Lethal toxin is a critical determinant of rapid mortality in rodent models of Clostridium sordellii endometritis

The toxigenic anaerobe Clostridium sordellii is an uncommon but highly lethal cause of human infection and toxic shock syndrome, yet few studies have addressed its pathogenetic mechanisms. To better characterize the microbial determinants of rapid death from infection both in vitro and in vivo studies were performed to compare a clinical strain of C. sordellii (DA-108), isolated from a patient who survived a disseminated infection unaccompanied by toxic shock syndrome, to a virulent reference strain (ATCC9714). Rodent models of endometrial and peritoneal infection with C. sordellii ATCC9714 were rapidly lethal, while infections with DA-108 were not. Extensive genetic and functional comparisons of virulence factor and toxin expression between these two bacterial strains yielded many similarities, with the noted exception that strain DA-108 lacked the tcsL gene, which encodes the large clostridial glucosyltransferase enzyme lethal toxin (TcsL). The targeted removal by immunoprecipitation of TcsL protected animals from death following injection of crude culture supernatants from strain ATCC9714. Injections of a monoclonal anti-TcsL IgG protected animals from death during C. sordellii ATCC9714 infection, suggesting that such an approach might improve the treatment of patients with C. sordellii-induced toxic shock syndrome.     

Comparative analysis of the extracellular proteomes of two Clostridium sordellii strains exhibiting contrasting virulence

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL⁺ reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin–nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.     

Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Inactivation of Bacillus Endospores in Envelopes by Electron Beam Irradiation

The anthrax incidents in the United States in the fall of 2001 led to the use of electron beam (EB) processing to sanitize the mail for the U.S. Postal Service. This method of sanitization has prompted the need to further investigate the effect of EB irradiation on the destruction of Bacillus endospores. In this study, endospores of an anthrax surrogate, B. atrophaeus, were destroyed to demonstrate the efficacy of EB treatment of such biohazard spores. EB exposures were performed to determine (i) the inactivation of varying B. atrophaeus spore concentrations, (ii) a D₁₀ value (dose required to reduce a population by 1 log₁₀) for the B. atrophaeus spores, (iii) the effects of spore survival at the bottom of a standardized paper envelope stack, and (iv) the maximum temperature received by spores. A maximum temperature of 49.2°C was reached at a lethal dose of ~40 kGy, which is a significantly lower temperature than that needed to kill spores by thermal effects alone. AD₁₀ value of 1.53 kGy was determined for the species. A surface EB dose between 25 and 32 kGy produced the appropriate killing dose of EB between 11 and 16 kGy required to inactivate 8 log₁₀ spores, when spore samples were placed at the bottom of a 5.5-cm stack of envelopes.     

Temperature, soluble solids and pH effect on Alicyclobacillus acidoterrestris viability in lemon juice concentrate

Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic, spore-forming bacterium detected in spoiled commercial pasteurized fruit juice. Apple, white grape and tomato are particularly susceptible. A. acidoterrestris spores are resistant to lemon juice pasteurization (2 min at 82°C), and they can germinate and grow causing spoilage. This contamination is characterized by a medicinal or disinfectant smell attributed to guaiacol (o-dihydroxybenzene) production and other taint chemicals. The aim of this work was to study the influence of temperature (82, 86, 92 and 95 °C), total soluble solids (SS) (6.20, 9.8, 50 and 68°Brix) and pH (2.28, 2.45, 2.80, 3.25, 3.5) on decimal reduction time (D) of the A. acidoterrestris in clarified and non-clarified concentrated lemon juice. Once D-value was determined, the resistance of A. acidoterrestris at the assayed temperatures was confirmed. SS and pH influence spore viability, because spore resistance increases with higher SS (50°Brix 22 min 82 °C–68°Brix 28 min 82 °C) and pH values (pH 2.28, 17 min–pH 4.00, 22 min). Bacterial growth was lower in clarified lemon juice, 26 min at 82 °C, than in non-clarified lemon juice, 51 min at 82 °C. Temperature was the parameter that had the greatest influence on the D value.     

