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.     

Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb³⁺ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.     

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.     

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.     

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.     

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.     

Fast sterility assessment by germinable-endospore biodosimetry

The increased demand for sterile products has created the need for rapid technologies capable of validating the hygiene of industrial production processes. Bacillus endospores are in standard use as biological indicators for evaluating the effectiveness of sterilization processes. Currently, culture-based methods, requiring more than 2 days before results become available, are employed to verify endospore inactivation. We describe a rapid, microscopy-based endospore viability assay (microEVA) capable of enumerating germinable endospores in less than 15 min. MicroEVA employs time-gated luminescence microscopy to enumerate single germinable endospores via terbium-dipicolinate (Tb-DPA) luminescence, which is triggered under UV excitation as 10(8) DPA molecules are released during germination on agarose containing Tb(3+) and a germinant (e.g., L-alanine). Inactivation of endospore populations to sterility was monitored with microEVA as a function of thermal and UV dosage. A comparison of culturing results yielded nearly identical decimal reduction values, thus validating microEVA as a rapid biodosimetry method for monitoring sterilization processes. The simple Tb-DPA chemical test for germinability is envisioned to enable fully automated instrumentation for in-line monitoring of hygiene in industrial production processes.     

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.     

Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history

Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats. We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T. sacchari is probably one of the oldest known living organisms. More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T. vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes. In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes. In varved sediment cores from three lakes, we found a temporal relationship between records of T. vulgaris endospores and the pollen of plants indicating agriculture. Endospores were very rare in sediments deposited before agriculture, ca. 1100 A.D. From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded. High endospore accumulation rates were found with the subsequent agricultural expansion. This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses. Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies.     

Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history.

Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats. We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T. sacchari is probably one of the oldest known living organisms. More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T. vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes. In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes. In varved sediment cores from three lakes, we found a temporal relationship between records of T. vulgaris endospores and the pollen of plants indicating agriculture. Endospores were very rare in sediments deposited before agriculture, ca. 1100 A.D. From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded. High endospore accumulation rates were found with the subsequent agricultural expansion. This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses. Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies.     

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.     

Ultrastructure of Coccidioides immitis after exposure to the imidazole antifungals miconazole and ketoconazole

Scanning and transmission electron microscopy was performed on the various phases of Coccidioides immitis, exposed for different periods of time to the imidazole antifungals miconazole and ketoconazole. The development of spherules into endospores, which takes place in cultures under normal growth conditions, was suppressed in the drug treated cultures. Typical ultrastructural changes were localized at the cell periphery and in the vacuolar system. The drugs did induce changes in mature, resting endospore cultures and in cultures incubated statically at room temperature. Aerobically growing endospores were not susceptible to either drug. The transformation of arthroconidia into mycelium was fully prevented after treatment. Mycelial cells were most susceptible to the antifungals for necrosis was induced in a substantial part of the hyphae after exposure for 24 h.     

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.     

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.     

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.     

Direct microscopy of bacillus endospore germination in soil microcosms

Antagonistic endospore-forming Bacillus spp. offer a large potential as seed inoculants for control of soil-borne pathogens. In the soil, however, inoculated Bacillus endospores may remain dormant without germination, and plant protection can therefore be inefficient and unpredictable. A method based on direct fluorescence microscopy in soil microcosms was used to determine whether low-cost organic additives incorporated into seed coating material could stimulate endospore germination. Complex organic additives supported a high level of endospore germination of the fungal antagonist Paenibacillus polymyxa CM5-5. Skim milk is a low-cost additive that may be incorporated into seed coating material for efficient induction of Bacillus endospore germination in soil.     

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).     

Assessment of bacterial endospore viability with fluorescent dyes

AIM: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. METHODS AND RESULTS: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). CONCLUSION: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.     

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.     

Aerobic methanotrophic communities in the bottom sediments of Lake Baikal

The results of the first methodical investigation into the aerobic methanotrophic communities inhabiting the bottom sediments of Lake Baikal are reported. Use of the radioisotopic method revealed methane consumption in 12 10- to 50-cm-long sediment cores. The maximum methane consumption rates (495-737 microl/(dm3 day) were recorded in sediments in the regions of hydrothermal vents and oil and gas occurrence. Methane consumption was most active in the surface layers of the sediments (0-4 cm); it decreased with the sediment depth and became negligible or absent at depths below 20 cm. The number of methanotrophic bacteria usually ranged from 100 to 1000 cells/cm3 of sediment and reached 1 million cells/cm3 in the regions of oil and gas occurrence. The 17 enrichment cultures obtained were represented mainly by morphotype II methanotrophs. Phylogenetic analysis of the enrichment cultures in terms of the amino acid sequence of the alpha subunit of the membrane-bound methane monooxygenase revealed the predominance of methanotrophs of the genus Methylocystis. The results obtained suggest the presence of an active aerobic methanotrophic community in Lake Baikal.