Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence.

Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined.

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.

Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium-hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water.

The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure's success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores' spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone's 1600-1700 cm-1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.

Bacterial spores are extremely resistant life-forms that play an important role in food spoilage and foodborne disease. The return of spores to a vegetative cell state is a three-step process, these being activation, germination, and emergence. High-pressure (HP) processing is known to induce germination in part of the spore population and even to inactivate a high number of Bacillus spores when combined with other mild treatments such as the addition of nisin. The aim of the present work was to investigate the mechanisms involved in the sensitization of spores to nisin following HP treatment at ambient temperature or with moderate heating leading to a heterogeneous spore response. Bacillus subtilis spores were subjected to HP treatment at 500 MPa at 20 and 50°C. The physiological state of different subpopulations was characterized. Then Fourier transform infrared (FTIR) microspectroscopy coupled to a synchrotron infrared source was used to explore the heterogeneity of the biochemical signatures of the spores after the same HP treatments. Our results confirm that HP at 50°C induces the germination of a large proportion of the spore population. HP treatment at 20°C generated a subpopulation of ungerminated spores reversibly sensitized to the presence of nisin in their growth medium. Regarding infrared spectra of individual spores, spores treated by HP at 50°C and germinated spores had similar spectral signatures involving the same structural properties. However, after HP was performed at 20°C, two groups of spores were distinguished; one of these groups was clearly identified as germinated spores. The second group displayed a unique spectral signature, with shifts in the spectral bands corresponding to changes in membrane fluidity. Besides, spores spectra in the amide region could be divided into several groups close to spectral properties of dormant, germinated, or inactivated spores. The part of the spectra corresponding to α-helix and β-sheet-structures contribute mainly to the spectral variation between spores treated by HP at 20°C and other populations. These changes in the lipid and amide regions could be the signature of reversible changes linked to spore activation.

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix.

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

Clubroot, caused by Plasmodiophora brassicae, is a severe soil-borne disease that restricts the production of cruciferous crops worldwide. A better understanding of biotic and abiotic factors regulating germination of P. brassicae resting spores in the soil is significant for developing novel control methods. Previous studies reported that root exudates can trigger P. brassicae resting spore germination, thus enabling a targeted attack of P. brassicae on host plant roots. However, we found that native root exudates collected under sterile conditions from host or non-host plants cannot stimulate the germination of sterile spores, indicating that root exudates may not be direct stimulation factors. Instead, our studies demonstrate that soil bacteria are essential for triggering germination. Through 16s rRNA amplicon sequencing analysis, we found that certain carbon sources and nitrate can reshape the initial microbial community to an inducing community leading to the germination of P. brassicae resting spores. The stimulating communities significantly differed in composition and abundance of bacterial taxa compared to the non-stimulating ones. Several enriched bacterial taxa in stimulating community were significantly correlated with spore germination rates and may be involved as stimulation factors. Based on our findings, a multi-factorial 'pathobiome' model comprising abiotic and biotic factors is proposed to represent the putative plant-microbiome-pathogen interactions associated with breaking spore dormancy of P. brassicae in soil. This study presents novel views on P. brassicae pathogenicity and lays the foundation for novel sustainable control strategies of clubroot.

Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.

The occurrence of bacterial pathogens in the food chain has caused a severe impact on public health and welfare in both developing and developed countries. Moreover, the existence of antimicrobial-tolerant persisting morphotypes of these pathogens including both persister-cells as well as bacterial spores contributes to difficulty in elimination and in recurrent infection. Therefore, comprehensive understanding of the behavior of these persisting bacterial forms in their environmental niche and upon infection of humans is necessary. Since traditional antimicrobials fail to kill persisters and spores due to their (extremely) low metabolic activities, antimicrobial peptides (AMPs) have been intensively investigated as one of the most promising strategies against these persisting bacterial forms, showing high efficacy of inactivation. In addition, AMP-based foodborne pathogen detection and prevention of infection has made significant progress. This review focuses on recent research on common bacterial pathogens in the food chain, their persisting morphotypes, and on AMP-based solutions. Challenges in research and application of AMPs are described.

