Repression of toxin production by tryptophan in Clostridium botulinum type E

Of the seven amino acids required by Clostridium botulinum type E, tryptophan is the most essential and may provide the cell with nitrogen. The addition of excess tryptophan (10–20 mM) or other nitrogenous nutrients to minimal growth medium markedly decreased toxin formation but did not affect growth in C. botulinum type E. On the other hand, the addition of an enzymatic digest of casein (NZ Case) stimulated toxin formation and overcame repression by tryptophan. Immunoblots of proteins in culture fluids using antibodies to type E toxin indicated that tryptophan-repressed cultures produced less neurotoxin protein. Inhibitors of neurotoxin did not accumulate in cultures grown in minimal medium supplemented with high tryptophan. The results suggest that tryptophan availability in foods or in the intestine may be important for toxin formation by C. botulinum type E.     

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.     

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non-toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

The effects of temperature on the respiration and production of the freshwater nematode Anonchus sp.

Summary Growth and respiration were measured in a species of Anonchus (Nematoda: Plectidae) at 5°C, 10°C, 15°C, 20°C and 25°C. At 5°C no growth was measurable but the organisms remained active. Maximum production occurred at 15°C, but the highest rate of growth occurred at 20°C. Thus, adult size attained is dependent on the temperature of growth. Respiratory energy losses derived from Cartesian diver microrespirometry, increased with temperature up to 25°C. Regression coefficients (b values) derived from a log log linear regression of weight against oxygen consumption varied between 0.574–1.793, the lowest value being attained at 5°C, the highest at 20°C. Based on Q₁₀, production and respiratory energy losses the optimum temperatures for Anonchus appears to lie between 10°C–15°C.     

Aerobic growth and toxigenicity ofClostridium botulinum types A and B

Clostridium botulinum types A and B cultured in association with avian skin flora, had similar growth patterns under both aerobic and anaerobic conditions. The selective “C. botulinum isolation” (CBI) medium was found to be especially useful for the recovery and quantitation of small numbers of type A or type B organisms from the mixed cultures. Enzyme immunoassay in conjunction with conventional mouse biossay provided a practical means for the quantitation of toxigenicity ofC. botulinum in avian skin cultures. The amount of toxin produced by type A was always higher than that produced by type B strains. The aerobically incubated type A or type B cultures appeared to be less toxigenic than cultures incubated anaerobically.     

Effects on Growth and Toxin Production of Exposure of Spores of Clostridium botulinum Type F to Sublethal Doses of Gamma Irradiation

Spores of the Langeland strain of Clostridium botulinum type F were grown at 30 or 10 C after exposure to 0.0, 0.1, or 0.2 megarad of cesium-137 gamma irradiation. When incubated at 30 C, cultures irradiated at the 0.2-megarad level reached the stationary growth phase 15 hr earlier than the 0.0 or 0.1 megarad-irradiated cultures. This was not the result of earlier or more frequent germination of the irradiated spores, the formation of larger individual cells, filament formation, or cell clumping. It appeared to result from elimination of a lytic phenomenon noted in 0.0 and 0.1 megarad-irradiated cultures after 26 and 29 hr of incubation, respectively, which was followed by a second exponential-growth response 5 hr later in these cultures. The time of toxin appearance in culture supernatant fractions was independent of prior irradiation treatment and occurred after 36 hr of incubation. Toxin release was essentially logarithmic until maximal titers were reached and maximal toxin titers were higher in irradiated than in unirradiated cultures. The higher toxin level was sustained over a period of 23 days of 30 C. Toxin produced in the 30 C cultures could not be trypsin-activated. An incubation temperature of 10 C resulted in no outgrowth of spores subjected to 0.2 megarad of irradiation, although spore germination did occur. At 10 C, outgrowth of the 0.1-megarad culture was faster with slightly higher quantities of a more stable toxin than was seen in the unirradiated control. At 10 C, trypsinization was necessary to demonstrate the toxin present in the cultures.     

Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 10³ spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060–5010 mg/kg dry wt of culture) at ambient temperature (16–29°C) in daylight than at ambient temperature (18–23 °C) in darkness or at controlled temperatures of 11 °C, 20 °C or 25 °C in darkness. Yields at 25 °C were low. Incubation at 11 °C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 °C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 °C (2460-21 360 mg/kg), 1 strain at 11 °C (6570 mg/kg). Yields at a temperature oscillating daily from 10–20 °C were less than at 15 °C. One of the 5 strains produced appreciable amounts of a-zearalenol (1645 mg/kg at 20°C) and 2 of nivalenol (340 and 499 mg/kg at 20 °C).     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.     

