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PubMed1983

[Clinical evaluation of immunologic reactivity of the organism and immunotherapy in purulent surgical infection].

The reaction of leukocytolysis with microbial standard allergens is found to be more sensitive than the intracutaneous test to detect the bacterial allergy in patients with a purulent surgical infection. The microbial allergization of the organism is followed by a considerable inhibition of the humoral and cell immunity. Immunotherapy must be included in the complex treatment of patients with purulent surgical infection for elimination of the specific sensibilization and improvement of the immune reactivity of the organism.

Antibody Formation↗

PubMedDate not supplied

[Detection of microorganisms in the environment and the preliminary appraisal of their physiological state by X-ray microanalysis].

The paper deals with the X-ray microanalysis of the elemental composition of bacteriomorphic particles in 170,000-year old Antarctic permafrost sediments and in indoor dust. A comparative analysis of the phosphorus, sulfur, calcium, and potassium contents and the Ca/K and P/S ratios in these particles and in reference microbial cells occurring in different physiological states showed that the absence of P and/or S peaks in the X-ray spectrum of an object may indicate that it is abiotic. Resting microbial forms can be revealed on the basis of the following characteristic features: an increased content of Ca, a high Ca/K ratio, and a low P/S ratio. Model experiments with nonviable bacterial and yeast micromummies with alterations in the structural and barrier functions of the cytoplasmic membrane showed that micromummies can be recognized by a super-high content of a marker element (e.g., P, K, or Si), accumulated due to facilitated diffusion along the deliberately created concentration gradient. Such an analysis of the permafrost sediment and dust made it possible to suggest the presence of mummified cells in these objects. The possibility of using X-ray microanalysis for the detection of microbial cells in natural habitats in order to enhance the efficiency of ecological monitoring of the environment is discussed.

Antarctic Regions↗

PubMed2003

Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids.

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.

Ammonia↗

PubMed2004

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

Bacteria↗

PubMed1983

Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur.

Sheep fed the forage Digitaria pentzii fertilized with sulfur were compared with those fed unfertilized forage for the rumen microbial population involved with fiber degradation. No differences were detected in the bacterial population as determined by anaerobic cultures on a habitat-simulating medium, xylan, or pectin, by 35S labeling techniques for microbial protein, or by transmission electron microscopic studies of bacterium-fiber interactions. Rumen volume and water flow from the rumen were not different for sheep fed each of the forages. Rumen fungi were prevalent in sheep fed sulfur-fertilized D. pentzii as shown by sporangia adhering to forage fiber and by colonies developing from zoospores in roll tubes with cellobiose plus streptomycin and penicillin. Fungi were absent or in extremely small numbers in sheep fed unfertilized forage. Nylon bag digestibility studies showed that the fungi preferentially colonized the lignified cells of blade sclerenchyma by 6 h and caused extensive degradation by 24 h. In the absence of bacteria in in vitro studies, extensive hyphal development occurred; other lignified tissues in blades (i.e., mestome sheath and xylem) were attacked, resulting in a residue with partially degraded and weakened cell walls. Nonlignified tissues were also degraded. Breaking force tests of leaf blades incubated in vitro with penicillin and streptomycin and rumen fluid from sheep fed sulfur-fertilized forage or within nylon bags in such sheep showed a residue at least twice as fragile as that from sheep fed unfertilized forage. In vitro tests for dry matter loss showed that rumen fungi, in the absence of actively growing bacteria, could remove about 62% of the forage material. The response of rumen fungi in sheep fed sulfur-fertilized D. pentzii afforded a useful in vivo test to study the role of these microbes in fiber degradation. Our data establish that rumen fungi can be significant degraders of fiber and further establish a unique role for them in attacking and weakening lignocellulosic tissues. The more fragile residues resulting from attack by fungi could explain the greater intake consistently observed by sheep eating sulfur-fertilized compared with unfertilized D. pentzii forage.

Animal Feed↗

PubMed2005

The microbial selenoproteome of the Sargasso Sea.

