嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

利用纤维素降解细菌和纤维素粘附的方法分别从新鲜牛粪、高温堆肥和本实验室保存的纤维素降解富集物中分离得到4株嗜热厌氧纤维素降解细菌。分离菌株为革兰氏染色阴性,直的或稍弯曲杆菌,菌体大小为0.4μm～0.6μm×3μm～15μm,严格厌氧,不还原硫酸盐,形成芽孢。多数芽孢着生于菌体顶端。分离菌株能利用纤维素滤纸、纤维素粉Whatman CFII、微晶纤维素、纤维素粉MN300和未经处理的玉米秆芯、甘蔗渣、水稻秸杆。分离菌株在pH6.2～8.9、温度45℃～65℃范围内利用纤维素,最适pH为7.0～7.5,最适温度为55℃～60℃,发酵纤维素产生乙醇、乙酸、H2和CO2。分离菌株还可利用纤维二糖、葡萄糖、果糖、麦芽糖、山梨醇作为碳源。部分长度的16S rDNA序列分析表明,分离菌株EVAI与Clostridium thermocellum具有99.8%相似性。     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions

Classical enumeration methods for anaerobes are time-consuming and require special conditions. Solid phase cytometry (SPC) is a recent laser scanning technique for the quantitative detection of fluorescently labelled bacteria on a membrane filter that eliminates the need for a growth phase. Fluorescent labelling of cells results from the cleavage by intracellular esterases of a fluorescein type ester to yield a free fluorescein derivative, which is retained only in cells with an intact cytoplasmic membrane. However, as the standard labelling procedure is carried out under the conditions of aerobiosis, labelling of anaerobic bacteria does not appear to be obvious. We have labelled eight strains of vegetative anaerobic bacteria (i.e. Bacteroides thetaiotaomicron, Clostridium bifermentans, C. butyricum, C. perfringens, Fusobacterium nucleatum, Porphyromonas canoris, P. gingivalis, Propionibacterium acnes) and two strains of spores (C. butyricum, C. perfringens,) within 4 h under aerobic conditions. However, anaerobiosis remained necessary for spores of C. sordellii, C. sporogenes, C. tyrobutyricum. For vegetative cells of all strains, plots of SPC versus plate counts were linear with slopes exceeding 1.0, indicating that SPC consistently yielded higher numbers of bacteria.     

Enumeration and isolation of cellulolytic and hemicellulolytic bacteria from human feces

The fibrolytic microbiota of the human large intestine was examined to determine the numbers and types of cellulolytic and hemicellulolytic bacteria present. Fecal samples from each of five individuals contained bacteria capable of degrading the hydrated cellulose in spinach and in wheat straw pretreated with alkaline hydrogen peroxide (AHP-WS), whereas degradation of the relatively crystalline cellulose in Whatman no. 1 filter paper (PMC) was detected for only one of the five samples. The mean concentration of cellulolytic bacteria, estimated with AHP-WS as a substrate, was 1.2 X 10(8)/ml of feces. Pure cultures of bacteria isolated on AHP-WS were able to degrade PMC, indicating that interactions with other microbes were primarily responsible for previous low success rates in detecting fecal cellulolytic bacteria with PMC as a substrate. The cellulolytic bacteria included Ruminococcus spp., Clostridium sp., and two unidentified strains. The mean concentration of hemicellulolytic bacteria, estimated with larchwood xylan as a substrate, was 1.8 X 10(10)/ml of feces. The hemicellulose-degrading bacteria included Butyrivibrio sp., Clostridium sp., Bacteroides sp., and two unidentified strains, as well as four of the five cellulolytic strains. This work demonstrates that many humans harbor intestinal cellulolytic bacteria and that a hydrated cellulose source such as AHP-WS is necessary for their consistent detection and isolation.     

Enumeration and isolation of cellulolytic and hemicellulolytic bacteria from human feces.

The fibrolytic microbiota of the human large intestine was examined to determine the numbers and types of cellulolytic and hemicellulolytic bacteria present. Fecal samples from each of five individuals contained bacteria capable of degrading the hydrated cellulose in spinach and in wheat straw pretreated with alkaline hydrogen peroxide (AHP-WS), whereas degradation of the relatively crystalline cellulose in Whatman no. 1 filter paper (PMC) was detected for only one of the five samples. The mean concentration of cellulolytic bacteria, estimated with AHP-WS as a substrate, was 1.2 X 10(8)/ml of feces. Pure cultures of bacteria isolated on AHP-WS were able to degrade PMC, indicating that interactions with other microbes were primarily responsible for previous low success rates in detecting fecal cellulolytic bacteria with PMC as a substrate. The cellulolytic bacteria included Ruminococcus spp., Clostridium sp., and two unidentified strains. The mean concentration of hemicellulolytic bacteria, estimated with larchwood xylan as a substrate, was 1.8 X 10(10)/ml of feces. The hemicellulose-degrading bacteria included Butyrivibrio sp., Clostridium sp., Bacteroides sp., and two unidentified strains, as well as four of the five cellulolytic strains. This work demonstrates that many humans harbor intestinal cellulolytic bacteria and that a hydrated cellulose source such as AHP-WS is necessary for their consistent detection and isolation.     

