Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Response of Clostridium perfringens Spores and Vegetative Cells to Temperature Variation

The vegetative cells and spores of four strains of Clostridium perfringens were examined to determine the effect of lowered and elevated temperatures. Spores were produced by following the method of Ellner, and vegetative cells were obtained from thioglycolate cultures. After exposure to freezing or refrigeration temperatures (-17.7 and 7.1 C, respectively), only small numbers of the vegetative cells were recovered. After similar treatment, 16 to 58% of the spores were recovered. Essentially no vegetative cells and few spores survived holding at 80 C for 10 min. Although all strains were isolated from food, only one strain of the four studied had its origin in a food-poisoning outbreak, and it had been carried on laboratory media for approximately 10 years.     

The influence of storage temperature on spore viability of Mattesia trogodermae (Protozoa: Neogregarinida)

The viability of Mattesia trogodermae spores stored at different temperatures was assessed by the percentage infection induced in 30-day-old Trogoderma glabrum larvae. Exposure to 73°C and higher temperatures for 30 min was lethal to the spores. Spores stored at ?19°C survived better than those stored at 26.7°, 3.5°, or ?30°C.     

Heat resistance of Paecilomyces variotii in sauce and juice

It has been demonstrated that some anamorphic fungi ( Paecilomyces variotii, Fusarium sp) could cause spoilage of food products after pasteurisation. Four food-borne and one clinical isolate of P. variotii were cultivated on one solid medium and three liquid media. Their survival after heating at 80–100˚C for 0.25–15 min in sterile distilled water and curry sauce or fruit juice was investigated. Heat resistance was determined by the thermal death method in a thermostatically-controlled oil bath. The most resistant spores of P. variotii from curry sauce cultivated on malt extract agar survived 100˚C for 0.5 min in sauce; cultivated in curry sauce survived 100˚C for 15 min in water and cultivated in malt broth survived 100˚C for 5 min in water and sauce. The most resistant spores of P. variotii from juice cultivated on malt extract agar were able to survive 100˚C for 15 min in water; cultivated in juice survived 100˚C for 0.5 min in juice and suspensions from cultivation in malt broth survived 100˚C for 1.5 min in juice. Spores of the clinical strain of P. variotiifrom malt extract agar survived 95˚C for 0.33 min in water, and orange juice cultures survived 96˚C for 10 min in orange juice. It was thus found that P. variotii strains cultivated in food were better adapted to heat stress, suggesting that fungal biomass suspensions were able to survive the higher temperatures for longer time intervals than spore suspensions. Journal of Industrial Microbiology & Biotechnology (2000) 24, 227–230. Received 02 June 1999/ Accepted in revised form 05 December 1999     

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.     

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self-administration. We have previously shown that street heroin induces apoptosis in neuronal-like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6-monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly-ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non-selective caspase inhibitor z-VAD-fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT-2 rho(0) cells. Nonetheless, in street heroin-treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl-2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.     

Effect of pH on Growth and Survival of Three Avian and One Saprophytic Mycoplasma Species

The ability of Mycoplasma meleagridis, M. laidlawii, an unnamed, nonpathogenic avian species, and three isolates of M. gallisepticum to grow and survive in liquid media of various pH values was investigated. All species grew over a pH range that was generally about two units. The most significant finding was that cultures initiated in media within a pH range of approximately 8.5 to 8.9 remained viable for 45 to 60 days at 37 C, whereas cultures initiated at lower values survived for shorter periods.     

Spore coat architecture of Clostridium novyi NT spores

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.     

Interactions between Clostridium perfringens spores and Raw 264.7 macrophages

Clostridium perfringens is the causative agent of a variety of histotoxic infections in humans and animals. Studies on the early events of C. perfringens infections have been largely focused on the interactions between their vegetative cells and macrophages. Consequently, in the current study we have examined the interactions between C. perfringens spores and Raw 264.7 macrophages. Raw 264.7 cells were able to interact and phagocytose Clostridium perfringens spores of a food poisoning isolate, strain SM101, and a non-food borne isolate, strain F4969, albeit to different extents. Phagocytosis and to a lesser extent, association, of C. perfringens spores by Raw 2647 macrophages was completely inhibited in presence of cytochalasin D. Complement increased association and phagocytosis of C. perfringens spores by Raw 264.7 macrophages. Survival of C. perfringens spores during macrophage infection seems to depend on the ability of spore germination during infection as: (i) F4969 spores germinated during infection with Raw 264.7 macrophages and subsequently killed by macrophages; and (ii) SM101 spores remained dormant inside Raw 264.7 macrophages and thus survived up to 24 h of infection. The in vitro spore-resistance factors, α/β-type SASP, SpmA/B proteins and spore's core water content, seems to play no role in mediating SM101 spore-resistance to macrophages. Collectively, these results might well have implications in understanding the initial stages of infections by C. perfringens spores.     