Respiratory energy losses in the protozoan predator Didinium nasutum Müller (Ciliophora)

Summary Respiration in Didinium nasutum, an active protozoan predator, was investigated in relation to cell weight at 10, 15, and 20° C by means of cartesian diver microrespirometry. Oxygen uptake increased progressively over the 10–20° C temperature range; a table of Q ₁₀ related to weight is presented. Regression coefficients of log weight versus log oxygen uptake (b) were 0.96 at 10° C, 0.98 at 15° C and 1.00 at 20° C. D. nasutum was shown to expend very much higher levels of respiratory energy than a mainly sedentary carnivorous ciliate of comparable weight.     

Isolation and improvement of a thermotolerant Saccharomyces cerevisiae strain

The ambient temperature is a drawback in industrial ethanol production in Jaffna due to heat killing of yeast during fermentation. Thus a search was initiated for thermotolerant organisms suitable for fermentation in hot climates. The screening of the best wild-type organisms was undertaken as the first step. Thermotolerant strains were selected from environments where there are chances of organisms being exposed to high temperature. The samples were enriched and screened for thermotolerant organisms which survived at 45 °C for 15 h. Among the yeast strains selected from different sources, thermotolerant strains with the capacity to withstand 45 °C for 15 h were found in samples collected from the compost heap and distillery environments. Three colonies from the distillery environment were selected for further studies and named p1, p2 and p3. Exponential phase (18 h) cultures of p1, p2 and p3 were subjected to 15 temperature treatment cycles (at 50 °C each for 3 h) and thermally adapted strains pt1, pt2 and pt3 were obtained, showing 100, 30 and 20% viability at 50 °C for 30 min respectively. The initial round of thermal adaptation cycles increased the duration of 100% viability from 20 h (p1) to 68 h (pt1) when incubated at 40 °C. Very little benefit was obtained when pt1 was treated with u.v. and ethyl methanesulphonate. The selected strain was identified and designated as Saccharomyces cerevisiae S1. The ethanol produced from 100 g glucose l^–1 by S. cerevisiae S1 was 46 g l^–1 (36 h), 38 g l^–1 (48 h) and 26 g l^–1 (48 h) at 40, 43 and 45 °C respectively in rich nutrient medium.     

Selection and Study of Potent Lactose-Fermenting Yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen strains from milk products, showing maximum potency, fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11–12, and 10–12%, respectively (4°C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30°C for 2–3 days, the ethanol concentration was 4–5%.     

Recovery of Clostridium botulinum from mud samples incubated at different temperatures

Summary In mud samples naturally contaminated with Clostridium botulinum type C, other types cannot be recovered when incubated at 37°C. At incubation temperatures equal to or lower than 30°C and in the presence of type E, Clostridium botulinum type C cannot always be detected. The use of two incubation temperatures to increase the probability of detecting all types of Clostridium botulinum can therefore be recommended.     

Low-Temperature Irradiation of Beef and Methods for Evaluation of a Radappertization Process

An inoculated, irradiated beef pack (1,240 cans) study was conducted for the determination of microbiological safety for unrestricted human consumption. Each can contained a mixture of 10⁶ spores of each of 10 strains of Clostridium botulinum (5 type A and 5 type B), or a total of 10⁷ spores/can. The cans were irradiated to various doses (100 cans/dose) with ⁶⁰Co gamma rays at -30 ± 10 C, incubated at 30 ± 2 C for 6 months, and examined for swelling, toxicity, and recoverable botulinal cells. The minimal experimental sterilizing dose based on nonswollen, nontoxic sterile cans was 2.2 < experimental sterilizing dose ≤ 2.6 Mrad. Using recoverable cells as the most stringent criterion of spoilage, and assuming the conventional simple exponential (without an initial shoulder) rate of spore kill, the “12D” dose was 3.7 Mrad when estimated on the basis of a mixture of 10 strains totaling 10⁷ spores/can, and 4.3 Mrad if it is assumed that each can of beef contained 10⁶ spores of a single most resistant strain and all of these spores were of identical resistances. However, an analysis of the data by extreme value statistics indicated with 90% confidence that the spore death rate was not a simple exponential but might be a shifted exponential (with an initial shoulder), Weibull, lognormal, or normal, with a “12D” equivalent of about 3.0 Mrad regardless of the initial spore density per can. There was an apparent antagonism between the irradiated type A and B strains in the cans. Some of the cans contained type B toxin but did not include type B viable cells. Other cans had a mixture of type A and B toxins, but a large number of these cans did not yield recoverable type B cells. However, type A viable cells could always be demonstrated in those cans containing type A toxin.     