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.     

Multilayer adhesion to filter paper of two mesophilic,cellulolytic clostridia

Adhesive properties of a recently isolated, mesophilic, cellulolytic Clostridium (strain A22) and ofClostridium cellulolyticum (ATCC 35319) were described. For study of these properties, a method with Whatman No. 1 filter paper was developed, allowing faster kinetic analysis measuring unbound cells. This method was confirmed with [methyl-³H] thymidine to measure directly bound cells. The results of kinetic analysis indicated for the two clostridia a two-step adhesion process. In the first step, cell-cellulose interaction was observed. In the second step, cell-cell interaction was determined with an aggregation of cells away from the surface. This result was confirmed by scanning electron microscopy. Competition between strain A22 andC. cellulolyticum for adhesion sites was investigated. The two clostridia have the same specific adhesion sites was investigated. The two clostridia have the same specific adhesion sites on cellulose (filter paper), and unspecific interactions between two strains occur.     

Detection, cloning, and sequence analysis of an indigenous plasmid from cellulolytic clostridial strain MCF1

Nucleotide sequence analysis of a 2451-bp plasmid (pMCF1) from a cellulolytic Clostridium revealed that the protein specified by the largest open reading frame (ORF1) was homologous to RepB of Clostridium butyricum plasmid pCB101. The data suggest that pMCF1 belongs to the pC194 family of rolling-circle replicating plasmids and the ORF1 protein functions as its replication protein.     

Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants.

A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.     

Effect of oxygen concentration on dinitrogen fixation and volatile fatty acid production by Clostridium butyricum growing in association with fungi on cellulose and on wheat straw

Clostridium butyricum was grown with cellulolytic fungi on the substrates wheat straw and cellulose at a range of oxygen concentrations. The straw was not sterile and was inoculated with Sodaria alcina while the cellulose was sterile and was inoculated with Trichoderma harzianum as examples of cellulolytic organisms. The straw subsequently developed a mixed fungal population. Dinitrogen fixation was only significant at a reduced oxygen concentration: optima being 9.3 and 2.3% O₂ for the straw and cellulose experiments, respectively. The efficiencies of N, fixation were 6 mg N per g straw decomposed and 2.3 mg N per g cellulose decomposed and N, fixation occurred only in the presence of significant cellulolysis. Both acetate and butyrate formation increased as the oxygen concentration decreased. With cellulose as a substrate, their formation correlated with a decrease in pH and an increase in final numbers of Cl. butyricum . The thickness of the anaerobic zone at the aerobic/anaerobic interface was linearly related to the square root of the oxygen concentration at the surface of the interface.     

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Mesophilic cellulolytic clostridia from freshwater environments

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.     

Inhibition of Spores of Clostridium spp. by Sodium Nitrite

S ummary . Sodium nitrite heated in a laboratory medium was more inhibitory to spores of Clostridium spp. than nitrite added as a filter-sterilized solution to the same medium. Most spores remained refractile after inhibition for >3 months and some proved viable when inoculated into fresh nitrite-free medium. The inhibitory activity of heated nitrite medium was not stable indefinitely, growth sometimes occurred on re-inoculation with vegetative cells.     

Intestinal Distribution and Intraluminal Localization of Orally Administered Clostridium butyricum in Rats

Clostridium butyricum has been used as a probiotic in animals and humans for years, however, its fate in the intestine has not been clarified yet. We investigated the intestinal fate of C. butyricum using a selective medium and a monoclonal antibody after orally administering C. butyricum spores to rats. The number of C. butyricum, both viable and dead cells, in the intestinal contents were counted by enzyme-linked immunosorbent assay (ELISA) at various times after a single oral administration. The total viable number of C. butyricum was counted using a selective medium, and viable resting spores were selectively detected by treating the samples with ethanol. To investigate the intraluminal localization of the C. butyricum cells, frozen intestinal tracts were imprinted onto slides and stained with immunogold-silver. Total viable spores exceeded the number of viable resting spores by more than 10-fold from the proximal to middle of the small intestine 30 min after administration. Vegetative cells of C. butyricum were first detected in the distal small intestine after 2 hr, and vegetative growth was observed from the cecum to the colon 5 hr after administration. Dead vegetative cells were detected 9 hr after administration, and C. butyricum cells were not detected in the intestine after 3 days. The C. butyricum cells in the intestinal imprints were stained specifically by immunogold-silver staining, and proliferative cells were observed in the cecum after 3 hr. These results suggest that the administered C. butyricum germinated in the upper small intestine, grew mainly from the distal small intestine to the colon and were excreted from the rat intestine within 3 days.     