Further comparison of temperature effects on growth and survival of Clostridium perfringens type A isolates carrying a chromosomal or plasmid-borne enterotoxin gene

Clostridium perfringens type A isolates can carry the enterotoxin gene (cpe) on either their chromosome or a plasmid, but food poisoning isolates usually have a chromosomal cpe gene. This linkage between chromosomal cpe isolates and food poisoning has previously been attributed, at least in part, to better high-temperature survival of chromosomal cpe isolates than of plasmid cpe isolates. In the current study we assessed whether vegetative cells and spores of chromosomal cpe isolates also survive better than vegetative cells and spores of plasmid cpe isolates survive when the vegetative cells and spores are subjected to low temperatures. Vegetative cells of chromosomal cpe isolates exhibited about eightfold-higher decimal reduction values (D values) at 4 degrees C and threefold-higher D values at -20 degrees C than vegetative cells of plasmid cpe isolates exhibited. After 6 months of incubation at 4 degrees C and -20 degrees C, the average log reductions in viability for spores of plasmid cpe isolates were about fourfold and about threefold greater, respectively, than the average log reductions in viability for spores from chromosomal cpe isolates. C. perfringens type A isolates carrying a chromosomal cpe gene also grew significantly faster than plasmid cpe isolates grew at 25 degrees C, 37 degrees C, or 43 degrees C. In addition, chromosomal cpe isolates grew at higher maximum and lower minimum temperatures than plasmid cpe isolates grew. Collectively, these results suggest that chromosomal cpe isolates are commonly involved in food poisoning because of their greater resistance to low (as well as high) temperatures for both survival and growth. They also indicate the importance of proper low-temperature storage conditions, as well as heating, for prevention of C. perfringens type A food poisoning.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

<Emphasis Type="Italic">Polytrichum commune</Emphasis> spores nucleate ice and associated microorganisms increase the temperature of ice nucleation activity onset

Moss spores disperse via wind and have been found previously in precipitation and air samples. Their presence in the atmosphere led to this study’s examining the potential of moss spores to contribute to ice nucleation, a process necessary for ice formation in clouds prior to precipitation. Ice nucleation assays were conducted using Polytrichum commune spores that were either associated with natural assemblages of microbes or extracted aseptically from capsules and subsequently confirmed to be free of culturable microbes. Liquid suspensions of capsule spores and non-sterile spores nucleated ice at temperatures as high as ?12 and ?7 °C, respectively. When capsule and non-sterile spores were heated at 95 °C for 10 min, which killed all culturable microbes on non-sterile spores, both nucleated ice from ?10 to ?13 °C. An additional non-sterile spore sample collected from partially opened capsules in a forested ecosystem (ID, USA) nucleated ice at temperatures as high as ?7 °C, similar to non-sterile P. commune spores. This is the first set of results to indicate that P. commune spores themselves are capable of nucleating ice at temperatures higher than many abiological particles such as mineral dust (≤?15 °C) and that natural assemblages of microbes can increase their ice nucleation efficiency. Future studies aimed at determining the abundance of moss spores in the atmosphere and the identity of ice-nucleating microbes associated with them will provide further insights into the ability of moss spores to impact precipitation patterns.     

Survival of the mite Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae) under starvation

The ability of a natural enemy to tolerate starvation increases its chances to survive in the absence of food, what is an important factor for its success in storage grain environment. The objective of the present work was to assess the survival of Acarophenax lacunatus (Cross & Krantz) in the absence of food. The experiment used individualized physogastric females of A. lacunatus placed in petri dishes (5 cm diameter) and maintained at 20, 25, 28, 30 and 32 degrees C, 50+/-5 % R.H. and 24h scotophase. The number of live mites was recorded every 6h thus assessing the progeny survival without food at different temperatures. The mites died within 60h at the temperatures 30 degrees C and 32 degrees C, while they survived for up to 108h at 20, 25 and 28 degrees C. The mean lethal time for death was 58.6h for the lowest temperatures and 39.3h for the highest temperatures. Thus, A. lacunatus subjected to starvation lived longer under lower temperatures, what is probably due to its lower metabolism. In contrast, the mites survived for about 90h at 28 degrees C, temperature commonly observed in tropical and subtropical climates, what may favor their use as control agents of stored product insects in these regions.     

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.     

Temperature-pH effect upon germination of bacterial spores.

Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed.     

Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C

The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.     

Agni's fungi: heat-resistant spores from the Western Ghats, southern India

This study concerns the thermotolerance of spores of mesophilic fungi isolated from a tropical semi-arid habitat subject to dry season fire in the Western Ghats, southern India. Among 25 species of Ascomycota isolated from leaf litter, nine were able to grow after incubation in a drying oven for 2h at 100°C; the spores of two of these species survived 2h incubation at 110°C, and one survived exposure to 115°C for 2h. The range of thermotolerance among mesophilic fungi isolated from the leaf litter was surprising: filamentous fungi from other habitats, including species that colonize scorched vegetation after fires and thermophilic forms occurring in self-heating plant composts, cannot survive even brief exposure to such high temperatures. It is possible that the exceptional heat resistance of the Indian fungi is related to adaptations to surviving fires. Genetic analysis of the physiological mechanisms of heat resistance in these fungi offers prospects for future biotechnological innovations. The discovery of extreme thermotolerance among common saprotrophs shows that this physiological trait may be more widespread than recognized previously, adding to concern about the evolution of opportunistic pathogens on a warmer planet. The fungi in this study are among the most heat-resistant eukaryotes on record and are referred to here as 'Agni's Fungi', after the Hindu God of Fire.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.