocol. Here, the variation in Raman spectra of bacterial endospores repeatedly prepared by the same procedure is compared to the variation between Raman spectra of spores prepared using different media. Bacillus cereus T strain (BcT) samples were correctly classified according to the medium used to induce sporulation for 100% of spores grown in a controlled manner by the same scientist using Raman spectroscopy and multivariate data analysis. The proof-of-concept results from BcT spores produced in 12 different sporulation media showed correct classification by medium for 98% of samples (with 100% classification accuracy for all but one sporulation medium in this data set). Spectral differences were discerned between spores that had been freshly prepared or freeze-dried and spores that had been frozen; however, the differences did not impact the classification of the sporulation medium. Latent variables reduced the classification accuracy of BcT sporulated in G medium by different scientists using different media lots and stored for different periods of time and requires further study.

High pressure treated Bacillus subtilis spores — Structural analysis by means of synchrotron and laboratory based soft X-ray microscopy

Abstract Measurements were carried out on high pressure treated (HPT) bacterial endospores of the Bacillus subtilis strain PS832 by means of laboratory (LTXM) and synchrotron based transmission soft X-ray microscopy (SynchTXM). Using LTXM at 500 eV HPT spores could be identified by a higher transparency, due to dipicolinic acid (DPA) release from the spore's core, and their shriveled appearance caused by air-drying. Dormant spores were investigated at different photon energies at SynchTXM. Inner structures were visualized and it was shown that at a photon energy of 280 eV the micrographs yielded bigger absorption differences, and thus better contrast between the core/coat and the peptidoglycan-rich cortex. Tilt series of dormant and HPT spores were reconstructed. Virtual sections revealed the dormant spores' core, cortex and coat without the need to physically slice the sample. It was also possible to visualize the inside of the HPT spores, which proved to be void of DPA. Industrial relevance Isostatic high pressure processing (HPP) is often regarded as the major recent technological innovation in food preservation. However, this technology is only applied in large scale for pasteurization and not for sterilization, which is largely due to the unknown inactivation mechanisms of highly resistant bacterial endospores. The structural analysis of high pressure treated endospores by X-ray microscopy could be used to study the progress of spore germination and inactivation under pressure and may add to the information already gathered in previous studies such as (Reineke et al., 2013) to establish the high pressure thermal sterilization at industrial scale. This technology in combination with different extrinsic factors like chemical or thermal preservation may open up new possibilities for a multi-target spore inactivation strategy for food sterilization.

Quantitative X-ray phase contrast waveguide imaging of bacterial endospores.

Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 105 Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

Decontamination of whole black pepper using different cold atmospheric pressure plasma applications

Whole black pepper is a dry product, which is often naturally contaminated with bacterial endospores and sometimes also with human pathogens like Salmonella. Dry pepper itself is a shelf-stable product, but if it is incorporated into high moisture minimally processed food, the microorganisms can reduce the shelf-life of the final product and/or can cause foodborne diseases. In this study the antimicrobial effect of two different atmospheric pressure plasma applications for the decontamination of whole black pepper was investigated. Naturally contaminated peppercorns and with Bacillus subtilis spores, Bacillus atrophaeus spores and Salmonella enterica inoculated ones were treated using a plasma jet or a microwave-driven remote plasma. Surface color and the content of essential oils and piperine was measured. S. enterica, B. subtilis spores and B. atrophaeus spores were reduced by 4.1, 2.4 and 2.8 log, respectively, after 30 min remote plasma treatment. Direct plasma jet treatment did not result in equivalent inactivation levels. However, both plasma applications did not considerable affect the quality parameters.

Bacillus and other spore-forming genera: variations in responses and mechanisms for survival.

The ubiquity of Bacilli endospores in soils facilitates their easy transfer routes to other environments, including cleanrooms and low-biomass sites required by many industries such as food production and processing. A bacterial endospore is a metabolically dormant form of life that is much more resistant to heat, desiccation, lack of nutrients, exposure to UV and gamma radiation, organic chemicals, and oxidizing agents than is a vegetative cell. For example, the heat tolerance of endospores depends on multiple factors such as sporulation temperature, core dehydration, and the presence of minerals and small, acid-soluble proteins (SASPs) in the core. This review describes our current understanding of the persistence mechanisms related to sporeformers' biochemical properties and discusses in detail spores' heat, radiation, and reactive chemical resistance. In addition, it discusses the impact of contamination with spores on many areas of human activity, spore adhesive properties, and biofilm contribution to resistance.

Fast and effective inactivation of Bacillus atrophaeus endospores using light-activated derivatives of vitamin B2.

Highly resistant endospores may cause severe problems in medicine as well as in the food and packaging industries. We found that bacterial endospores can be inactivated quickly with reactive oxygen species (ROS) that were generated by a new generation of flavin photosensitizers. Flavins like the natural compound vitamin B2 are already known to produce ROS but they show a poor antimicrobial photodynamic killing efficacy due to the lack of positive charges. Therefore we synthesized new flavin photosensitizers that have one (FLASH-01a) or eight (FLASH-07a) positive charges and can hence attach to the negatively charged surface of endospores. In this study we used standardized Bacillus atrophaeus endospores (ATCC 9372) as a biological surrogate model for a proof-of-concept study of photodynamic inactivation experiments using FLASH-01a and FLASH-07a. After incubation of spores with different flavin concentrations, the flavin derivatives were excited with blue light at a light dose of 70 J cm(-2). The inactivation of spores was investigated either in suspension or after attachment to polyethylene terephthalate (PET) surfaces. Incubation of spores suspended in Millipore water with 4 mM FLASH-01a for 10 seconds and irradiation with blue light for 10 seconds caused a biologically relevant decrease of spore survival of 3.5 log10 orders. Using FLASH-07a under the same conditions we achieved a decrease of 4.4 log10 orders. Immobilized spores on PET surfaces were efficiently killed with 7.0 log10 orders using 8 mM FLASH-07a. The total treatment time (incubation + irradiation) was as short as 20 seconds. The results of this study show evidence that endospores can be fastly and effectively inactivated with new generations of flavin photosensitizers that may be useful for industrial or medical applications in the future.