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.     

A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.     

Membrane potential changes after infection of monocytes by Toxoplasma gondii.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.     

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.     

RAPID DETECTION OF GERMINATING BACILLUS CEREUS CELLS USING FLUORESCENT IN SITU HYBRIDIZATION

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.
B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus -specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 10³ cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.     

The Effects of Heat Activation on Bacillus Spore Germination,with Nutrients or under High Pressure,with or without Various Germination Proteins

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores'' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the l-asparagine–glucose–fructose–K⁺ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores'' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores'' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.     

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

The use of voltage-sensitive dyes to monitor signal-induced changes in membrane potential-ABA triggered membrane depolarization in guard cells

The plant membrane potential reports on the activity of electrogenic plasma membrane transport processes. The membrane potential is widely used to report for early events associated with changes in light regime, hormone action or pathogen attacks. The membrane potentials of guard cells can be precisely measured with microelectrodes, but this technique is not well suited for rapid screens with large sample numbers. To provide the basis for large-scale membrane potential recordings, we took advantage of voltage-sensitive dyes. Using the fluorescent dyes bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC(4)(3)) and the FLIPR Membrane Potential Assay Kit (FMP) dye we followed changes in the membrane potential in guard cells and vacuoles. Based on the fluorescence of DiBAC(4)(3) a method was established for quantification of the membrane potential in guard cell protoplasts which should be considered as an excellent system for high-throughput screening of plant cells. In the absence of abscisic acid (ABA), one-third of the guard cell protoplast population spontaneously oscillated for periods of 5-6 min. Upon application of ABA the hyperpolarized fraction ( approximately 50%) of the guard cell protoplast population depolarized within a few minutes. Membrane potential oscillations were terminated by ABA. Oscillations and ABA responses were found in cell populations with active anion channels. Thus time- and voltage-dependent anion channels likely represent the ABA-sensitive conductance and part of the membrane potential oscillator. The suitability of membrane potential dyes was tested on vacuoles, too. Dye-based vacuolar membrane polarization was monitored upon ATP exposure. We conclude that voltage-sensitive dyes provide an excellent tool for the study of changes in the membrane potential in vacuole as well as guard cell populations.     

Morphology of mitochondrial nucleoids, mitochondria, and nuclei during meiosis and sporulation of the yeast Saccharomycodes ludwigii

The morphology of mitochondrial nucleoids (mt-nucleoids), mitochondria, and nuclei was investigated during meiosis and sporulation of the diploid cells of the ascosporogenic yeast Saccharomycodes ludwigii. The mt-nucleoids appeared as discrete dots uniformly distributed in stationary-phase cells as revealed by 4',6-diamidino-2-phenylindole (DAPI) staining. Throughout first and second meiotic divisions, the mt-nucleoids moved to be located close to the dividing nuclei with the appearance of dots. On the other hand, mitochondria, which had tubular or fragmented forms in stationary-phase cells, increasingly fused with each other to form elongated mitochondria during meiotic prophase as revealed by 3,3' -dihexyloxacarbocyanine iodide [DiOC(6)(3)] staining. Mitochondria assembled to be located close to dividing nuclei during first and second meiotic divisions, and were finally incorporated into spores. During the first meiotic division, nuclear division occurred in any direction parallel, diagonally, or perpendicular to the longitudinal axis of the cell. In contrast, the second meiotic division was exclusively parallel to the longitudinal axis of the cell. The behavior of dividing nuclei explains the formation of a pair of spores with opposite mating types at both ends of cells. In the course of this study, it was also found that ledges between two spores were specifically stained with DiOC(6)(3).     

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.     

Study of calcium dipicolinate release during bacterial spore germination by using a new, sensitive assay for dipicolinate.

The release of calcium and dipicolinic acid from spores of Bacillus megaterium KM during L-alanine-induced triggering of germination has been studied using a new, simple, and rapid assay for dipicolinic acid capable of detecting a concentration of 0.5 micron. The release of both calcium and dipicolinate started within seconds of exposure of the spores to L-alanine, thus preceding other measurable changes associated with germination. From the earliest times, the two substances were released in equimolar quantities, although later in germination calcium predominated.     

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis

AIMS: To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. METHODS AND RESULTS: Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. CONCLUSIONS: Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

The effects of alterations in the suspending medium on low temperature activation of spores of Bacillus stearothermophilus NGB101

The effects of eight different sodium salts on the activation of spores of Bacillus stearothermophilus NGB101 at 30°C were examined. Sodium nitrite was a potent activator spores of NGB101. Complete activation of spore populations was obtained after 6 h or less at 30°C. Activation of spores of NGB101 in solutions of sodium nitrite, like activation in distilled water, was temperature dependent, with optimal activation at 30°C. While a potent activator of spores of NGB101 at 30°C, sodium nitrite was ineffective as an initiator of germination at 65°C. Activation of spores of NGB101 produced marked increases in colony forming spores compared with nonactivated populations. Spore populations activated in solutions of sodium nitrite gave higher plate counts compared with spores activated in distilled water.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

Polymeric glycoconjugates protect and activate macrophages to promote killing of <Emphasis Type="Italic">Bacillus cereus</Emphasis> spores during phagocytosis

Diseases caused by Bacillus spores might be attenuated if macrophages were able to kill the spores on exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for modulation of phagocytosis of B. cereus spores. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores during phagocytosis. In the presence of glycoconjugates, however, the macrophages efficiently killed the organisms. The glycoconjugates were shown to have a protective influence, sparing macrophages from spore-induced cell death. Very low concentrations of the glycoconjugates prevented macrophage cell death, as shown by lactate dehydrogenase (LDH) release and trypan blue assays. Increased levels of inducible nitric oxide (NO) production by the macrophages in the presence of glycoconjugates suggested that the glycoconjugates provide an activation signal to the macrophages. These results suggest that glycoconjugates promote the killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level. Polymeric glycoconjugates may suggest novel approaches to improve existing vaccines as well as prevent and treat infections incurred through either B. cereus or B. anthracis spores.     

Heat Injury of Bacillus subtilis Spores at Ultrahigh Temperatures

The following three criteria indicated that Bacillus subtilis A spores were injured, but not completely inactivated, by ultrahigh temperature treatment. (i) Significant reductions in survivors were observed when spores were enumerated with a standard medium but not when the medium contained added CaCl(2) and sodium dipicolinate. (ii) After a damaging heat treatment, more survivors were enumerated with the standard medium after incubation at 32 C than at 45 C, which was opposite to the result with untreated or slightly heated spores. (iii) Apparent numbers of survivors increased during the initial period of 3 C storage when enumerated with the standard medium at 45 C. No injury was evident when survivors were enumerated at either incubation temperature with the medium containing added CaCl(2) and sodium dipicolinate. Heat activation of the spores did not significantly influence the appearance of heat injury. The data suggested that the heat injury occurred in a germination system which was required in the absence of CaCl(2) and sodium dipicolinate.     

Heat activation of Streptomyces viridochromogenes spores.

The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.