The Architectonics of Colonies of Bacillus subtilis 2335

Colonies grown from vegetative Bacillus subtilis 2335 cells had a standard structure, with bacillar cells occupying the whole colony volume. At the same time, the colonies of this bacterium grown from germinated spores had an abnormal structure characterized by the location of cells in a surface layer 100–200 m thick at the colony boundary with the air. The glycocalyx of the colonies grown from spores was characterized by a wetting angle _e of 120°–160°, whereas that of the colonies grown from vegetative cells had an angle _eas low as 5°–30°. It is suggested that spores and vegetative cells follow different strategies of substrate colonization and that the architectonics of bacterial colonies is determined by the physicochemical properties of the glycocalyx.     

Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes

Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell-chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

The growth of potential food poisoning organisms on chicken and pork muscle surfaces

Muscle surfaces of pork were inoculated with a mixture of Yersinia enterocolitica and Staphylococcus aureus, and chicken muscle with Campylobacter jejuni or a mixture of Salmonella typhimurium and Staph. aureus. The surface growth at 20 degrees C was followed microscopically. Organisms grew as discrete colonies bound together by a glycocalyx which differed between bacterial species. On prolonged incubation colonies spread peripherally and tended to coalesce, while still retaining their colony structure. Staphylococcus aureus colonies were very small and remained so. The glycocalyx was considered critical in maintaining the dense populations of bacteria on the meat surfaces.     

Sporulation in single-spore isolates from amitrole-induced multispored asci of Saccharomyces cerevisiae.

Amitrole treatment causes multispored ascus production by cells of a yeast strain whose asci normally contain two diploid spores. Single spores were isolated from asci containing two to eight spores and their ability to germinate was determined. Cells in colonies grown from single spores sporulated in the same manner as the parent strain indicating that amitrole had not induced meiotic division in the developing asci.     

The growth of potential food poisoning organisms on chicken and pork muscle surfaces

Muscle surfaces of pork were inoculated with a mixture of Yersinia enterocolitica and Staphylococcus aureus , and chicken muscle with Campylobacter jejuni or a mixture of Salmonella typhimurium and Staph. aureus . The surface growth at 20°C was followed microscopically. Organisms grew as discrete colonies bound together by a glycocalyx which differed between bacterial species. On prolonged incubation colonies spread peripherally and tended to coalesce, while still retaining their colony structure. Staphlycoccus aureus colonies were very small and remained so. The glycocalyx was considered critical in maintaining the dense populations of bacteria on the meat surfaces.     

The effects of incubation of marrow in tissue fragments in vitro on the growth of adherent cells from this marrow

Femoral marrow was either cultured as a single cell suspension immediately following collection from the donor mouse or following 4 day incubation in vitro of the whole marrow shaft. Several parameters of growth of adherent, i.e. composed of fibroblastoid cells and macrophages, colonies were determined following 14 day culture in Dulbecco medium at 37 degrees C. These included: number and diameter of macroscopic colonies, number of macrophages per fibroblastoid cell inside the colonies and per eyefield in intercolony spaces, number of cells in supernatant from the culture. The 4 day incubation of marrow fragments in vitro (Dulbecco medium, 37 degrees C) doubled the number of adherent colonies grown from this marrow and, moreover, the colonies formed were increased in size. Other parameters of cell growth in these cultures were unchanged. These data suggest that under conditions of in vitro incubation of marrow shaft (close cell-to-cell contact) marrow fibroblastoid colony forming units (MF-CFU) are stimulated to self-renewal.     

Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores.

The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C. Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures. Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied. More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl. The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors. At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C). However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C. Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism. If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair. If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C. Thus, repair appeared to be associated with outgrowth. Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

Differentiation of Clostridium botulinum Types A, B, and E by Pyrolysis-Gas-Liquid Chromatography

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.     

Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis

The surface hydrophobicity of nine Bacillus subtilis strains in different states (spores, vegetative cells, and dead cells) was assessed by water contact angle measurements, hydrophobic interaction chromatography (HIC) and bacterial adhesion to hydrocarbon (BATH). Electrokinetic properties of B. subtilis strains were characterized by zeta potential measurements and found to differ appreciably according to the strain. Correlations between HIC data, BATH data and zeta potential showed that HIC and BATH are influenced by electrostatic interactions. Water contact angle measurements thus provide a better estimate of cell surface hydrophobicity. The water contact angle of B. subtilis varied according to the strain and the state, the spores tending to be more hydrophobic than vegetative cells.     

Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

The pattern of sporulation of Bacillus popilliae in colonies

Spores accumulate periodically in colonies of Bacillus popilliae after 3 days of vegetative growth on solid medium. Sporulation occurs on the surface and primarily in a ring near the periphery, causing slight changes in colony contour. The formation of mature spores and their acquisition of resistance to drying and to heat occur in a stepwise manner. A high level of prespore forms persists in mature colonies. Sporulation in colonies is as efficient as early stages of sporulation in larvae, but efficiency in vivo must increase as milky disease progresses.     

Growth of heat-injured Vibrio parahaemolyticus in media supplemented with various cations.

Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.     

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.     

Ecology,development and pathogenicity of Buddenbrockia plumatellae Schröder, 1910 (Myxozoa,Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae

Buddenbrockia plumatellae, an enigmatic worm-like myxozoan, was observed as continuously writhing free and attached 'worms' and as free mature spores in the coelom of the freshwater bryozoans Plumatella fungosa, Hyalinella punctata, and Fredericella sp. 'Worm' numbers could double every three days. 'Worms' and spores could be expelled from colonies by external pressure. Some mature 'worms' exited actively, entraining release of free spores, and gradually ceased movement outside the host. Bryozoans sealed off infected regions of the colony. Infected colonies grew slowly, produced no statoblasts, and eventually regressed and died. Transmission was not achieved and prevalence was low. Electron microscopy of 'worms' revealed a single layer of mural cells on a fibrous basal lamina overlying four longitudinal muscle blocks and an inner sheet of two types of proliferating cells, an organization indicative of the bilaterian ancestry of the Myxozoa. Primary type A cells were attached directly by striated tubules to mural cells at positions between muscle blocks. Secondary type A cells had a secretory function. Type B cells underwent meiosis and subsequently developed to typical malacosporean myxozoan spores filling the internal cavity of the 'worms'. External tubes were formed during capsulogenesis in 'worms' from Fredericella sp. Tetracapsula bryozoides is synonymised with Buddenbrockia plumatellae and a new genus is proposed for Tetracapsula bryosalmonae.