Phages Bind to Vegetative and Spore Forms of Paenibacillus larvae and to Vegetative Brevibacillus laterosporus

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most destructive bacterial infection in honeybees. Even antibiotic-sensitive strains of P. larvae can produce recurrent AFB months to weeks post-antibiotic treatment due to the survival of bacterial spores. Recently, phages that infect P. larvae have been shown to effectively combat AFB in the field. Here, we present evidence that phages not only bind to vegetative P. larvae but also bind to P. larvae spores. Spore binding was observed in the results of three specific experiments: (1) bacteria counted by flow cytometry generated quantitative data of FITC-labeled phages that were bound to vegetative bacteria as well as those bound to spores, (2) electron microscopy captured images of phages bound to the surface of spores in both horizontal and vertical positions, and (3) phages incubated with P. larvae spores bound to the spores and created plaques in vegetative bacteria under conditions not conducive to spore activation, indicating that binding to spores is reversible and that the phages are still active. Identification of phages with reversible spore-binding capability for use in phage therapy may improve treatment of sporulating bacterial infections.

Closely related budding yeast species respond to different ecological signals for spore activation

Spore activation is one of the most important developmental decisions in fungi as it initiates the transition from dormant and stress resistant cells to vegetative cells. Because in many species mating follows spore activation and germination, signals that trigger this developmental transition can also contribute to species reproductive barriers. Here we examine the biochemical signals triggering spore activation in a natural species complex of budding yeast, Saccharomyces paradoxus (lineages SpA, SpB, SpC and SpC*). We first demonstrate that we can quantitatively monitor spore activation in these closely related lineages. Second, we dissect the composition of culture media to identify components necessary and/or sufficient to activate spores in the four lineages. We show that, contrary to expectation, glucose is necessary but not sufficient to trigger spore activation. We also show that two of the North American lineages (SpC and SpC*) diverge from the other North American (SpB) and European (SpA) lineages in terms of germination signal as their spore activation requires inorganic phosphate. Our results show that the way budding yeast interpret environmental conditions during spore activation diverged among closely related and incipient species, which means that it may play a role in their ecological differentiation and reproductive isolation.

Impact of Strain, pH and Ethanol Concentration on Ethanol Activation Method of Bacillus Spores Using a Single Spore Approach

Aims: This work aims to determine the ideal conditions for ethanol activation of spores during their enumeration and compare to thermal activation which is the reference method. Place and duration: Department of Microbiology of the University of Yaoundé I between May2016 and June 2018. Methodology: Bacillus cereus and Bacillus subtilis spores were activated according to an experimental central composite design with three-factor. The factors considered were exposure time, ethanol concentration, pH and activation temperature according to the types of activation. Germination yield was carried out by individually monitoring each spore of a population on solid medium in order to determine the population germination kinetic parameters (time and rate of colony appearance within a population during germination) and germination yields. These parameters were compared with those obtained after thermal activation known as a reference method. Results: The factors strain and pH, significantly influenced the rate of spore germination within the population after ethanol activation. In the case of thermal activation, the specie and activation temperature were the most influential factors. The best germination yields were obtained for alcoholic activation of spores at 30% ethanol for 60min exposure at pH7, while for thermal activation the best yields varied from one strain to another depending on the activation conditions. Conclusion: Ethanol activation can be considered as a good substitute of thermal activation during spore enumeration provided activation conditions are well controlled. This is in our opinion the first detailed study comparing ethanol activation to heat activation of Bacillus spores. It will impact future revisions of spore enumeration protocols proposed by norms that take into consideration spore activation and reduce bias in spore enumeration.

Clospore: A Liquid Medium for Producing High Titers of Semi-purified Spores of Clostridium difficile

Clostridium difficile continues to cause infections in healthcare and other settings. Its spores survive well indoors and require sporicidal chemicals for infection control. However, proper testing of disinfectants is impeded due to difficulties in obtaining viable spores of high enough quality and titers to meet current regulations for sporicidal claims. A new liquid medium (Clospore) has been developed, based on a systematic review of the compositions of 20 other available media. C. difficile spores grown in the new medium and treated with a mixture of lysozyme and trypsin yielded final suspensions with >109 CFU/mL of viable spores, with a purity of >91 as tested by spore-staining and phase-contrast microscopy. The spores showed a biological decay rate of about 0.1 log10/month when dried on metal disks and stored indoors (air temperature 23 2C; relative humidity 52.76 15.08). Heating the purified spore suspensions to 70C for 10 min to inactivate any vegetative cells showed no spore activation or inactivation. The spores could be stored for at least 14 months either refrigerated (4C) or frozen (20 or 80C) in 50 (v/v) ethanol with virtually no loss in viability. The resistance of the enzyme-treated spores to three levels of sodium hypochlorite (1000, 3000, and 5000 ppm), using a standardized quantitative carrier test, was almost identical to that of the spores concentrated by centrifugation alone. The described procedure has been successfully applied to four standard (ATCC) and six clinical strains of C. difficile.

