Endospore Production Research Articles

Fishmeal-Based Media Supports Growth and Endospore Production of Locally-Isolated Lysinnibacillus sphaericus and Induces its Toxicity to 3rd Instar Aedes aegypti Larvae in Laboratory Conditions

The aim of this study was to determine whether fishmeal-based media could be used to grow L. sphaericus and induce its toxicity against Aedes aegypti larvae. Three concentrations (10, 20 and 30%) of fishmeal-based media were used to grow L. sphaericus isolate Bs2-1-2. Cell growth and endospore production were observed every 12 hours for 72 hours. The lethal concentration was measured every 24 hours for 72 hours of fermentation. The highest cell concentration was found in L. sphaericus grown on the media with 30% fishmeal concentration (3.03x1008 cells/mL), followed by 20% concentration (2.43x1008 cells/mL) and the lowest at 10% concentration (2.20x1008 cells/mL). At the end of fermentation, the highest concentration was found in L. sphaericus grown on 30% fishmeal-based media (1.51x1008 cells/mL), followed by 20% media (6.95x1007 cells/mL) and 10% media (3.21x1007 cells/mL). After 72-hour incubation, the highest endospore concentration was achieved by L. sphaericus grown on 20% (2.51x1008 cells/mL) and 10% (2.19x1008 cells/mL) fishmeal-based media. Initial larval toxicity of L. sphaericus showed the highest mortality on 20 and 30% fishmeal-based media (both reaching 53.33%), while 10% fishmeal-based media gave only 26.67% larval mortality. The LC50 value at 72 hours was achieved by L. sphaericus cultured on 30% fishmeal-based media (2.47 x 1008 cells/mL), followed by 20% concentration (4.82 x 1008 cells/mL) and 10% concentration (9.01 x 1009 cells/mL). The conclusion of this study was all concentrations of fishmeal-based media could support cell growth, endospore production and larval toxicity induction of L. sphaericus.

Differential responses of Meloidogyne spp. to Pasteuria isolates over crop cycles

In the present study examining the interaction between Meloidogyne spp. and Pasteuria isolates over three crop cycles, significant differences were observed in eggmass production, root galling, plant root weight, endospore production, and parasitism rates. Higher eggmass production was noted in the Meloidogyne blend treatment, whereas M. incognita showed a marked decline after the third cycle. Root galling varied significantly with nematode populations and Pasteuria isolates, showing notable interaction effects (P0.01). The Meloidogyne blend treatment exhibited the highest gall rating, while M. javanica and M. incognita treatments with Pp3 and PpEcu isolates resulted in lower galling. Fresh root weights of tomato plants differed significantly in the presence of Meloidogyne spp. and Pasteuria isolates, with significant interactions observed (P0.05). Greater root weights were recorded in M. javanica and M. incognita treatments with Pp3 and PpEcu isolates. The production of Pasteuria endospores in Meloidogyne females also showed significant variability across crop cycles (P0.01), with M. incognita and PpEcu treatments yielding the highest endospore numbers. Moreover, parasitism of Meloidogyne females by Pasteuria was significantly influenced by nematode population and isolate presence (P0.01). Higher numbers of infected M. incognita females were found in PpEcu treatments. Final female populations of Meloidogyne spp. varied significantly under Pasteuria influence, with the Meloidogyne blend treatment showing the highest population and M. incognita under PpEcu the lowest. The results highlight the complex interactions between nematodes and the bacteria, emphasizing the potential of Pasteuria isolates to influence various aspects of Meloidogyne spp. development.

Endospore production of Bacillus spp. for industrial use.

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium.

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%-90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.

Exploitation of indigenous fluorescent pseudomonads against stem and pod rot of groundnut caused by <i>Sclerotium rolfsii</i>

