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Survey of microorganism for the production of extracellular phytase.

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.

6-Phytase↗

Some factors affecting fermentation capacity and net growth of rumen microorganisms.

The fermentation rate of rumen microorganisms obtained from a sheep fed on hay was determined at different hours after feeding. The highest rate of fermentation was obtained immediately after feeding whereas the lowest rate was always before feeding. The effect of adding glucose, sucrose, and starch alone or in combination with urea, sodium citrate, or succinic acid on the maximal fermentation rate was studied. In no case was the maximal fermentation rate greater than that obtained in the presence of concentrate and straw. An investigation on the effect of C/N ratios, dilution, and substrate concentration on the net growth of rumen microorganisms was undertaken. The highest net growth value was obtained with a C/N ratio of 16.5 to 17.5, which is the ratio found for the ration fed to the animal. It was possible to increase net growth approximately threefold by using the proper dilution and fivefold by using the proper feed concentration.

Animal Feed↗

Observation of microorganisms in soil and other natural habitats.

A procedure is described for visually observing and following the activities and interactions of bacteria, actinomyctes, fungi, protozoa, nematodes, and plant roots in masses of soil. Specific microscope components and objectives are used, and the numerical apertures are adjusted such that light diffraction colors are produced to allow differentiation of the various biological entities and their habitat materials. Strains or other alterations in the organisms and their habitat are not employed, and time-lapse photography can be used to follow the activities of soil microorganisms and plant roots. As a result of the use of this technique, it is apparent that in situ indigenous soil microorganisms differ from similar organisms grown in the laboratory, but that, under the proper conditions, the state of the organism in either habitat can be altered to match that which occurs in the contrasting habitat.

Bacteria↗

Whole microorganisms studied by pyrolysis-gas chromatography-mass spectrometry: significance for extraterrestrial life detection experiments.

Pyrolysis-gas chromatography-mass spectrometric studies of two microorganisms, Micrococcus luteus and Bacillus subtilis var. niger, indicate that the majority of thermal fragments originate from the principal classes of bio-organic matter found in living systems such as protein and carbohydrate. Furthermore, there is a close qualitative similarity between the type of pyrolysis products found in microorganisms and the pyrolysates of other biological materials. Conversely, there is very little correlation between microbial pyrolysates and comparable pyrolysis studies of meteoritic and fossil organic matter. These observations will aid in the interpretation of a soil organic analysis experiment to be performed on the surface of Mars in 1975. The science payload of this landed mission will include a combined pyrolysis-gas chromatography-mass spectrometry instrument as well as several "direct biology experiments" which are designed to search for extraterrestrial life.

Journal Article↗

Microorganisms in unamended soil as observed by various forms of microscopy and staining.

A light-diffraction microscope was modified to allow sequential viewing of the microorganisms in a soil smear by transmitted, reflected, and reflected-polarized incandescent light and by reflected ultraviolet light. Observations were also made by conventional incandescent and ultraviolet transmitted-light microscopy. All results for the various forms of bright-field microscopy with stained and unstained soils were in agreement, but they differed from the results obtained for two types of ultraviolet-fluorescence microscopy. The latter proved to be nonspecific for in situ soil microorganisms. Capsule-like areas were noted surrounding many of the resident microbial cells of soil when viewed by the various forms of bright-field microscopy. These areas could not be stained or removed by a variety of treatments, but they apparently often did take up stain after in situ soil growth had been initiated. It was concluded that these areas are not capsules but may represent a structural component of nonmultiplying microbial cells in soil.

Acridines↗

Pressure transducer method for measuring gas production by microorganisms.

A simple method for measuring gas production by microorganisms by using a pressure transducer to sense pressure buildup was developed and tested with members of the coliform group. The test system consisted of a 5.0 lb/in(2) pressure transducer and a pressure equalizer valve attached to the metal cap of a test tube (20 by 150 mm); gas pressure was recorded on a strip-chart recorder. Gas pressure response curves consisted of (i) a lag period with no marked increase in pressure, (ii) a rapid pressure buildup period, and (iii) a leveling-off period. A linear relationship was established between inoculum size and length of the lag period. Cultures shaken at 200 oscillations/min showed a marked increase in rate of gas release over stationary cultures. Cell concentrations at the time of rapid buildup in pressure were 10(8)/ml. Mean maximum pressure recordings, lb/in(2) per 10 ml of broth, were: Enterobacter aerogenes, 3.70; Citrobacter intermedium, 2.70; and Escherichia coli, 2.10. Mean CO(2) concentrations, ppm of headspace gas, for E. coli were: (i) 2,000 at time of inoculation, (ii) 25,000 at time of rapid buildup in pressure, and (iii) 150,000 at maximum pressure. These results indicate the potential application of the pressure transducer method for rapidly detecting coliforms and other gas-producing microorganisms in clinical samples and in sterility testing of foods.

