Determination of the enzymatic traits of anaerobic gram-negative nonsporulating bacteria

The method requiring the use of a semi-liquid medium with the reduced content of protein hydrolysates and with bromo-thymol blue added as indicator and permitting the determination of sugar fermentation by bacteroids and fusobacteria is presented. The determination is made by a change in the color of the indicator and the intensity of microbial growth (opacity) after the incubation of the inoculated medium under free access of air. A study has been made for comparing the above method with the electrometric method for the determination of fermentative features in anaerobic microorganisms.     

一株瘤胃厌氧菌Actinomyces ruminicola的分离鉴定

目的从健康奶牛瘤胃液中分离、筛选出1株以产乙酸为主Actinomyces ruminicola。方法无菌采取装有瘤胃瘘奶牛的瘤胃液,按照厌氧茵分离步骤,通过Actinomyces ruminicola的特异性培养基进行筛选,提取分离菌的基因组DNA,克隆其16SrRNA基因,进行序列测定,分离出1株Actinomyces ruminicola。结果通过形态学观察、生化反应和序列分析证实所分离的1株产乙酸的杆菌为Actinomyces ruminicola。结论从健康牛瘤胃液中成功分离出1株Actinomyces ruminicola,为进一步研究其对瘤胃发酵的影响奠定了基础。     

Reclassification of Corynebacterium pyogenes (Glage) in the genus Actinomyces, as Actinomyces pyogenes comb.nov

Corynebacterium pyogenes (Glage) differs to such an extent from the type species of Corynebacterium, Corynebacterium diphtheriae (Lehmann and Neumann), that it cannot be retained in this genus. Numerical phenetic and chemical data indicate a close relationship between Corynebacterium pyogenes and the species Actinomyces bovis (Harz). It is proposed that Corynebacterium pyogenes be reclassified in the genus Actinomyces, as Actinomyces pyogenes (Glage) comb.nov.     

Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria

Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria.

Treatment of whole cell suspensions of $\text{Eubacterium aerofaciens}$ and $\text{Bacteroides fragilis}$ with lysozyme resulted in a marked increase (>100-fold) in the rates of biotransformation of cholate to 7-ketodeoxycholate (7-KD) in the former but only a 2-fold increase in the latter bacterium. In $\text{B. fragilis}$ the total activity of both NAD-dependent 7-α-hydroxysteroid dehydrogenase (7-α-OHSDH) and bile salt hydrolase (BSH) increase markedly during the stationary growth phase. Both enzymes were found in the spheroplast lysate and the Triton-soluble washed membrane fractions but only BSH was found in the spheroplast medium.     

Effect of solvents on obligately anaerobic bacteria

Growth of Acetobacterium woodii and Clostridium sporogenes was studied in the presence of water-immiscible solvents. Nitrogen purging, vacuum distillation or distillation under nitrogen were all suitable as methods to remove oxygen from the solvents, since growth rates and yields of A. woodii were unaffected in the presence of tetradecane which had been degassed by these methods. Varying the solvent volume from 20% to 80% of the culture volume had little effect on growth rate of A. woodii. A.woodii was relatively sensitive to organic solvents since growth was inhibited by alkanes with logP(octanol/water) values below 7.1. C. sporogenes was less solvent sensitive, since it grew without inhibition when the logP of the solvent was > or = 6.6. Nevertheless, both A. woodii and C. sporogenes were more sensitive to solvent polarity than aerobic bacteria.     

Binding of alpha 2-macroglobulin and haptoglobin to Actinomyces pyogenes

All 25 cultures of Actinomyces pyogenes tested in the present study bound 125I-labelled human alpha 2-macroglobulin with a mean binding of 65.6%. Thirteen cultures also bound 125I-labelled human haptoglobin with a mean of 51.5%. None interacted with fibrinogen, fibronectin, immunoglobulin G, or albumin. Twenty-eight cultures representing other species of actinomycetaceae did not show any interaction with alpha 2-macroglobulin, haptoglobin, and other plasma proteins tested. The binding of alpha 2-macroglobulin and haptoglobin to A. pyogenes was saturable and could be completely inhibited by the respective unlabelled plasma proteins. The binding of alpha 2-macroglobulin could not be inhibited by unlabelled haptoglobin. On the other hand, alpha 2-macroglobulin blocked the binding of haptoglobin, possibly by steric hindrance. Treatment of the bacteria with trypsin reduced their binding activities for alpha 2-macroglobulin and haptoglobin indicating the protein nature of the binding sites. Exposure to heat (1 h, 80 degrees C) significantly diminished the binding activity for haptoglobin, but not that for alpha 2-macroglobulin. The binding of alpha 2-macroglobulin and haptoglobin could be an important feature in the classification of A. pyogenes among the members of actinomycetaceae.     