Au/CuS core/shell nanoparticles (NPs) were designed as a new type of transducer agent for photothermal therapy (PTT), with attractive features of easy preparation, low cost and small size for targeting. This paper studied for the first time the intrinsic antimicrobial activity of Au/CuS NPs to B. anthracis spores and cells in addition to its PTT effect.

For the first time, this study addresses the intensification of supercritical carbon dioxide (SC-CO2) treatments using high-power ultrasound (HPU) for the inactivation of fungal (Aspergillus niger) and bacterial (Clostridium butyricum) spores in oil-in-water emulsions. The inactivation kinetics were analyzed at different pressures (100, 350 and 550 bar) and temperatures (50, 60, 70, 80, 85 °C), depending on the microorganism, and compared to the conventional thermal treatment. The inactivation kinetics were satisfactorily described using the Weibull model. Experimental results showed that SC-CO2 enhanced the inactivation level of both spores when compared to thermal treatments. Bacterial spores (C.butyricum) were found to be more resistant to SC-CO2 + HPU, than fungal (A.niger) ones, as also observed in the thermal and SC-CO2 treatments. The application of HPU intensified the SC-CO2 inactivation of C.butyricum spores, e.g. shortening the total inactivation time from 10 to 3 min at 85 °C. However, HPU did not affect the SC-CO2 inactivation of A.niger spores. The study into the effect of a combined SC-CO2 + HPU treatment has to be necessarily extended to other fungal and bacterial spores, and future studies should elucidate the impact of HPU application on the emulsion's stability.

Clostridium sordellii is an often-lethal bacterium causing human and animal disease. Crucial to the infectious cycle of C. sordellii is its ability to produce spores, which can germinate into toxin-producing vegetative bacteria under favorable conditions. However, structural details of the C. sordellii spore are lacking. Here, we used a range of electron microscopy techniques together with superresolution optical microscopy to characterize the C. sordellii spore morphology with an emphasis on the exosporium. The C. sordellii spore is made up of multiple layers with the exosporium presenting as a smooth balloon-like structure that is open at the spore poles. Focusing on the outer spore layers, we compared the morphologies of C. sordellii spores derived from different strains and determined that there is some variation between the spores, most notably with spores of some strains having tubular appendages. Since Clostridium difficile is a close relative of C. sordellii, their spores were compared by electron microscopy and their exosporia were found to be distinctly different from each other. This study therefore provides new structural details of the C. sordellii spore and offers insights into the physical structure of the exosporium across clostridial species. IMPORTANCEClostridium sordellii is a significant pathogen with mortality rates approaching 100%. It is the bacterial spore that is critical in initiating infection and disease. An understanding of spore structures as well as spore morphology across a range of strains may lead to a better understanding of C. sordellii infection and disease. However, the structural characteristics of the C. sordellii spores are limited. In this work, we have addressed this lack of detail and characterized the C. sordellii spore morphology. The use of traditional and advanced microscopy techniques has provided detailed new observations of C. sordellii spore structural features, which serve as a reference point for structural studies of spores from other bacterial species.