Bacteria↗

Stress: a factor to be considered in heterotrophic microorganism enumeration from aquatic environments.

Heterotrophic microorganisms in water samples are susceptible to the transient stress of warmed agar used in the standard methods pour plate procedure, causing significantly decreased recoveries in comparison with a spread plate technique. Microbial starvation can increase susceptibility to a transient warming stress. The standard plate count procedure, as presently described, should not be considered for quantitation of microorganisms from aquatic environments.

Agar↗

Specimen holder to critical-point dry microorganisms for scanning electron microscopy.

Critical-point drying of microorganisms for scanning electron microscopy can be rapidly and effectively accomplished by use of a newly described specimen holder. Up to eight different samples of spores or vegetative cells are placed between polycarbonate membrane filters in the holder and processed through solvent dehydration and critical-point drying using carbon dioxide without loss or cross contamination of microorganisms. Yeasts, molds, bacteria, and actinomycetes have been successfully processed.

Ascomycota↗

Agar plate method for detection and enumeration of alkylbenzenesulfonate-degrading microorganisms.

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.

Aerobiosis↗

Seroprevalence of antibodies to microorganisms known to cause arterial and myocardial damage in patients with or without coronary stenosis.

Infections are assumed to play a role in coronary artery disease (CAD) and cardiomyopathies. It is unknown whether the seroprevalence of antibodies to these microorganisms is higher in patients with than without CAD. The seroprevalence of antibodies to Bartonella henselae, Borrelia burgdorferi, Chlamydia pneumoniae, Coxiella burnetii, Helicobacter pylori, human granulocytic Ehrlichia, Leptospira, Rickettsia conorii, and Treponema pallidum was assessed prospectively in patients with exertional dyspnea or anginal chest pain who underwent coronary angiography because of suspected CAD. Patients with normal angiograms (NA) were those in whom no more than 50% stenosis of any coronary artery was found. Patients with CAD were patients who underwent percutaneous transluminal coronary angioplasty. There were 50 patients with CAD (9 female) and 62 with NA (25 female), with a mean age of 62 years. All patients had antibodies to at least one microorganism: to B. henselae, 8% of CAD patients and 5% of NA patients; to B. burgdorferi IgG, 14% CAD and 6% NA; to B. burgdorferi IgM, 6% CAD and 3% NA; to C. pneumoniae lipopolysaccharide (LPS) IgA, 76% CAD and 77% NA; to C. pneumoniae LPS IgG, 80% CAD and 90% NA; to C. burnetii, 0% CAD and 5% NA; to H. pylori, 92% CAD and 68% NA; to human granulocytic Ehrlichia, 8% CAD and 3% NA; to Leptospira IgG, 4% CAD and 2% NA; to R. conorii, 10% in both groups; and to T. pallidum, 2% CAD and 0% NA. The seroprevalence of antibodies to micro-organisms known to induce arterial and myocardial damage does not differ between patients with CAD and NA.

Adult↗

Microorganisms resistant to free-living amoebae.

Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

Amoeba↗

Automated systems for identification of microorganisms.

Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided.

Bacteria↗

Adsorption of lysozyme from human whole saliva by Streptococcus sanguis 903 and other oral microorganisms.

Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supernatant. Bacteria were incubated in saliva for 60 min at 37 degrees C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90 degrees C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37 degrees C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 mug of adsorbed lysozyme per 10(7) cells) at 20 mug of lysozyme added per ml. Salivary lysozyme adsorption by several other oral microorganisms (A. viscosus WVU 626 and WVU 627, S. sanguis 73x11, S. mutans BHT, and S. salivarius NG) was similar to that of S. sanguis 903 in sensitivity to ionic strength. Lysozyme adsorption by S. sanguis 903 from either a buffer solution or a saliva supernatant was more sensitive to ionic strength at 0 than at 37 degrees C. On the basis of results from experiments in saliva versus buffer, we concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength. A comparison of pH and ionic strength effects on lysozyme adsorption by S. sanguis 903 with literature reports of lysozyme lysis of whole cells and hydrolysis of cell walls, peptidoglycan, and (GlcNAc)(4) suggested that adsorption by S. sanguis 903 was more dependent on electrostatic interactions than was lysozyme catalysis. The possibility is discussed that anionic bacterial surface components mediate lysozyme adsorption and temper the potential effects of lysozyme on the microorganisms.