BACKGROUND: Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project. RESULTS: We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted redox proteins containing catalytic selenocysteines. Several bacterial selenoproteins previously thought to be restricted to eukaryotes were detected by analyzing eukaryotic and bacterial SECIS elements, suggesting that eukaryotic and bacterial selenoprotein sets partially overlapped. The Sargasso Sea microbial selenoproteome was rich in selenoproteins and its composition was different from that observed in the combined set of completely sequenced genomes, suggesting that these genomes do not accurately represent the microbial selenoproteome. Most detected selenoproteins occurred sporadically compared to the widespread presence of their cysteine homologs, suggesting that many selenoproteins recently evolved from cysteine-containing homologs. CONCLUSIONS: This study yielded the largest selenoprotein dataset to date, doubled the number of prokaryotic selenoprotein families and provided insights into forces that drive selenocysteine evolution.

Algorithms↗

PubMed2006

Evaluation of freeze-dried kefir coculture as starter in feta-type cheese production.

The use of freeze-dried kefir coculture as a starter in the production of feta-type cheese was investigated. Maturation of the produced cheese at 4 degrees C was monitored for up to 70 days, and the effects of the starter culture, the salting method, and the ripening process on quality characteristics were studied. The use of kefir coculture as a starter led to increased lactic acid concentrations and decreased pH values in the final product associated with significantly higher conversion rates compared to salted rennet cheese. Determination of bacterial diversity at the end of the ripening process in salted kefir and rennet cheeses by denaturing gradient gel electrophoresis technology, based on both DNA and RNA analyses, suggested a potential species-specific inhibition of members of the genera Staphylococcus and Psychrobacter by kefir coculture. The main active microbial associations in salted kefir cheese appeared to be members of the genera Pseudomonas and Lactococcus, while in salted rennet cheese, Oxalobacteraceae, Janthinobacterium, Psychrobacter, and Pseudomonas species were noted. The effect of the starter culture on the production of aroma-related compounds responsible for cheese flavor was also studied by the solid-phase microextraction-gas chromatography-mass spectrometry technique. Kefir coculture also appeared to extend the shelf life of unsalted cheese. Spoilage of kefir cheese was observed on the 9th and 20th days of preservation at 10 and 5 degrees C, respectively, while spoilage in the corresponding rennet cheese was detected on the 7th and 16th days. Microbial counts during preservation of both types of unsalted cheese increased steadily and reached similar levels, with the exception of staphylococci, which were significantly lower in unsalted kefir cheese. All types of cheese produced with kefir as a starter were approved and accepted by the panel during the preliminary sensory evaluation compared to commercial feta-type cheese.

Animals↗

PubMed2001

Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces.

AIMS: The objectives of this work were to develop a selective and/or differential medium able to efficiently recover Dekkera/Brettanomyces sp. from wine-related environments and to determine the relationship between these yeasts and the 4-ethylphenol content in a wide range of wines. METHODS AND RESULTS: The selectivity of the developed medium was provided by the addition of ethanol, as single carbon source, and cycloheximide. The inclusion of bromocresol green evidenced acid-producing strains. The inclusion of p-coumaric acid, substrate for the production of 4-ethylphenol, enabled the differentiation by smell of Dekkera/Brettanomyces sp. from all other yeast species growing in the medium. The medium was used either by plating after membrane filtration or by the Most Probable Number (MPN) technique. In 29 white and 88 red randomly collected wines, these yeasts were found only in red wines at levels up to 2500 MPN ml-1, but constituted less than 1% of the total microbial flora. In red wines, 84% showed detectable amounts of 4-ethylphenol up to 4430 microg l-1 while 28% of the white wines showed detectable levels up to 403 microg l-1. CONCLUSION: The use of the medium proposed in this work evidenced the presence of low relative populations of Dekkera/Brettanomyces sp. even in wines contaminated by fast-growing yeasts and moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: Further ecological studies on Dekkera/Brettanomyces sp. should take into account the use of highly specific culture media in order to establish their true occurrence in nature.

Cell Division↗

PubMed1991

Methylated sulfur compounds in microbial mats: in situ concentrations and metabolism by a colorless sulfur bacterium.

