Sorting through the Wealth of Options: Comparative Evaluation of Two Ultraviolet Disinfection Systems

Background

Environmental surfaces play an important role in the transmission of healthcare-associated pathogens. Because environmental cleaning is often suboptimal, there is a growing demand for safe, rapid, and automated disinfection technologies, which has lead to a wealth of novel disinfection options available on the market. Specifically, automated ultraviolet-C (UV-C) devices have grown in number due to the documented efficacy of UV-C for reducing healthcare-acquired pathogens in hospital rooms. Here, we assessed and compared the impact of pathogen concentration, organic load, distance, and radiant dose on the killing efficacy of two analogous UV-C devices.

Principal Findings

The devices performed equivalently for each impact factor assessed. Irradiation delivered for 41 minutes at 4 feet from the devices consistently reduced C. difficile spores by ∼ 3 log₁₀CFU/cm², MRSA by>4 log₁₀CFU/cm², and VRE by >5 log₁₀CFU/cm². Pathogen concentration did not significantly impact the killing efficacy of the devices. However, both a light and heavy organic load had a significant negative impacted on the killing efficacy of the devices. Additionally, increasing the distance to 10 feet from the devices reduced the killing efficacy to ≤3 log₁₀CFU/cm² for MRSA and VRE and <2 log₁₀CFU/cm² for C.difficile spores. Delivery of reduced timed doses of irradiation particularly impacted the ability of the devices to kill C. difficile spores. MRSA and VRE were reduced by >3 log₁₀CFU/cm² after only 10 minutes of irradiation, while C. difficile spores required 40 minutes of irradiation to achieve a similar reduction.

Conclusions

The UV-C devices were equally effective for killing C. difficile spores, MRSA, and VRE. While neither device would be recommended as a stand-alone disinfection procedure, either device would be a useful adjunctive measure to routine cleaning in healthcare facilities.     

Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. Control of C. difficile is challenging because the spores are resistant to killing by alcohol-based hand hygiene products, antimicrobial soaps, and most disinfectants. Although initiation of germination has been shown to increase susceptibility of spores of other bacterial species to radiation and heat, it was not known if triggering of germination could be a useful strategy to increase susceptibility of C. difficile spores to radiation or other stressors.

Principal Findings

Here, we demonstrated that exposure of dormant C. difficile spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.

Conclusions

Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents.     

Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.Spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of stress factors (31, 32). While spores can remain dormant for long periods, if given the proper stimulus, they can rapidly “return to life” in the process of spore germination followed by outgrowth (30). Since spores are generally present in significant amounts on many foodstuffs and growing cells of a number of Bacillus species are significant agents of food spoilage and food-borne disease (32), there is continued applied interest in spore resistance and germination. While dormant spores can be killed by a treatment such as wet heat, this requires high temperatures that are costly and detrimental to food quality. Consequently, there has long been interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. However, this strategy has been difficult to apply because of the significant heterogeneity in germination rates between individual spores in populations. One reflection of this heterogeneity is the extremely variable lag times following addition of germinants but prior to initiation of germination events; while these lag times can vary from 10 to 30 min for most spores in populations, some spores have lag times of many hours or even many days (2, 12, 13, 15, 25). The spores that are extremely slow to germinate have been termed superdormant spores, and populations of superdormant spores have recently been isolated from three Bacillus species, and their germination properties characterized (9, 10). These superdormant spores germinate extremely poorly with the original germinants used to remove dormant spores from spore populations, thus allowing superdormant spore isolation, and also poorly with a number of other germinants, in particular, germinants that target nutrient germinant receptors different than those activated to isolate the superdormant spores. However, the superdormant spores germinate reasonably well with mixtures of nutrient germinants that target multiple germinant receptors. All reasons for spore superdormancy are not known, but one contributing factor is the number of nutrient germinant receptors in the spore''s inner membrane that trigger spore germination by binding to nutrient germinants (9). The levels of these receptors are most likely in the tens of molecules per spore (24), and thus stochastic variation in receptor numbers might result in some spores with such low receptor numbers that these spores germinate very poorly (23). Indeed, 20- to 200-fold elevated levels of at least one nutrient germinant receptor greatly decreases yields of superdormant spores of Bacillus subtilis (9).Spores of Bacillus species generally exhibit a requirement for an activation step in order to exhibit maximum germination (17). Usually this activation is a sublethal heat treatment that for a spore population exhibits an optimum of 60 to 100°C depending on the species. Spores are also extremely resistant to wet heat, generally requiring temperatures of 80 to 110°C to achieve rapid spore killing, with the major factor influencing the wet-heat resistance of spores of mesophilic strains being the spore core''s water content, which can be as low as 30% of wet weight as water in a fully hydrated spore (8, 19, 27, 28, 31). Invariably, increases in core water content are associated with a decrease in spore wet-heat resistance (8, 19, 22, 25). While spore populations most often exhibit log-linear kinetics of wet-heat killing, the observation of tailing in such killing curves at high levels of killing is not uncommon, suggesting there is significant heterogeneity in the wet-heat resistances of individual spores in populations (27, 28). While there has been no comparable work suggesting that there is also heterogeneity in the temperature optima for heat activation of individual spores in populations, this certainly seems possible and indeed was suggested as one cause of spore superdormancy, as yields of superdormant spores from spore populations that are not heat activated are much higher (9, 10). Consequently, the current work was initiated to test the hypothesis that superdormant spores require heat activation temperatures that are higher than those of the original dormant spores. Once this was found to be the case, the wet-heat resistance and core water content of the superdormant and original dormant spores were compared, and the environment of the spore core''s major small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) was assessed by Raman spectroscopy of individual spores.     