Requirements for Germination of Clostridium sordellii Spores In Vitro

ABSTRACT Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, d-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of l-phenylalanine and l-alanine but not in that of bicarbonate or l-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.

Transcriptional Transitions during Dictyostelium Spore Germination

ABSTRACT Many protozoa form spores in response to adversity; therefore, spore germination is a key process in their life cycle. Dictyostelium discoideum sporulates in response to starvation following a developmental program. Germination is characterized by two visible changes, spore swelling and the emergence of amoeba from the spore capsule. Several studies have indicated that an additional process termed spore activation is also required, but the physiological changes that characterize the three phases are largely uncharacterized. We used microarrays to monitor global transcriptional transitions as a surrogate measure of the physiological changes that occur during germination. Using two independent methods to induce germination, we identified changes in mRNA levels that characterized the germination process rather than changes that resulted from the induction method. We found that germination is characterized by three transitions. The first transition occurs during activation, while the spores appear dormant, the largest transition occurs when swelling begins, and the third transition occurs when emergence begins. These findings indicate that activation and swelling are not passive occurrences, such as dilution of inhibitors or spore rehydration, but are active processes that are accompanied by dramatic events in mRNA degradation and de novo transcription. These findings confirm and extend earlier reports that genes such as celA are regulated during spore germination. We also found by mutation analysis that the unconventional myosin gene myoI, which is induced during early germination, plays roles in the maintenance of dormancy and in spore swelling. This finding suggests that some of the observed transcriptional changes are required for spore germination.

Alicyclobacillus in Orange Juice: Occurrence and Heat Resistance of Spores

Spore suspensions of a pure culture of Alicyclobacillus acidoterrestris DSM 2498 were submitted to different heat treatments (60°C for 60 min, 60°C for 30 min, 70°C for 20 min, 80°C for 5 min, 80°C for 10 min, 80°C for 30 min, and boiling for 5 min) to determine the best activation conditions in orange juice. The best treatment for spore activation was shown to be 70°C/20 min. Seventy-five samples of concentrated orange juice from 11 different suppliers were examined for the presence of thermophilic acid-tolerant spore formers by the most probable number technique using Bacillus acidocaldarius medium (BAM broth) and incubation at 44°C for 5 days after a prior spore activation. After incubation, isolation was carried out using BAM agar medium incubating at 44°C for 5 days. Typical colonies were submitted to a microscopic examination, evaluation for the presence of spores, and various biochemical tests. Of the orange juice samples examined, 14.7% were found to be positive for Alicyclobacillus. The thermal death time open tube method was used to determine the heat resistance of the spores of strains confirmed as being Alicyclobacillus. The D-values determined were in the range from 60.8 to 94.5 min at 85°C, 10.0 to 20.6 min at 90°C, and 2.5 to 8.7 min at 95°C. The z-values were between 7.2°C and 11.3°C. The results demonstrated the occurrence of Alicyclobacillus in orange juice and the high heat resistance of the spores that could survive the heat treatments normally applied in the processing of orange juice.

The role of Ca2+ during spore germination in Dictyostelium: autoactivation is mediated by the mobilization of Ca2+ while amoebal emergence requires entry of external Ca2+

One of the developmental pathways used by the social amoeba Dictyostelium discoideum produces dormant spores. As with any temporary resistant stage, these spores must be able to germinate rapidly in response to positive environmental stimuli. One such stimulus is the autoactivator, an endogenous, diffusible molecule that is secreted by spores. Previous work has shown that three phases of germination, autoactivation, spore swelling and amoebal emergence, require the activity of the Ca(2+)-dependent, regulatory protein calmodulin, implicating Ca2+ as an essential cation during germination. In this study we used a pharmacological approach coupled with the direct measurement of Ca2+ levels in germinating spore populations by atomic adsorption to examine Ca(2+)-dependent signal transduction during spore activation and germination in D. discoideum. Inhibitors of both phospholipase C and internal Ca2+ release inhibited autoactivation while exogenously added Ins(1,4,5)P3, acted synergistically with the autoactivator. The antagonists specifically affected spore activation as mediated by the autoactivator, since neither had any effect on heat-activated spores. In contrast, La3+, an inhibitor of Ca2+ uptake, had little or no effect on either autoactivation or the swelling of autoactivated spores. However, an inhibition of Ca2+ influx by La3+ inhibited both the swelling of heat-activated spores and amoebal emergence following each period of autoactivation or heat activation. Ca2+ levels change in the spore population during germination. During activation and swelling, Ca2+ efflux occurs from the spores. Both of the activating stimuli used here, the autoactivator and heat, caused this Ca2+ efflux. The efflux is reversed during emergence when there is a net Ca2+ uptake by the spores and cells from the medium. Together these data provide the first evidence that autoactivation is mediated by Ca(2+)-dependent signal transduction, leading to Ca2+ efflux, and that the late event of germination, amoebal emergence, requires Ca2+ uptake to proceed. The data also suggest that the responses of the spore to the each of autoactivator and heat, i.e. Ca2+ movements and germination, are mediated by different mechanisms.