Inactivation of Geobacillus stearothermophilus Spores by High-Pressure Carbon Dioxide Treatment

High-pressure CO₂ treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO₂ treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO₂ treatment. We also investigated the influence of temperature on CO₂ inactivation of G. stearothermophilus. Treatment with CO₂ and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO₂ treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO₂ treatment of G. stearothermophilus spores, CO₂ treatment at 95°C was more effective than treatment at 95°C alone.     

Clostridium difficile survives minimal temperature recommended for cooking ground meats

We quantified the thermal inhibitory effect of 71 °C (recommended for cooking ground meats), and re-heating at 85 °C, on food- and food-animal-derived Clostridium difficile spores. All C. difficile strains tested (n = 20) survived 71 °C for 2 h, but 90% died within 10 min when re-heated at 85 °C. Current cooking recommendations would need revision to include C. difficile.     

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H₂O₂ and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

Factors influencing the pathogenicity and development of Mattesia trogodermae infecting Trogoderma glabrum larvae

Factors that regulate development of Mattesia trogodermae in Trogoderma glabrum were defined, and their quantitative effects were determined. The rate of and the extent to which spore formation proceeds is strictly governed by temperature. More spores are produced at 30° than at 25°C and very low numbers of spores are formed when the incubation temperature is 35°C. When insects are incubated at 35°C for 1–10 days and transferred to 30°C for the remainder of the 30-day experiment, spore production capacity gradually declines with increasing time at 35°C. Two hypotheses are proposed for this phenomenon. Larval size also regulates the extent of spore production, larger larvae having greater potential for spore development. This is not influenced by dosage. Spore production in pupae and adults was always retarded.Dosage and environmental conditions which influence the virulence of M. trogodermae were investigated. These studies show that rates of mortality are higher at higher temperatures. Low doses of spores result in longer LT50's than do high doses at 25° and 30°C. No differences in rates of mortality were found between different doses at 35°C.     

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with K_D ~ 1 μM. Proteins co-localize in the soluble fraction of the cell at 30–37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpA_His6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30–37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.     

Activation ofDictyostelium discoideum spores with guanidine and methyl derivatives of urea

A majority ofDictyostelium discoideum spores were activated with guanidine hydrochloride and tetramethylurea treatments. Dimethylurea could be utilized over a wide range of concentrations to activate spores. The minimal concentration was 2 M dimethylurea employed for 45–60 min, and the maximal concentration was 5 M dimethylurea employed for 20–30 min. Moderate overstimulation with dimethylurea resulted in an increase in the postactivation lag time, while severe overstimulation caused lysis and death of the spores. Partial spore deplasmolysis was a requirement for activation with dimethylurea at 23,5°C; deplasmolysis and activation did not occur at 0°C. The time required to produce an LD₅₀ was twice the time required for optimal activation when spores were treated with high concentrations of urea derivatives. A correlation was found for the hydrophobicity of the urea family of compounds and the molar concentration required for maximal activation with a 30-min treatment (2 M tetramethylurea, 5 M dimethylurea, and 8 M urea).     

Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Constant and fluctuating temperature acclimations have similar effects on phenotypic plasticity in springtails

Much interest exists in the extent to which constant versus fluctuating temperatures affect thermal performance traits and their phenotypic plasticity. Theory suggests that effects should vary with temperature, being especially pronounced at more extreme low (because of thermal respite) and high (because of Jensen's inequality) temperatures. Here we tested this idea by examining the effects of constant temperatures (10 to 30 °C in 5 °C increments) and fluctuating temperatures (means equal to the constant temperatures, but with fluctuations of ±5 °C) temperatures on the adult (F2) phenotypic plasticity of three thermal performance traits – critical thermal minimum (CT_min), critical thermal maximum (CT_max), and upper lethal temperature (ULT₅₀) in ten species of springtails (Collembola) from three families (Isotomidae 7 spp.; Entomobryidae 2 spp.; Onychiuridae 1 sp.). The lowest mean CT_min value recorded here was -3.56 ± 1.0 °C for Paristoma notabilis and the highest mean CT_max was 43.1 ± 0.8 °C for Hemisotoma thermophila. The Acclimation Response Ratio for CT_min was on average 0.12 °C/°C (range: 0.04 to 0.21 °C/°C), but was much lower for CT_max (mean: 0.017 °C/°C, range: -0.015 to 0.047 °C/°C) and lower also for ULT₅₀ (mean: 0.05 °C/°C, range: -0.007 to 0.14 °C/°C). Fluctuating versus constant temperatures typically had little effect on adult phenotypic plasticity, with effect sizes either no different from zero, or inconsistent in the direction of difference. Previous work assessing adult phenotypic plasticity of these thermal performance traits across a range of constant temperatures can thus be applied to a broader range of circumstances in springtails.     