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.     

Adhesive Properties of a Symbiotic Bacterium from a Wood-Boring Marine Shipworm

Adhesive properties of a cellulolytic, nitrogen-fixing bacterium isolated from a marine shipworm by Waterbury et al. (J. B. Waterbury, C. B. Calloway, and R. D. Turner, Science 221:1401-1403, 1983) are described. ³⁵S-labeled cells of the shipworm bacterium bound preferentially to Whatman no. 1 cellulose filter paper, compared with its binding to other cellulose substrata or substrata lacking cellulose. The ability of the bacteria to bind to Whatman no. 1 filter paper was significantly reduced by glutaraldehyde or heat treatment of cells. Pretreatment of cells with azide, valinomycin, gramicidin-D, bis-hexafluoroacetylacetone (1799), or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone inhibited adhesion activity. Cells pretreated with pronase or trypsin also exhibited reduced binding activity, but chymotrypsin and peptidase had no effect on adhesion activity. Cellodextrins and methyl cellulose 15 inhibited the adhesion of shipworm bacteria to filter paper, whereas glucose, cellobiose, and soluble carboxymethyl cellulose had no significant effect. The divalent cation chelators EDTA and EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′N′-tetraacetic acid] had little or no effect on adhesive properties of shipworm bacteria. Also, preabsorbing the substratum with extracellular endoglucanase isolated from the shipworm bacterium or 1% bovine serum albumin had no apparent effect on bacterial binding. Low concentrations (0.01%) of sodium dodecyl sulfate solubilized a fraction from whole cells, which appeared to be involved in cellular binding activity. After removal of sodium dodecyl sulfate, several proteins in this fraction associated with intact cells. These cells exhibited up to 50% enhanced binding to filter paper in comparison to cells which had not been exposed to the sodium dodecyl sulfate-solubilized fraction.     

Correlation of the DNA nucleotide makeup with the physiological and cytological characteristics of spore-forming anaerobic bacteria]

The nucleotide composition of DNA from 12 studied species of anaerobic bacteria belongs to AT type, with G+C varying from 28.4 to 36.8 mole%. In the anaerobic group of Clostridium bifermentans, a correlation has been established between the nucleotide composition of DNA, the type of appendages on spores, and some physiologo-biochemical characteristics. The nucleotide composition of DNA in the spores of four anaerobic species is shifted toward GC type as compared to DNA in the vegetative cells. Data on the content of GC pairs in DNA of the spores may sometimes be of a higher taxonomic value than the corresponding evidence on DNA of the vegetative cells.     

The kinetics of Bacillus subtilis spore inactivation on filter paper by u.v. light and u.v. light in combination with hydrogen peroxide

Inactivation of spores of Bacillus subtilis (ATCC 6633) on two different grades of cellulose filter paper (Whatman Grades 2 and 6), by ultraviolet light (u.v.), at an intensity of approximately 4·5 Wm⁻² and at fluences of up to 2 × 10³ Jm⁻², and u.v. in the presence of hydrogen peroxide, is described in terms of multi-target and single hit–single target kinetic expressions. Wet spores were inactivated at rates ranging from 6·7 to 10·6 higher than that of dry spores on both grades of filter paper. In addition, spore inactivation was up to 5·6 times more rapid on Grade 2 filter paper. Synergistic inactivation was seen to occur when spores were irradiated in the presence of 1% (w/v) hydrogen peroxide with rates up to 5·3 times higher than with treatment solely by u.v. The results obtained are discussed in general terms with particular reference to surface characteristics which might provide shielding to micro-organisms from incident u.v. light.     

Antibacterial Effect of Cysteine-Nitrosothiol and Possible Percursors Thereof

The postulated intermediate of nitrite-myoglobin reaction, cysteine-nitrosothiol, was prepared and its antibacterial effect was tested on Salmonella strains, Streptococcus faecium, and spores and vegetative cells of Clostridium sporogenes. Cysteine-nitrosothiol showed a higher inhibitory effect than nitrite. Preliminary results on the effect of simultaneous use of nitrite and cysteine on Clostridium sporogenes spores were also presented.