Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.     

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

Validation of a Clostridium endospore viability assay and analysis of Greenland ices and Atacama Desert soils

A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb(3+) and d-alanine triggers Clostridium spore germination and the concomitant release of ～10(8) molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb(3+)-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil).     

An automated front-end monitor for anthrax surveillance systems based on the rapid detection of airborne endospores

A fully automated anthrax smoke detector (ASD) has been developed and tested. The ASD is intended to serve as a cost effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb(3+)) luminescence. Anthrax attacks were simulated using aerosolized Bacillus atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne endospores collected. A concentration dependence of 10(2)-10(6) spores/mg of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores/L when 250 L of air were sampled. Simulated attacks were detected in less than 15 min.     

Effective terminal sterilization using supercritical carbon dioxide

Gentle alternatives to existing sterilization methods are called for by rapid advances in biomedical technologies. Supercritical fluid technologies have found applications in a wide range of areas and have been explored for use in the inactivation of medical contaminants. In particular, supercritical CO(2) is appealing for sterilization due to the ease at which the supercritical state is attained, the non-reactive nature, and the ability to readily penetrate substrates. However, rapid inactivation of bacterial endospores has proven a barrier to the use of this technology for effective terminal sterilization. We report the development of a supercritical CO(2) based sterilization process capable of achieving rapid inactivation of bacterial endospores while in terminal packaging. Moreover, this process is gentle; as the morphology, ultrastructure, and protein profiles of inactivated microbes are maintained. These properties of the sterilization process suit it for possible use on a wide range of biomedical products including: materials derived from animal tissues, protein based therapies, and other sensitive medical products requiring gentle terminal sterilization.     

Detecting the dormant: a review of recent advances in molecular techniques for assessing the viability of bacterial endospores

Due to their contribution to gastrointestinal and pulmonary disease, their ability to produce various deadly exotoxins, and their resistance to extreme temperature, pressure, radiation, and common chemical disinfecting agents, bacterial endospores of the Firmicutes phylum are a major concern for public and environmental health. In addition, the hardy and dormant nature of endospores renders them a particularly significant threat to the integrity of robotic extraterrestrial life-detection investigations. To prevent the contamination of critical surfaces with seemingly ubiquitous bacterial endospores, clean rooms maintained at exceedingly stringent cleanliness levels (i.e., fewer than 100,000 airborne particles per ft³) are used for surgical procedures, pharmaceutical processing and packaging, and fabrication and assembly of medical devices and spacecraft components. However, numerous spore-forming bacterial species have been reported to withstand typical clean room bioreduction strategies (e.g., UV lights, maintained humidity, paucity of available nutrients), which highlights the need for rapid and reliable molecular methods for detecting, enumerating, and monitoring the incidence of viable endospores. Robust means of evaluating and tracking spore burden not only provide much needed information pertaining to endospore ecophysiology in different environmental niches but also empower decontamination and bioreduction strategies aimed at sustaining the reliability and integrity of clean room environments. An overview of recent molecular advances in detecting and enumerating viable endospores, as well as the expanding phylogenetic diversity of pathogenic and clean room-associated spore-forming bacteria, ensues.     

Endospore dipicolinic acid detection during Bacillus thuringiensis culture

Aims: The aim of this work was to detect Bacillus thuringiensis endospore production during fermentation under conditions hindering endospore detection, i.e. in a complex undefined industrial medium with a high content of solids in suspension. Methods and Results: Bacterial endospore production was measured using the photoluminescence of dipicolinate (DPA) with Tb³⁺. The high temperature and pressure of a conventional autoclave was used to release DPA from the endospores. The endospore was obtained from B. thuringiensis var. kurstaki HD‐73 fermentations in industrial‐type media with 25·1 and 54·1 g l^?1 glucose, 4·4 and 35·3 g l^?1 soybean meal, 5·8 g l^?1 yeast extract, 9·2 g l^?1 corn steep solids and mineral salts. Conclusions: In this study, we successfully determined the DPA concentrations during the culture of B. thuringiensis in high‐concentration soybean meal media. A good correlation was found between microscope endospore counting and DPA measurements in the cultures. Significance and Impact of the Study: Because of synergy between Cry protein and endospore in B. thuringiensis bioinsecticides formulation, it is important to be able to determine endospore development during B. thuringiensis industrial‐type fermentation, in order to ascertain the beginning of sporulation.     

Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Bacillus endospore resistance to gas dynamic heating

Aim: To develop a novel laboratory procedure for the study of shock wave‐induced damage to Bacillus endospores. Methods and Results: Bacillus atrophaeus endospores are nebulized into an aerosol, loaded into the stanford aerosol shock tube and subjected to shock waves of controlled strength. Endospores experience uniform test temperatures between 500 and 1000 K and pressures ranging from 2 to 7 atm, for a relatively short time (2–3 ms). During this process, the bioaerosol is observed using in situ laser absorption and scattering diagnostics. Additionally, shock‐treated samples are extracted for ex situ analysis including viability plating, flow cytometry and SEM imaging. Measurements indicate that endospores lose the ability to form colonies when heated to test temperatures above 500 K while significant breakdown in morphology is observed at test temperatures above 750 K. Conclusion: These results demonstrate the disruption of essential biochemical pathways or biomolecules prior to the onset of significant endospore morphological deterioration. Significance and Impact of the Study: This novel laboratory approach to study the interaction of endospores with shock waves provides an experimental means to investigate the mechanisms of endospore resistance to rapid heating. In addition, this methodology allows for the direct simulation of a blast wave–bioaerosol interaction in an atmospheric environment.     

