脆弱类杆菌属菌株的培养及免疫血清的制备

本文报告了从国外引进的五种类杆菌培养,鉴定及其抗血清的制备。结果表明这些类杆菌在牛心脑培养基中容易生长。均为厌氧性革兰氏阴性无芽孢多形态杆菌。经生化反应鉴定结果发现脆弱类杆菌对红霉素(60μg)、利福平(15μg)敏感,而对多粘菌素B(10μg)、青霉素(2IU)、卡那霉素(1000μg),万古霉素(5μg)耐受。用不同接种途径免疫家兔所获得的脆弱类杆菌抗血清的结果表明皮下加不完全佐剂组血清中的抗体滴度明显高于一般皮下组及静脉注射组。     

成年牙周炎病人龈下菌斑优势菌群的探讨

本文用2种非选择性培养基和12种选择性培养基对17名北京市成年牙周炎病人和9名健康对照者龈下及龈沟菌斑菌群进行了培养、分离和鉴定。结果,牙周炎患者龈下菌斑的菌群主要由专性厌氧的革兰氏阴性杆菌组成(60.8％),优势菌为牙龈拟杆菌等;而对照组的菌群主要由兼性厌氧的革兰氏阳性球菌组成(69.2％),优势菌为链球菌属等。这种菌群构成,两组样本存在显著性差异,对于我们认识牙周炎的病原及有关微生态学问题可能有重要意义。     

拟杆菌的研究及应用

拟杆菌大量存在于宿主体内,对宿主的健康起着重要的作用。当宿主微生态平衡被破坏时它又是条件致病菌,使宿主患病。主要总结了最近10年来国内外学者对拟杆菌的研究状况,重点对拟杆菌的致病及益生机理进行了阐述。此外,还介绍了几株有重要应用价值的拟杆菌,并就以后的研究趋势作了展望。     

A rapid method for preparation of rough and smooth lipopolysaccharide from Bacteroides, Porphyromonas and Prevotella

Abstract We describe a new method for lipopolysaccharide (LPS) preparation by water extraction at 100°C and subsequent digestion with proteinase K. The crude LPS could be reliably used for immunoblotting since it retained a high level of antigenicity, and was free of SDS and proteinase K, both of which can cause problems. Two monoclonal antibodies which failed to react with LPS prepared by two conventional methods reacted well with our preparation. We used the new method to prepare LPS from 44 strains of bacteria formerly classified as Bacteroides , some of which have been reclassified as Porphyromonas or Prevotella . In general, yields were good, and electrophoretic profiles obtained with SDS-PAGE and silver staining enabled strains to be rated rough, semi-rough, or smooth.     

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms

H₂-producing microorganisms are a promising source of sustainable biohydrogen. However, most H₂-producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H₂ exist, common H₂ sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity (TC) sensors and developed a parallelized, online H₂ monitoring for time-efficient characterization of H₂ production by anaerobes. Since TC sensors are nonspecific for H₂, the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO₂ concentrations. The systems' measurement range was validated with two anaerobes: a high H₂-producer (Clostridium pasteurianum) and a low H₂-producer (Phocaeicola vulgatus). Online monitoring of H₂ production in shake flask cultivations was demonstrated, and H₂ transfer rates were derived. Combined with online CO₂ and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H₂ production with the model anaerobes was gained. The presented online H₂ monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.     

Effect of oxygen and peroxide on Bacteroides fragilis cell and phage survival after treatment with DNA damaging agents

Abstract Bacteroides fragilis Bf-2 cells were more sensitive to far-UV radiation, N -methyl- N '-nitrosoguanidine, ethylmethane sulphonate, acriflavine and mitomycin C under aerobic conditions than under anaerobic conditions. The opposite effect was observed with H₂O₂-treated cells and exposure to O₂ enhanced the survival of H₂O₂-treated cells. Pretreatment of cells with sublethal concentrations of H₂O₂ also increased the survival of H₂O₂-treated cells. Reactivation of UV- and X-irradiated and methylmethane sulphonate and H₂O₂-treated phage b-1 was induced by O₂ and H₂O₂ in B. fragilis .     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Sensitive detection of RNA using strand-specific M13 probes

We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.