Stem and pod rot of groundnut is an economically important soil borne disease caused by Sclerotium rolfsii Sacc. The present study aimed to evaluate the indigenous bacterial bio control agent Pseudomonas fluorescens strains having multiple traits related to bio control and plant growth promoting activity. Healthy rhizospheric soil from groundnut field was collected from different North Karnataka districts. Nineteen strains of P. fluorescens were isolated by serial dilution technique on King’s B medium. The antagonistic potential of nineteen strains was tested against S. rolfsii by using dual culture technique. The per cent inhibition was highly variable among the nineteen strains of P. fluorescens ranging from 2.59 to 75.18 per cent. Maximum mycelial inhibition of S. rolfsii was recorded by the strain PF-2 (75.18%) followed by PF-3 (72.96%), PF-6 (69.62%) and least inhibition was recorded by PF-13 (2.59%). Five superior strains of P. fluorescens showed fluorescens under UV light, yellowish green pigmentation, rod shaped cells under microscope. Five superior strains were subjected for various biochemical tests and all the isolates were positive for biochemical characterization such as Gram staining, endospore production, catalase, starch hydrolysis, urease test, casein hydrolysis and gelatin liquefy action and negative for KOH and in dole test. Further, these strains were subjected for plant growth promoting traits such as HCN production, IAA production, siderophore production and volatile compounds production. Among these, the strainsviz., PF-2 and PF-3 were scored as strong with respect to antagonism and growth promotion. The strainsPF-6, PF-7 and PF-10 were scored as moderate with light brown colour. Among the tested strains of P. fluorescens, the isolates PF-2 and PF-3 were recorded higher production of siderophore, isolates PF-6, PF-7 and PF-10 showed moderate production of siderophore. The strain PF-2 of P. fluorescens showed highest per cent of mycelial inhibition of S. rolfsii indicating higher production of volatile compounds, followed by PF-3 and PF-6. Whereas, the least mycelial inhibition was recorded by the isolate PF-10 indicating less production of volatile compounds.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Endospore Production in a Regulated Bioprocess Using an Optimized Chemically Defined Medium

Endospore Production in a Regulated Bioprocess Using an Optimized Chemically Defined Medium

EVALUATION OF EFFICACY OF PASTEURIA PENETRANS ALONE AND IN COMBINATION WITH VERTICILLIUM CHLAMYDOSPORIUM AGAINST MELOIDOGYNE JAVANICA

The potential control of Meloidogyne javanica using Pasteuria penetrans (Pp) alone and in combination with Verticillium chlamydosporium (Vc) was tested in earthen pots following a cropping sequence "tomato-tomato-tomato” over three crop cycles. After the final harvesting, analysis of variance showed significant effect of treatments (P0.01) regarding number of eggmasses, galls and nematode female populations. Similarly, significant effect of treatments (P0.01) was also recorded in case of infected nematode females with Pasteuria and number of eggs/eggmass while no significant effect (P 0.0 5) was observed in case of endospore production. Higher numbers of eggmasses (360) and root galling (6.2) was observed where biocontrol agents were absent (control). The treatments showed 46.58, 58.85 and 33.13 percent reduction in number of galls, eggmasses and nematodes in Pp alone and 43.34, 55.21 and 30.09 percent reductions in Vc+Pp treatments respectively. Numbers of females infected with the endospores of P. penetrans were recorded higher in Pp-3 alone treatment (13.2) followed by Vc+Pp combined treatment (13.0) and maize rotated treatment (10.4) respectively. Significantly lesser number of eggmasses, galls and nematodes were recorded in pots where tomato was rotated with maize (treatment 3) compared with control. Thus rotation prevented the buildup of nematode population and resulted in a 72% decrease in numbers of eggmasses, 38% in root galling and 46% regarding female populations over the control after the final harvest. Maximum colony forming units of V. chlamydosporium per gram of soil were recorded after its addition to the soil. The fungus established in the soil during the first crop and soil colonization of the fungus was also observed after final crop.

Formulación de un medio de cultivo para la producción de δ-endotoxinas de Bacillus thuringiensis

Objetivo: Formular un medio de cultivo para la producción de proteínas parasporales de Bt.Método: Se evaluaron 5 medios de cultivo, contrastando su formulación con la producción de biomasa. Se extrajo la proteína total de cada uno de los cultivos y se cuantificó por espectrofotometría.Resultados: Se encontró que el caldo glucosado modificado con proporciones 2,4:1 de fuentes de nitrógeno orgánicas (extracto de levadura) e inorgánicas ((NH4)2SO4) mejoró hasta en 70 veces la producción de proteína con concentraciones entre 19,38 y 154,41 mg/mL.Conclusión: Este estudio contribuye a la afirmación de que la proporción entre Nitrógeno Orgánico e Inorgánico tiene efectos importantes sobre la fase III del crecimiento de Bt, contrastando con la producción de endosporas y en consecuencia proteínas totales en las cuales e incluyen el aumento de las δ-endotoxinas.