Superdormant Spores of Bacillus Species Germinate Normally with High Pressure,Peptidoglycan Fragments,and Bryostatin

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.Spores of Bacillus species are formed in sporulation, a process that is generally triggered by starvation for one or more nutrients (13, 19). These spores are metabolically dormant and extremely resistant to a large variety of environmental stresses, including heat, radiation, and toxic chemicals, and as a consequence of these properties, these spores can remain viable in their dormant state for many years (13, 18, 19). However, spores are constantly sensing their environment, and if nutrients return, the spores can rapidly return to growth through the process of spore germination (17). Spore germination is generally triggered by specific nutrients that bind to nutrient germinant receptors, with this binding alone somehow triggering germination. However, spore germination can also be triggered by many non-nutrient agents, including cationic surfactants such as dodecylamine, a 1:1 complex of Ca²⁺ with pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA], a major spore small molecule), very high pressures, specific peptidoglycan fragments, and bryostatin, an activator of Ser/Thr protein kinases (17, 19, 20). For nutrient germinants in particular, spore germination is also potentiated by a prior sublethal heat treatment termed heat activation (17).While normally the great majority of spores in populations germinate relatively rapidly in response to nutrient germinants, a small percentage of spores germinate extremely slowly. These spores that are refractory to nutrient germination have been termed superdormant spores and are a major concern for the food industry (8). Recently superdormant spores of three Bacillus species have been isolated by repeated germination of spore populations with specific nutrient germinants and isolation of remaining dormant spores (5, 6). These superdormant spores germinate extremely poorly with the nutrient germinants used in superdormant spore isolation, as well as with other nutrient germinants. All of the specific defects leading to spore superdormancy are not known, although an increased level of receptors for specific nutrient germinants decreases levels of superdormant spores obtained with the nutrients that are ligands for these receptors (5). Superdormant spores also have significantly higher temperature optima for heat activation of nutrient germination than the spore population as a whole (7).In contrast to the poor germination of superdormant spores with nutrient germinants, superdormant spores germinate normally with dodecylamine and Ca-DPA (5, 6). This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11, 15, 17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2, 3, 11, 15, 20, 22, 23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.The spores used in this work were from Bacillus subtilis PS533 (16), a derivative of strain 168 that also carries plasmid pUB110, providing resistance to kanamycin (10 μg/ml), and Bacillus cereus T (originally obtained from H. O. Halvorson). Spores of these strains were prepared and purified as described previously (6, 10, 12). Superdormant spores of B. subtilis were prepared by germination following heat activation at 75°C for 30 min by two germination treatments at 37°C with 10 mM l-valine for 2 h, followed by isolation of remaining dormant spores, all as described previously (5, 10, 12). These superdormant spores germinated extremely poorly with 10 mM valine at 37°C, giving ≤10% germination in 2 h at 37°C, while the initial spore population exhibited >95% germination under the same conditions (data not shown). Superdormant B. cereus spores were isolated similarly, although heat activation was at 65°C for 30 min and the germinant was 5 mM inosine as described previously (6). These superdormant B. cereus spores exhibited <5% germination with inosine in 2 h at 37°C compared to the >95% germination of the initial dormant spores under the same conditions (data not shown).     

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

Background

Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe^®) would reduce the burden of spores on skin.

Methods

Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths.

Results

Spore removal from hands was enhanced with Vashe soak (>2.5 log₁₀ reduction) versus soap and water wash or soak (~2.0 log₁₀ reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log₁₀ spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin.

Conclusions

Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.     

Levels of Germination Proteins in Bacillus subtilis Dormant,Superdormant, and Germinating Spores

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (T_lag and T_release times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT_release times (T_release?T_lag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔT_release times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in T_lag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T_lag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.     