Inducing sporulation in Alternaria solani I. Effect of water treatment

The sporulation was induced when fully grown cultures were given dip or spray treatment with distilled water (cold or hot) and thereafter, kept partially covered at different temperatures. Cultures dipped in cold water (4° C) for 4 minutes or sprayed with cold water (4° C) or hot water (58° C) and thereafter incubated at room temperature (13–26° C) in diffused sunlight, produced maximum number of spores within 60 hours. Incubating water treated cultures in diffused sunlight or complete darkness and age and scraping of the cultures had a considerable effect upon intensity of sporulation. The cultures yield a number of subsequent crops of spores when scraped and given dip treatment with cold or hot water, after obtaining each crop of spores.     

Minimal Growth Temperature, Sodium Chloride Tolerance, pH Sensitivity, and Toxin Production of Marine and Terrestrial Strains of Clostridium botulinum Type C

Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 × 10⁶ viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 × 10⁶ unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.     

Radiation Sterilization of Bacon for Military Feeding

Sliced, cured bacon, packed in cans and seeded with 6 × 10⁵ spores per can of Clostridium botulinum strains 33A or 41B, or with 3 × 10⁶ spores per can of strains 36A, 12885A, 9B, or 53B, was irradiated to various dose levels with γ radiation. Evidence provided by swelling, toxicity, and recoverable C. botulinum with 2,200 inoculated, irradiated cans demonstrated that: (i) 4.5 Mrad were more than adequate as a sterilization dose; (ii) the experimental minimal sterilizing dose was 2.0 Mrad, and the theoretical 12-log reduction dose was 2.65 or 2.87 Mrad depending on the method of calculation; (iii) some spoilage occurred at dose levels below 2.0 Mrad; (iv) all visible spoilage of irradiated bacon was due to strains 33A and 12885A only, whose D values were, respectively, 0.141 and 0.177 Mrad based on spoilage data, and 0.221 and 0.188 Mrad, respectively, when based on recovery data; (v) toxic cans did not always result in swelling, nor did swollen cans always produce toxic spoilage; and (vi) viable C. botulinum can exist for at least 8 months in storage at 30 C without producing visible or toxic spoilage at doses below 2.0 Mrad.     

Influence of Environmental Factors on the Chemotaxis of Bradyrhizobium japonicum

Dependence of motility and chemotaxis was studied in two strains of Bradyrhizobium japonicum upon several environmental factors. In both strains, chemotaxis was found to increase with an increasing concentration of the attractant (glucose) to 5.5 × 10^–2 M. Both motility and chemotaxis reached their maximum in the two- to three-day cultures at neutral pH. The maximum motility of these bacteria occurred at 40°C. The maximum values of chemotaxis in these microorganisms were, however, observed at 20–25°C. Chemotaxis in acidic or alkaline media and at low temperatures was found to be markedly weaker. Nonoptimal values of these parameters in soil may be a limiting factor for the interaction of the given bacteria with soybean roots.     

Studies on the rust,Maravalia cryptostegiae,a potential biological control agent of rubber-vine weed,Cryptostegia grandiflora (Asclepiadaceae: Periplocoideae), in Australia,II: Infection

The parameters which govern infection of rubber-vine weed by the rustMaravalia cryptostegiae were investigated. The infection process, from appressorial formation to sporulation, is described and illustrated. Uredinioid teliospores have an optimum temperature range for germination at 22–27 °C, both in vitro and in vivo. However, germination on the rubber-vine leaf was more than double (81–92%) that in the absence of the host, and appressoria were formed only in vivo. An optimum temperature of 20–22°C and a dew period of 12 hours or more gave the highest level of infection as measured by sporulation density. The latent period from inoculation to pustule formation decreased with increasing temperature; the shortest period (8–11 days) being recorded at 25–27°C. At the lower temperatures (18°C), this was significantly extended (19–21 days). Four successive inoculations significantly reduced plant height and dry weight, although a compensatory growth flush occurred after the third inoculation. The addition of cryoprotectants had a negative affect on spore viability and subsequent infectivity. Cooling dry spores to –196°C at the rate of 10°C min^–1 gave the best results, with high germination (93–65%) up to 8 days after thawing.