Actinomyces↗

Nonspecific induction of immunoglobulin M antibodies to periodontal disease-associated microorganisms after polyclonal human B-lymphocyte activation by Fusobacterium nucleatum.

The production of antibodies to oral bacteria was determined in lymphocyte cultures stimulated with sonicated Fusobacterium nucleatum, a potent inducer of polyclonal B-cell activation. After 9 days the cultures were examined by a microenzyme-linked immunosorbent assay for immunoglobulin M (IgM) antibodies to F. nucleatum, Bacteroides gingivalis, Actinomyces viscosus, and Streptococcus sanguis. Antibodies to these four bacteria were detected in cultures stimulated with polyclonal B-cell activation-inducing concentrations of F. nucleatum. However, significant concentrations of antibodies to F. nucleatum, but not to the other three microorganisms, were produced in cultures that received suboptimal polyclonal B-cell activation-inducing doses of F. nucleatum. Absorption studies indicated the specificity of the antibodies to each of the bacteria tested. IgM antibody production induced by F. nucleatum was enhanced by the addition of T cells. The production of IgM antibodies to the bacteria was reproducible in cultures from a single person tested on 3 consecutive days. The concentration of antibodies in replicate cultures, however, fluctuated greatly. To obtain consistent responses on successive days, multiple replicate cultures were required. These results suggest that F. nucleatum, which is frequently present in subgingival plaque, could induce the production of antibodies not only to F. nucleatum, but also to other microorganisms associated with periodontal diseases.

Antibody Specificity↗

Human immune responses to oral microorganisms: patterns of systemic antibody levels to Bacteroides species.

Human systemic antibody levels to oral members of the Bacteroides genus were assessed with an enzyme-linked immunosorbent assay. Antibody levels to B. gingivalis, two homology groups of B. intermedius, B. melaninogenicus, B. denticola, B. loescheii, B. corporis, B. oralis, B. buccae, and B. gracilis were determined in subjects with localized juvenile periodontitis, advanced destructive periodontitis, or adult periodontitis and in normal persons. Significantly elevated serum immunoglobulin G (IgG) antibody levels to B. gingivalis were seen in adult and advanced destructive periodontitis patients. Serum IgM and IgA antibodies were increased in diseased versus normal subjects, whereas negligible levels of serum IgE antibody were detected to this microorganism. Serum IgG antibody levels to B. intermedius were increased in advanced destructive periodontitis patients; however, the frequency of elevated responses were similar among the groups. Extreme antibody levels to the other Bacteroides spp. were occasionally observed in this population. Additionally, all of the elevated levels were found in diseased patients. Distribution analyses of the antibody levels indicated that most patients exhibited a pattern of elevated antibodies to a limited number of the oral Bacteroides spp. The results suggested that elevated systemic antibody levels to oral Bacteroides spp. are more frequently found in periodontal disease patients. These antibody responses presumably reflect a colonization of the patients. The distribution of the responses may indicate the potential pathogenicity of the microorganisms and is consistent with distinctive host-parasite interactions in this disease.

Adolescent↗

Quantitative comparisons of potentially cariogenic microorganisms cultured from noncarious and carious root and coronal tooth surfaces.