Effect of alkyldimethylamine oxides on anaerobic sporulating bacteria of genusClostridium

Antibacterial activity of alkyldimethylamine oxides and lodaminox was determinedin vitro in 11 strains of clostridia. The most efficient was (1-methyldodecyl)dimethylamine oxide (MIC=7.8–78 μmol/L). Iodaminox was about twice as efficient (MIC=3.9–31 μmol/L). The bactericidal activity of both compounds was tested at different periods of exposure, ambient pH and cultivation temperature on three species of clostridia. The activity of both compounds was the highest at pH 6 and 40°C.     

Studies on anaerobic bacteria

Summary A new chromogenic anaerobe, Clostridium roseum nov. spec., has been found. It is characterized by: red-orange pigment, turning purplish on oxidation; gelatin liquefaction and other evidence of proteolysis; nitrate reduction; fermentation of various carbohydrates including pectin; close resemblance to Cl. acetobutylicum in corn mash fermentation, with the same neutral products, acetone, ethyl alcohol and butyl alcohol, in nearly the same ratios; agglutinative specificity and separation from Cl. acetobutylicum and Cl. felsineum, as well as several less nearly physiologically related butyric anaerobes.     

Effect of selective media on recovery of obligately anaerobic gram-negative rods from human faeces

Total numbers and gross composition of the anaerobic human faecal flora were compared using non-selective and selective media. Combinations of selective agents to suppress the gram-negative part of the flora such as vancomycin and neomycin, vancomycin and kanamycin, or kanamycin and bile were found to reduce total numbers of recovered obligately anaerobic gram-negattive rods by 50–75%. With reference to experiments with penicillin as selective agent, underlying mechanisms for this phenomenon are discussed. It is concluded that selective media should not be used for quantitative enumeration of anaerobic gram-negative rods from the faecal flora.     

Effect of the medium redox potential on the growth and metabolism of anaerobic bacteria

Based on the available literature data on a decrease in the redox potential of medium to low negative values and a decrease in pH during the growth of sugar-fermenting anaerobic bacteria, it was concluded that these processes cannot be described by the theory of redox potential. A theory was developed according to which the regulation of bacterial metabolism is accomplished through changes in the redox potential. The theory considers the redox potential as a factor determining the growth of anaerobic bacteria, which is regulated by oxidizers and reducers. The assumption is put forward that, under anaerobic conditions, bacteria are sensitive to changes in the redox potential and have a redox taxis. The effect of the redox potential on the transport of protons and other substances through membranes and the activity of membrane-bound enzymes, including the proton F1-F0-ATPase, whose mechanisms of action involve changes in the proton conductance of the membrane, the generation of proton-driving force, and dithiol-disulfide transitions in proteins was studied.     

Enumeration of cellulolytic anaerobic bacteria from the bovine rumen: Comparison of three methods

Three methods—the most probable number technique, a cellulose agar overlay on basal carbohydrate plates, and carboxymethylcellulose in basal carbohydrate plates—were compared for ease of preparation, interpretation of results, and agreement in estimation of size of the cellulolytic bacterial population in digesta samples from the rumen. The most probable number method yielded consistent detection of cellulose hydrolysis in liquid medium but required at least a 10-day incubation, and its mean was associated with wide 95% confidence intervals. The cellulose overlay method was the least consistent, and zones of hydrolysis often were difficult to see. The carboxymethylcellulose method was the easiest method for preparation and required only a 2-day incubation. The three methods estimated the same population size (all within one-half log unit of each other), but the carboxymethylcellulose method had the lowest coefficient of variation.     

Effect of radiation dose on the recovery of aerobic and anaerobic bacteria from mice

The presence of aerobic and anaerobic bacteria in the blood, spleen, and liver was investigated in mice that were exposed to 7, 8, 9, or 10 Gy 60Co radiation. Microorganisms were detected more often in animals exposed to higher doses of radiation. The number of mice that were culture positive and the number of isolates in one site increased with increasing dose. Bacteria were recovered in mice killed at various times after radiation, in 3 of 100 mice exposed to 7 Gy, in 13 of 100 irradiated with 8 Gy, in 23 of 90 exposed to 9 Gy, and in 34 of 87 irradiated with 10 Gy. The predominant organisms recovered were Escherichia coli, anaerobic Gram-positive cocci, Proteus mirabilis, Staphylococcus aureus, and Bacteroides spp. Escherichia coli and anaerobes were more often isolated in animals exposed to 10 Gy, while S. aureus was more often recovered in those irradiated with 9 Gy. These data demonstrate a relationship between the dose of radiation and the rate of infection due to enteric aerobic and anaerobic bacteria.