Isolation and Characterization of Superdormant Spores of Bacillus Species

Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores'' defect was not genetic. The superdormant spores did, however, germinate with Ca²⁺-dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores.Spores of various Bacillus species are formed in sporulation and are metabolically dormant and very resistant to environmental stress factors (21, 37). While such spores can remain in this dormant, resistant state for long periods, they can return to life rapidly through the process of germination, during which the spore''s dormancy and extreme resistance are lost (36). Spore germination has long been of intrinsic interest, and continues to attract applied interest, because (i) spores of a number of Bacillus species are major agents of food spoilage and food-borne disease and (ii) spores of Bacillus anthracis are a major bioterrorism agent. Since spores are much easier to kill after they have germinated, it would be advantageous to trigger germination of spores in foods or the environment and then readily inactivate the much less resistant germinated spores. However, this simple strategy has been largely nullified because germination of spore populations is heterogeneous, with some spores, often called superdormant spores, germinating extremely slowly and potentially coming back to life long after treatments are applied to inactivate germinated spores (8, 9, 16). The concern over superdormant spores in populations also affects decisions such as how long individuals exposed to B. anthracis spores should continue to take antibiotics, since spores could remain dormant in an individual for long periods and then germinate and cause disease (3, 11).In many species, spore germination can be increased by a prior activation step, generally a sublethal heat treatment, although the changes taking place during heat activation are not known (16). Spore germination in Bacillus species is normally triggered by nutrients such as glucose, amino acids, or purine ribosides (27, 36). These agents bind to germinant receptors located in the spore''s inner membrane that are specific for particular nutrients. In Bacillus subtilis, the GerA receptor responds to l-alanine or l-valine, while the GerB and GerK receptors act cooperatively to respond to a mixture of l-asparagine (or l-alanine), d-glucose, d-fructose and K⁺ ions (AGFK [or Ala-GFK]) (1, 27, 36). There are even more functional germinant receptors in Bacillus megaterium spores, and these respond to d-glucose, l-proline, l-leucine, l-valine, or even salts, such as KBr (6). Glucose appears to trigger germination of B. megaterium spores through either of two germinant receptors, GerU or GerVB, while l-proline triggers germination through only the GerVB receptor, and KBr germination is greatly decreased by the loss of either GerU or GerVB (6). Nutrient binding to the germinant receptors triggers the release of small molecules from the spore core, most notably the huge depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) present in spores predominantly as a 1:1 diluted chelate with Ca²⁺ (Ca-DPA) (35, 36). Ca-DPA release then triggers the activation of one of two redundant cortex lytic enzymes (CLEs) that degrade the spore''s peptidoglycan cortex, and cortex degradation completes spore germination and allows progression into outgrowth and then vegetative growth (27, 33, 36).Spore germination can also be triggered by nonnutrient agents, including Ca-DPA and cationic surfactants (27, 33, 36). With B. subtilis spores, Ca-DPA triggers germination by activating one particular CLE, termed CwlJ, and bypasses the spore''s germinant receptors. Germination by the cationic surfactant dodecylamine also bypasses the germinant receptors, and this agent appears to release small molecules including Ca-DPA from the spore core either by opening a normal channel in the spore''s inner membrane for Ca-DPA and other small molecules or by creating such a channel (31, 38, 39).Almost all work on the specifics of the germination of spores of Bacillus species has focused on the majority of spores in populations, and little detailed attention has been paid to that minority of spores that either fail to germinate or germinate extremely slowly. However, it is these latter spores that are most important in unraveling the cause of superdormancy and perhaps suggesting a means to germinate and thus easily inactivate such superdormant spores. Consequently, we have undertaken the task of isolating superdormant spores from spore populations, using buoyant density centrifugation to separate dormant spores from germinated spores. The properties of these purified superdormant spores were then studied, and this information has suggested some reason(s) for spore superdormancy.     

Clostridium difficile Spore-Macrophage Interactions: Spore Survival

Background

Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment.

Methodology/Principal Findings

In this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1.

Conclusions/Significance

These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.     

Monitoring of Commitment,Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores'' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T_release) greater than the time of germinant removal (T_b) increased, while the time T_lag − T_b, where T_lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔT_release = T_release − T_lag) were increased; Factors affecting average T_release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

Background

Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores.

Results

Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP₃) levels and the effect of added IP₃ uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP₅ phosphatase leading to high IP₃ levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP₃ levels; addition of exogenous IP₃ to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP₃ levels remain high, also at unfavourable environmental conditions.