Potentially cariogenic microorganisms cultured from noncarious and carious root and coronal (enamel) surfaces were quantitatively compared in patients 22 to 84 years of age (mean, 52 years). We collected 150 plaque specimens from 26 in situ teeth with initial root lesions and from 25 extracted teeth with advanced root lesions. The frequencies of isolation of Streptococcus mutans, Actinomyces viscosus, and Lactobacillus spp. were, respectively, 94, 72, and 51% at the noncarious root site; 98, 71, and 54% at the root lesion; 84, 61, and 44% at the noncarious enamel site; and 100, 66, and 90% at the enamel lesion. The streptococci made up the largest mean proportion of the total anaerobic cultivable microflora, ranging from 31.2% at the noncarious enamel site to 37.6% at the root lesion, while S. mutans varied between 18% at the noncarious enamel and root surfaces and approximately 24% at both the enamel and root lesions. The proportion of actinomyces ranged from 12.3% at the root lesion to 23.6% at the noncarious root site, while A. viscosus varied from 7.8% at the root lesion to 15.1% at the noncarious root site. The largest mean proportion of lactobacilli (4.2%) was recovered at the enamel lesion site. Proportions of Candida spp. made up less than 0.1% at all sites. Proportions of microorganisms did not differ significantly between noncarious enamel and root sites, but the noncarious coronal and root sites had higher (P less than 0.05) proportions of actinomyces than did the root lesion. Also, enamel lesions had a greater (P less than 0.05) percentage of Lactobacillus spp. than did root lesions. The number of streptococci recovered from the root lesion was greater (P less than 0.01) than the number of actinomyces at the same site. S. mutans was recovered from initial root lesions in greater numbers (P less than 0.001) than were actinomyces and lactobacilli. The number of S. mutans recovered at the initial root lesions was greater (P less than 0.01) than that recovered from the advanced root lesions.

Actinomyces↗

Relative availability of transferrin-bound iron and cell-derived iron to aerobactin-producing and enterochelin-producing strains of Escherichia coli and to other microorganisms.

A method is described for determination of the relative availability of transferrin-bound iron and cell-derived iron to microbial iron-scavenging mechanisms. This involved incubation of parallel cultures of microorganisms in dialysis tubes placed in RPMI 1640 tissue culture medium containing 30%-iron-saturated transferrin and K562 erythroleukemia cells. In one culture the transferrin was labelled with 59Fe and in the other the cells were labelled, and the relative uptake of radioiron by the microorganisms determined. The results showed that Staphylococcus epidermidis and Staphylococcus aureus acquired iron predominantly from cells, while Candida albicans and the enteropathogenic Escherichia coli NCTC 8623 tended to acquire iron from transferrin. E. coli K-12 strains W3110 and LG1705, which (like NCTC 8623) produce the siderophore enterochelin but not aerobactin, acquired predominantly transferrin-bound iron, whereas the related E. coli strains LG1315 and LG1628, which produce aerobactin but not enterochelin, showed a preference for cell-derived iron. When the cells were incubated in the presence of 59Fe-labelled transferrin and 55Fe-labelled ferritin, no difference in relative availability of iron to E. coli was observed, suggesting that differences in the ability of aerobactin and enterochelin to remove iron from intracellular ferritin were not responsible for this preference. These results may help to explain why production of aerobactin, despite its relatively low affinity for iron, is more closely associated with invasiveness in E. coli than is enterochelin production. Reduced availability of cell-bound iron during inflammation may contribute to antimicrobial defenses.

Candida albicans↗

Effects of fecal microorganisms and their chloroform-resistant variants derived from mice, rats, and humans on immunological and physiological characteristics of the intestines of ex-germfree mice.

In order to elucidate the nature of intestinal flora affecting the immunological and physiological parameters of the intestine, we produced several kinds of ex-germfree mice associated with fecal organisms and their chloroform-resistant variants derived from mice, rats, and humans. The phenotypes of intraepithelial lymphocytes were changed to those in conventional mice, particularly the increased positive percentage of alpha beta T-cell-receptor and Thy-1-bearing T cells, on association of the microorganisms (MF) and their chloroform resistant variants (MChl) derived from mice, but not rats and humans, with germfree mice. The cytolytic activity of intraepithelial lymphocytes was expressed only in the MF and MChl groups. The induction of the synthesis of fucosyl asialo GM1 glycolipid, the expression of major histocompatibility complex class II molecule, an increase in the mitotic indices of colonic epithelial cells, and a decrease in lactase activity of the small intestinal epithelial cells also occurred only in the two groups. On the other hand, the cecal size (cecal weight/body weight ratio) was reduced in the mice of all groups examined here, there being approximately the same amount and composition of organic acids, such as acetic acid, butyric acid, and propionic acid, in the cecal contents. Taken together, the results suggest that mouse-specific and chloroform-resistant microorganisms, which are difficult to cultivate at present, may contribute to alteration of the immunological and epithelial characteristics of the mouse intestine. Another factor derived from the intestinal flora, for example, bacterial metabolites such as organic acids, may also affect the cecal size.

Animals↗