Resistant bacterial spores are a major concern in industrial decontamination processes. An approach known as pressure-mediated germination-inactivation strategy aims to artificially germinate spores by isostatic pressure to mitigate their resistance to inactivation processes. The successful implementation of such a germination-inactivation strategy relies on the germination of all spores. However, germination is heterogeneous, with some "superdormant" spores germinating extremely slowly or not at all. The present study investigated potential underlying reasons for moderate high-pressure (150 MPa; 37°C) superdormancy of Bacillus subtilis spores. The water and dipicolinic acid content of superdormant spores was compared with that of the initial dormant spore population. The results suggest that water and dipicolinic acid content are not major drivers of moderate high-pressure superdormancy. A proteomic analysis was used to identify proteins that were quantified at significantly different levels in superdormant spores. Subsequent validation of the germination capacity of deletion mutants revealed that the presence of protein YhcN is required for efficient moderate high-pressure germination and that proteins MinC, cse60, and SspK may also play a role, albeit a minor one. IMPORTANCE Spore-forming bacteria are ubiquitous in nature and, as a consequence, inevitably enter the food chain or other processing environments. Their presence can lead to significant spoilage or safety-related issues. Intensive treatment is usually required to inactivate them; however, this treatment harms important product quality attributes. A pressure-mediated germination-inactivation approach can balance the need for effective spore inactivation and retention of sensitive ingredients. However, superdormant spores are the bottleneck preventing the successful and safe implementation of such a strategy. An in-depth understanding of moderate high-pressure germination and the underlying causes of superdormancy is necessary to advance the development of mild high pressure-based spore control technologies. The approach used in this work allowed the identification of proteins that have not yet been associated with reduced germination at moderate high pressure. This research paves the way for further studies on the germination and superdormancy mechanisms in spores, assisting the development of mild spore inactivation strategies.

Bacterial spores are one of the most resilient life forms on earth and are involved in many human diseases, such as infectious diarrhea, fatal paralytic illnesses and respiratory infections. Here, we investigated the mechanisms involved in the death of Bacillus pumilus spores after exposure to electric arcs in water. Cutting-edge microscopies at the nanoscale did not reveal any structural disorganization of spores exposed to electric arcs. This result suggested the absence of physical destruction by a propagating shock wave or an exposure to an electric field. However, Pulsed-Field Gel Electrophoresis (PFGE) revealed genomic DNA damage induced by UV radiation and Reactive Oxygen Species (ROS). UV induced single-strand DNA breaks and thymine dimers while ROS were mainly involved in base excision. Our findings revealed a correlation between DNA damage and the treatment of spores with electrical discharges.

Milk is a source of essential nutrients for infants and adults, and its production has increased worldwide over the past years. Despite developments in the dairy industry, premature spoilage of milk due to the contamination by Bacillus cereus continues to be a problem and causes considerable economic losses. B. cereus is ubiquitously present in nature and can contaminate milk through a variety of means from the farm to the processing plant, during transport or distribution. There is a need to detect and quantify spores directly in food samples, because B. cereus might be present in food only in the sporulated form. Traditional microbiological detection methods used in dairy industries to detect spores show limits of time (they are time consuming), efficiency and sensitivity. The low level of B. cereus spores in milk implies that highly sensitive detection methods should be applied for dairy products screening for spore contamination. This review describes the advantages and disadvantages of classical microbiological methods used to detect B. cereus spores in milk and milk products, related to novel methods based on molecular biology, biosensors and nanotechnology.

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

Microsporidia are a group of widespread eukaryotic spore-forming intracellular parasites of great economic and scientific importance. Since microsporidia cannot be cultured outside of a host cell, the search for new antimicrosporidian drugs requires an effective antiseptic to sterilize microsporidian spores to infect cell lines. Here, we show that a new polyhexamethylene guanidine derivative M250, which is active against fungi and bacteria at a concentration of 0.5-1 mg/L, is more than 1000 times less effective against spores of the microsporidium Nosema bombycis, a highly virulent pathogen of the silkworm Bombyx mori (LC50 is 0.173%). Treatment of N. bombycis spores that were isolated non-sterilely from silkworm caterpillars with 0.1% M250 solution does not reduce the rate of spore polar tube extrusion. However, it completely prevents contamination of the Sf-900 III cell culture medium by microorganisms in the presence of antibiotics. The addition of untreated spores to the medium results in contamination, whether antibiotics are present or not. Since 0.1% M250 does not affect spore discharging, this compound may be promising for preventing bacterial and fungal contamination of microsporidia-infected cell cultures.