Conclusions

The results imply that environmental conditions regulate PLC activity and that IP₃ induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP₃-forming pathway.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore''s DPA once rapid DPA release had begun (ΔT_release) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ΔT_release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination ≥15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T_lag, between mixing spores with nutrient germinants and the beginning of ΔT_release. (iv) A number of factors decrease spores'' T_lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25, 29, 30). The germinants that most likely trigger spore germination in the environment are low-molecular-weight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore''s inner membrane that recognize their cognate germinants in a stereospecific manner (17, 24, 25, 29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6, 29, 30). Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B. subtilis spore germination by binding of l-alanine or l-valine to the GerA GR. However, many GRs cooperate such that binding of germinants by ≥2 different GRs is needed to trigger germination (2, 29): for example, the triggering of B. subtilis spore germination by the binding of components of a mixture of l-asparagine, d-glucose, d-fructose, and K⁺ ions (AGFK) to the GerB and GerK GRs. The binding of nutrient germinants to GRs triggers subsequent events in germination, although how this is accomplished is not known.The first readily measured biochemical event after addition of nutrient germinants to Bacillus spores is the rapid release of the spore''s large depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus its chelated divalent cations, predominantly Ca²⁺ (Ca-DPA), from the spore core (25, 29). Ca-DPA release then results in the activation of two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, which hydrolyze the spore''s peptidoglycan cortex layer (16, 22, 27, 29). CwlJ is activated by Ca-DPA as it is released from the spore while SleB is activated only after most DPA is released (17, 20, 22, 26, 27). Cortex hydrolysis ultimately allows the spore core to expand and take up more water, raising the core water content from the 35 to 45% of wet weight in the dormant spore to the 80% of wet weight characteristic of growing cells. Full hydration of the spore core then allows enzyme action, metabolism, and macromolecular synthesis to resume in the now fully germinated spore.Germination of spores in populations is very heterogeneous, with some spores germinating rapidly and some extremely slowly (4, 5, 9, 11, 13-15, 19, 26, 31, 32). Where it has been studied, the reason for this heterogeneity has been suggested to be due to a variable lag period (T_lag) between the time of mixing spores with a germinant and the time at which rapid DPA release begins, since once rapid DPA release begins, the time required for release of almost all DPA as well as for subsequent cortex hydrolysis is generally rather short compared to T_lag values in individual spores (5, 11, 13-15, 19, 26, 31, 32). The times required for DPA release and cortex hydrolysis are also similar in wild-type spores with both very short and long T_lag values (5, 15, 19, 27). The reasons for the variability in T_lag times between individual spores in populations are not known, although there are reports that both activation of spores for germination by a sublethal heat treatment (heat activation) as well as increasing concentrations of nutrient germinants can shorten T_lag values (12, 14, 15, 18, 32). However, there has been no detailed study of the causes of the variability in T_lag values between very large numbers of individual spores in populations.In order to study the heterogeneity in spore germination thoroughly, methods are needed to follow the germination of hundreds of individual spores over several hours. Initial studies of the germination of individual spores examined a single spore in a phase-contrast microscope and followed the germination of this spore by changes in the core''s refractive index due to DPA release and core swelling (14, 15, 32, 34). However, this method is labor-intensive for gathering data with hundreds of individual spores. More recently, confocal microscopy and then surface adsorption and optical tweezers have been used to capture single spores, and germination events have been followed by methods such as Raman spectroscopy to directly measure DPA release, as well as phase-contrast microscopy and elastic light scattering (3, 5, 9, 10, 19, 26). While the latter recent advances have allowed accumulation of much information about germination, collection of this type of data for large numbers of individual spores is still labor-intensive, although use of dual optical traps (35) and perhaps multiple traps in the future may alleviate this problem. However, phase-contrast microscopy plus appropriate computer software has recently allowed the monitoring of many hundreds of individual spores for several hours, with automated assessment of various changes in the cells during the period of observation (19). In the present work, we have used both phase-contrast and differential interference contrast (DIC) microscopy to monitor the germination of many hundreds of individual spores of three Bacillus species adhered on either an agarose pad or a glass coverslip for 1 to 2 h. This work, as well as examination of times needed for release of most DPA once rapid DPA release has begun during germination of individual spores under a variety of conditions, has allowed detailed examination of the effects of heat activation, nutrient germinant concentration, GR numbers per spore, and individual CLEs on spore germination heterogeneity and on values of T_lag for individual spores.     

Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.     

Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, T_lag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T₁; (ii) the period of slow DPA leakage (ΔT_leakage = T_lag − T₁) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T₁ times but increased values of ΔT_leakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T₁ times of less than 10 min, suggesting that T₁ is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T₁ and T_lag times but not the amount of DPA released in ΔT_leakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔT_leakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.