A new medium for spore production of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> using response surface methodology

Optimization of the medium components which enhance sporulation of the two mating types of the fungus Blakeslea trispora ATCC 14271 and ATCC 14272 (a heterothallic Zygomycota producing carotene) was achieved with the aid of response surface methodology (RSM). Glucose, corn steep liquor, yeast extract, and ammonium sulfate were investigated as carbon and nitrogen sources in a basal medium. RSM was adopted to optimize the medium in order to obtain a good growth of the fungus as a prerequisite for enhanced sporulation. In the second step, the basal medium was supplemented with different trace elements which significantly affect sporulation (i.e. CuSO₄·5H₂O, FeCl₃·6H₂O, Co(NO₃)₂·6H₂O, and MnCl₂·4H₂O). Central composite design proved to be valuable in optimizing a chemically defined solid medium for spore production of B. trispora. The composition of the new solid medium to enhance spore production by B. trispora (ATCC 14271) is as follows (per liter): 7.5 g glucose, 3.2 g corn steep liquor, 1.7 g yeast extract, 4.1 g ammonium sulfate, 6 mg CuSO₄·5H₂O, 276 mg FeCl₃·6H₂O, 2 mg Co(NO₃)₂·6H₂O, and 20 g agar (pH 6.0). Practical validation of this optimum medium gave spore number of 1.2 × 10⁸ spores/dish which is 77% higher than that produced in Potato Dextrose Agar (PDA). In the case of B. trispora (ATCC 14272) the new solid substrate for enhanced sporulation consists of (per l) 6.4 g glucose, 3.3 g corn steep liquor, 1.4 g yeast extract, 4.3 g ammonium sulfate, 264 mg CuSO₄·5H₂O, 485 mg FeCl₃·6H₂O, 223 mg MnCl₂^.4H₂O, and 20 g agar (pH 6.0). Spore numbers of 2 × 10⁷ spores/dish were obtained on the new medium by B. trispora (ATCC 14272), which is 95% higher than that produced on PDA. The results corroborated the validity and the effectiveness of the models. The new media considerably improved sporulation of both strains of B. trispora compared to the production of spores on PDA, which is the medium usually used for sporulation of the fungus.     

Effect of heat treatment on lactoperoxidase activity in caprine milk

Using isothermal conditions, inactivation of lactoperoxidase (LPO) in caprine milk was studied in a temperature range of 69–73 °C. In order to evaluate the effect of temperature on the reaction rate, the Arrhenius and thermal death time models were used for data analysis. Thermal inactivation of LPO can be accurately described by a first-order kinetic model, as indicated by the relationships obtained by plotting the retention values as a function of treatment time on a semi-logarithmic scale and confirmed by the high R²-values obtained. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The corresponding Z- and E_a-values calculated from the slope of the semi-logarithmic plots of D and k as a function of temperature were 9.45 °C and 225.98 kJ/mol, respectively.     

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores'' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores'' germination kinetics were heterogeneous; (ii) individual spores'' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T_lag) prior to a period of the rapid release (ΔT_release) of the spores'' dipicolinic acid in a 1:1 chelate with Ca²⁺ (CaDPA); (iii) spore germination at 50 MPa had longer average T_lag values than that at ∼150 MPa, but the ΔT_release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔT_release values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T_lag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores'' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.     

Heat activation and germination–promotion of Bacillus subtilis spores by infrared irradiation

The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m^?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.