Does soil warming affect the interaction between Pasteuria penetrans and Meloidogyne javanica in tomato plants?

A system to grow tomato plants infected by Meloidogyne javanica under constant temperatures of 18, 21, 24, 27 and 30 °C was developed and used to assess how temperature and the application of the biological control bacterium Pasteuria penetrans affected plant growth, the nematode population and endospore production. Each plant was inoculated with 300 second‐stage juveniles (J2) with four or five spores of P. penetrans attached to their cuticles or with 300 nematodes without P. penetrans. Increasing soil temperature increased tomato growth, the number of endospores per female, and the number of galls of M. javanica at the end of 38 days. Increasing temperatures up to 27 °C also increased the number of egg masses produced by M. javanica. Presence of P. penetrans reduced the numbers of galls and egg masses at all temperatures by up to 52.2% and 61.4% at 27 and 30 °C, respectively. Pasteuria penetrans reduced the M. javanica population even at soil temperatures of 18 and 21 °C. However, temperatures of 27 and 30 °C enhanced nematode control and the production of P. penetrans endospores is faster. The system developed in this work is simple and efficient for growing plants under constant temperatures and can be used for different purposes.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature.

Biocontrol products based on Bacillus amyloliquefaciens CPA-8 using fluid-bed spray-drying process to control postharvest brown rot in stone fruit

Two products based on the biocontrol agent Bacillus amyloliquefaciens CPA-8 have been developed as an effective alternative to chemical applications to control postharvest brown rot in stone fruit. As part of the production and formulation processes, the effects of three different nitrogen sources on growth media and the effects of different carrier materials and protectants on fluid-bed spray-drying were studied. CPA-8 populations achieved 109 CFU/mL after 72 h of culture. However, the protein PROSTAR 510A at 20 g/L provided better growth curves compared to the boiled extract from Defatted Soy Flour and protein PROSTAR 510A at 10 g/L. Furthermore, culture ages of 72 h were needed to obtain high endospore production and therefore, suitable heat tolerance of CPA-8. The use of the protectants 20% sucrose plus 10% skimmed milk resulted in the best formulations when either carrier material, maltodextrin or potato starch, was used. These two products were then selected for assays of shelf life and efficacy. CAP-8 viability was unchanged after 15 months of storage at 4 and 22 °C, maintaining concentrations between 7.8 × 109 and 1.2 × 1010 CFU/g. Finally, the efficacy of the CPA-8 products against Monilinia spp. was confirmed (>44.4% disease reduction) on peaches, nectarines, flat peaches, cherries, apricots and plums.

Alkaliphilic Bacillus species show potential application in concrete crack repair by virtue of rapid spore production and germination then extracellular calcite formation.

Characterization of alkaliphilic Bacillus species for spore production and germination and calcite formation as a prelude to investigate their potential in microcrack remediation in concrete. Conditions, extent and timing of endospore production was determined by dark-field light microscopy; germination induction and kinetics were assessed by combining reduction in optical density with formation of refractile bodies by phase-contrast microscopy. Bacillus pseudofirmus was selected from several species as the most suitable isolate. Levels and timing of calcium carbonate precipitated invitro by B. pseudofirmus were evaluated by atomic absorption spectroscopy and structural identity confirmed as calcite and aragonite by Raman spectroscopy and FTIR. The isolate produced copious spores that germinated rapidly in the presence of germinants l-alanine, inosine and NaCl. Bacterial cells produced CaCO3 crystals in microcracks and the resulting occlusion markedly restricted water ingress. By virtue of rapid spore production and germination, calcium carbonate formation invitro and insitu, leading to sealing of microcracks, B. pseudofirmus shows clear potential for remediation of concrete on a commercial scale. Microbial sealing of microcracks should become a practicable and sustainable means of increasing concrete durability.