A highly sensitive HPLC method for determination of nanomolar concentrations of dipicolinic acid, a characteristic constituent of bacterial endospores

A high-performance liquid chromatographic method with indirect fluorescence detection has been developed for quantification of dipicolinic acid, a major constituent of bacterial endospores. After separation on a reversed-phase column, a post-column reagent of sodium acetate at 1 mol l(-1) with 50 micromol l(-1) terbium chloride was added for complexation of dipicolinic acid. Terbium monodipicolinate complexes formed were quantified by measuring the fluorescence emission maximum at 548 nm after excitation with UV light at 270 nm wavelength. Parameters of post-column complexation were optimized to achieve a detection limit of 0.5 nmol DPA l(-1), corresponding to about 10(3) Desulfosporosinus orientis endospores per ml. The method was applied to the analysis of spore contamination in tuna and for estimating the endospore numbers in marine sediments.     

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.     

Antibodies from Chicken Eggs as Probes for Antigens from Pasteuria penetrans Endospores

The bacteria Pasteuria spp. have been identified as among the most promising of several microbial organisms currently under investigation as biological control agents of plant-parasitic nematodes. As part of our goal to develop methods to discriminate isolates of Pasteuria penetrans with different host preferences, we investigated the potential of developing antibody probes to identify endospores of different isolates of P. penetrans. Polyclonal IgY antibodies were raised in chickens against endospores of P. penetrans isolates P20 and P100. Hens were injected with P20 or P100 endospore suspensions and boosted at 14 days. Anti-spore titers were determined with ELISA on yolk extracts of individual eggs as a function of time. The highest titers were found in eggs produced at 22 to 35 days after initial injections. Yolk extracts showing the highest titers were combined and processed to provide partially purified IgY preparations. SDS-PAGE and immunoblot analyses identified protein antigens with Mr values of 23-24, 46, and 57-59 KDa common to both P20 and P100 endospores. One protein antigen with an Mr value of 62 KDa was unique to the PI00 endospores. The IgY antibodies reduced the attachment of Pasteuria endospores to their nematode hosts, indicating antibody interaction with antigens on the endospore surface that are involved in the recognition and attachment processes.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo‐inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo‐inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo‐inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium‐like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium‐like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter‐species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium‐producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo‐inactivation properties.     

Spore dipicolinic acid contents used for estimating the number of endospores in sediments

Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.     

Role of superoxide in the germination of Bacillus anthracis endospores

The spore forming Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Germination of endospores during their internalization within the myeloid phagocyte, and the ability of those endospores to survive exposure to antibacterial killing mechanisms such as superoxide (O(2)*-, is a key initial event in the infective process. We report herein that endospores exposed to fluxes of O(2)*- typically found in stimulated phagocytes had no effect on viability. Further endospores of the Sterne strain of B. anthracis were found to scavenge O(2)*-, which may enhance the ability of the bacterium to survive within the hostile environment of the phagolysosome. Most intriguing was the observation that endospore germination was stimulated by a flux of O(2)*- as low as 1 microM/min. Data presented herein suggest that B. anthracis may co-opt O(2)*- which is produced by stimulated myeloid phagocytes and is an essential element of host immunity, as a necessary step in productive infection of the host.     

Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.     

Spin labelling of Bacillus anthracis endospores: a model for in vivo tracking by EPR imaging

Anthrax is caused by the gram-negative bacterium, Bacillus anthracis. Infection by this microbe results from delivery of the endospore form of the bacillus through direct contact, either topical or inhalation. With regard to the latter route of administration, it is proposed that endospores of B. anthracis enter the lungs and are phagocytized by host alveolar macrophages. Thereafter, it is unclear as to how endospores travel to distal loci and what tissues are the targets. Herein, this study describes the spin labelling of endospores through two different approaches with various aminoxyls. Indeed, after exposure to RAW 264.7 cells, these aminoxyl-containing endospores were phagocytized, as demonstrated by EPR spectroscopy of the infected macrophage, thus providing a potential tool for EPR imaging in animals.     

The use of flow cytometry to study the germination of Bacillus cereus endospores.

BACKGROUND: At present the study of endospore germination is conducted using microbiological methods which are slow and yield data based on the means of large heterogeneous populations. Flow cytometry (FCM) offers the potential to rapidly quantify and identify germination and outgrowth events for large numbers of individual endospores. METHODS: Standard methods were employed to arrest the germination of Bacillus cereus endospores at defined stages. Endospores were then stained with SYTO 9 alone or carboxyfluorescein diacetate (CFDA) together with Hoechst 33342 and analysed using FCM. Comparisons were made between FCM as a method to measure germination rate and standard microbiological techniques. RESULTS: Germinating endospores displayed increases in permeability to SYTO 9 and hydrolysis of CFDA compared with controls. Statistically significant correlations were found between the standard plate count method and both FCM methods for measuring the percentage of germinating and outgrowing endospores up to 75 min after addition of germinant. CONCLUSIONS: Using FCM, the percentage of germinating or outgrowing endospores at various time points during germination and/or outgrowth can be quantified. FCM with CFDA/Hoechst 33342 staining may be used to estimate overall germination rate, whereas FCM with SYTO 9 staining may be used to quantify ungerminated, germinating and outgrowing endospores.