Fast and effective inactivation of Bacillus atrophaeus endospores using light-activated derivatives of vitamin B2
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Abstract Highly resistant endospores may cause severe problems in medicine as well as in the food and packaging industries. We found that bacterial endospores can be inactivated quickly with reactive oxygen species (ROS) that were generated by a new generation of flavin photosensitizers. Flavins like the natural compound vitamin B2 are already known to produce ROS but they show a poor antimicrobial photodynamic killing efficacy due to the lack of positive charges. Therefore we synthesized new flavin photosensitizers that have one (FLASH-01a) or eight (FLASH-07a) positive charges and can hence attach to the negatively charged surface of endospores. In this study we used standardized Bacillus atrophaeus endospores (ATCC 9372) as a biological surrogate model for a proof-of-concept study of photodynamic inactivation experiments using FLASH-01a and FLASH-07a. After incubation of spores with different flavin concentrations, the flavin derivatives were excited with blue light at a light dose of 70 J cm −2 . The inactivation of spores was investigated either in suspension or after attachment to polyethylene terephthalate (PET) surfaces. Incubation of spores suspended in Millipore water with 4 mM FLASH-01a for 10 seconds and irradiation with blue light for 10 seconds caused a biologically relevant decrease of spore survival of 3.5 log 10 orders. Using FLASH-07a under the same conditions we achieved a decrease of 4.4 log 10 orders. Immobilized spores on PET surfaces were efficiently killed with 7.0 log 10 orders using 8 mM FLASH-07a. The total treatment time (incubation + irradiation) was as short as 20 seconds. The results of this study show evidence that endospores can be fastly and effectively inactivated with new generations of flavin photosensitizers that may be useful for industrial or medical applications in the future.Spore germination
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Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.
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S ummary . The recovery of Bacillus subtilis spores was studied after different heat treatments at 95° and incubation at different temperatures in roll tubes in a gradient temperature incubator. Plate count agar and brain–heart infusion agar were used in the roll tubes. Unheated spores showed similar recoveries at 16–48° whereas heated spores had an optimum recovery temperature of c. 30.9. The rate of germination of untreated spores was greatest at c. 41° and ceased at 50°. Heated spores germinated at 52°5°, suggesting that recovery of heat‐treated spores is not limited by their ability to germinate. Outgrowth of spores at different incubation temperatures was similar for germinated and ungerminated spores. Accordingly it is outgrowth rather than germination which is sensitive to temperature.
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The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.
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Summary. Maximum colony counts of unheated and heated spores of Bacillus stearothermophilus were obtained after incubation at 50‐65°C and 45‐50°C, respectively. the composition of the plating medium was found to have a marked effect upon recovery of unheated and heated spores. the effects of diluent and incubation time on recovery have also been investigated.
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The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202-2209, 2016, http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed.Clostridium difficile spores are recognized as the morphotype of persistence and transmission of C. difficile infections. Spores of C. difficile are intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires a detailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidence that two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, are formed in the same C. difficile sporulating culture or during biofilm development.
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Effect of 60Co gamma-rays on inactivation of Bac. subtilis spores was studied. In the course of investigation, the dose of gamma-rays was divided in to two parts with an interval of 4 or 24 hours between irradiation. Fractional irradiation was found to be more effective for decontamination of Bac. subtilis spores than single irradiation.
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