Evaluation of Bacillus strains isolated from solanaceous phylloplane for biocontrol of Alternaria early blight of tomato

Bacillus biological control agents are powerful alternatives to chemical fungicides for suppressing plant diseases caused by fungi. In this work, 93 strains of spore-forming bacteria isolated from solanaceous phylloplane, were screened for in vitro antibiotic activity against Alternaria alternata, causal agent of tomato early blight. The twenty most active strains were characterized for their morphological and physiological traits (Gram reaction, production of endospores, antifungal volatile compounds and siderophores) and by M13-PCR DNA fingerprinting. Planta bioassays performed with four selected strains, were capable to decrease severity of Alternaria disease on tomato. These antagonistic bacteria were identified by 16S-rRNA partial gene sequencing, and results assigned them to strains related to Bacillus species. The evident inhibition zone observed in dual culture plates, suggested an antibiosis-like mechanism. While, API-ZYM enzymatic profiles indicated that strains could be potential ecological competitors. Consistently, light microscopy revealed the occurrence of Bacillus-induced malformations in the fungus’s vegetative structures, probably caused by secreted compounds.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i> subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt); and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h; while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores; malachite green-saffranin stained both δ-crystals and endospores; and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

Dual production of amylase and δ-endotoxin by Bacillus thuringiensis subsp. kurstaki during biphasic fermentation

This study examined the efficacy of a Bacillus thuringiensis (Bt) strain in producing amylase (EC 3.2.1.1) as a by-product without affecting its unique ability for producing δ-endotoxin, thus to establish a cultivation strategy for the dual production and recovery of both δ-endotoxin and amylase from the fermented medium with an industrial perspective. LB medium was individually supplemented (5 to 100%, wt/vol) with flour from six naturally available starchy stored foods (banana, Bengal gram, jack seed, potato, taro or tapioca); after initial fermentation (12 h), the supernatant in the medium obtained by centrifugation (1000 g, 10 min) was used for harvesting amylase and the resultant pellet was further incubated aseptically for the production of endospores and δ-endotoxin by solid-state fermentation. Maximum crude amylase activity (867 U/gram dry substrate, 12 h) was observed in potato flour-supplemented medium (10% wt/vol, 12 h), while the activity in LB control was only 4.36 U/mL. SDS-PAGE profile of the crude (supernatant), as well as partially purified (40–60% (NH4)2SO4 fractionation) amylase showed that its apparent molecular mass was 51 kDa, which was further confirmed by native PAGE. The harvest of industrially significant extracellular amylase (probably α-amylase) produced as a byproduct during early growth phase would boost the economics of the Bt-based bio-industry engaged in δ-endotoxin production.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12h submerged fermentation (SmF) at 37°C and 125rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10min); the resultant pellet subsequently incubated statically (37°C) for the production of B.thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48h (as against 72h from M1), thus the gestation period of M2 was reduced by 24h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Endospore production allows using spray-drying as a possible formulation system of the biocontrol agent Bacillus subtilis CPA-8

The role of endospore production by Bacillus subtilis CPA-8 on survival during spray-drying was investigated by comparison with a non-spore-forming biocontrol agent Pantoea agglomerans CPA-2. Endospore formation promoted heat resistance in CPA-8 depending on growth time (72 h cultures were more resistant than 24 h ones). The survival of CPA-8 and CPA-2 after spray-drying was determined after being grown in optimised media for 24 and 72 h. Spray-dried 72 h CPA-8 had the best survival (32%), while CPA-2 viability was less than 2%. CPA-8 survival directly related with its ability to produce endospores. Spray-dried CPA-8 reduced Monilinia fructicola conidia germination similarly to fresh cells, demonstrating that spray-drying did not adversely affect biocontrol efficacy. Endospore production thus improves CPA-8 resistance to spray-drying. These results can provide a reliable basis for optimising of the spray-drying formulation process for CPA-8 and other microorganisms.

The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type

Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these ‘epidemic’ types generally sporulate more prolifically than ‘non-epidemic’ strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.

Clostridium difficile spore germination: an update

Endospore production is vital for the spread of Clostridium difficile infection. However, in order to cause disease, these spores must germinate and return to vegetative cell growth. Knowledge of germination is therefore important, with potential practical implications for routine cleaning, outbreak management and potentially in the design of new therapeutics. Germination has been well studied in Bacillus, but until recently there had been few studies reported in C. difficile. The role of bile salts as germinants for C. difficile spores has now been described in some detail, which improves our understanding of how C. difficile spores interact with their environment following ingestion by susceptible individuals. Furthermore, with the aid of novel genetic tools, it has now become possible to study the germination of C. difficile spores using both a forward and reverse genetics approach. Significant progress is beginning to be made in the study of this important aspect of C. difficile disease.