The concentrations of the volatile organic sulfur compounds methanethiol, dimethyl disulfide, and dimethyl sulfide (DMS) and the viable population capable of DMS utilization in laminated microbial ecosystems were evaluated. Significant levels of DMS and dimethyl disulfide (maximum concentrations of 220 and 24 nmol cm3 of sediment-1, respectively) could be detected only at the top 20 mm of the microbial mat, whereas methanethiol was found only at depth horizons from 20 to 50 mm (maximum concentration of 42 nmol cm3 of sediment-1). DMS concentrations in the surface layer doubled after cold hydrolysis of its precursor, dimethylsulfoniopropionate. Most-probable-number counts revealed 2.2 x 10(5) cells cm3 of sediment-1, in the 0- to 5-mm depth horizon, capable of growth on DMS as the sole source of energy. An obligately chemolithoautotrophic bacillus designated strain T5 was isolated from the top layer of the marine sediment. Continuous culture studies in which DMS was the growth-limiting substrate revealed a maximum specific growth rate of 0.10 h-1 and a saturation constant of 90 mumol liter-1 for aerobic growth on this substrate.

Disulfides↗

PubMed1991

Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways.

CD4+ T cells are equipped to detect two major classes of ligands. Infectious microbial agents, including bacteria and retroviruses, carry a class of proteins termed superantigens that are recognized by the T-cell receptor in association with class II products of the major histocompatibility complex. Proteins expressed by other cells and organisms are processed by macrophages into peptides that are presented to CD4+ T cells by class II molecules. We have examined CD4+ T-cell clones that proliferate vigorously in response both to conventional peptide antigens and to bacterial or retroviral superantigens. The response to peptide antigen is characterized by a rapid and sustained increase in the levels of intracellular free Ca2+ and a vigorous cytokine response. In contrast, the proliferative response of these clones to bacterial or retroviral superantigen is not accompanied by detectable increases in intracellular Ca2+ or by significant cytokine production. Further analysis of T-cell activation indicates that interaction of a single T-cell receptor with the two types of ligand may be coupled to functionally distinct signaling pathways that interact in a synergistic fashion to achieve T-cell activation.

Animals↗

PubMed1984

High-performance liquid chromatography of microbial acid metabolites.

The use of high-performance liquid chromatography with a cation-exchange column and effluent monitoring at 210 nm has been evaluated for the profiling of selected microbial metabolites including aliphatic, dicarboxylic, and phenolic acids, as an adjunct to the identification of selected bacteria, detection of bacterial metabolites in foods, and the monitoring of industrial microbial fermentations. Advantages of the technique include the simultaneous profiling of different classes of organic acids without derivatization. Most applications require only qualitative or semi-quantitative data. For others, data are given on the day-to-day reproducibility for several acids.

Bacteria↗

PubMed2004

Oligonucleotide microarrays in microbial diagnostics.

Oligonucleotide microarrays offer a fast, high-throughput alternative for the parallel detection of microbes from virtually any sample. The application potential spreads across most sectors of life sciences, including environmental microbiology and microbial ecology; human, veterinary, food and plant diagnostics; water quality control; industrial microbiology, and so on. The past two years have witnessed a rapid increase of research in this field. Many alternative techniques were developed and validated as seen in 'proof-of-concept' articles. Publications reporting on the application of oligonucleotide microarray technology for microbial diagnostics in microbiology driven projects have just started to appear. Current and future technical and bioinformatics developments will inevitably improve the potential of this technology further.

Ecosystem↗

PubMed1998

Rapid chair-side DNA probe assay of Bacteroides forsythus and Porphyromonas gingivalis.

This study compared a rapid, colorimetric DNA probe assay designed to be performed in a dental office within 40 min, with anaerobic culture and indirect immunofluorescence microscopy (IFM) for detection of Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque samples. The DNA probe assay used the Periodontal Microbial Identification Test (Saigene Corporation, Bothell, Washington, USA). B. forsythus was detected in 46 (52%), 49 (55%) and 39 (44%) of the samples by DNA probe, culture (at levels > or = 10(5)) and IFM, respectively. P. gingivalis was detected in 24 (27%), 18 (20%) and 29 (33%) of the samples by DNA probe, culture (at levels > or = 10(5)) and IFM, respectively. Results from the DNA probe assay were compared to culture. Culture negative, probe positive samples were re-evaluated by IFM, and IFM positive samples were considered positive in "resolved" data. Using resolved data. DNA probe detection sensitivity and specificity values for B. forsythus were 81% and 91% and for P. gingivalis were 80% and 95%, respectively. DNA probe test results were further compared with culture and IFM. For samples negative by both culture and IFM, probe specificity was 92% in 25 B. forsythus samples and 95% in 57 P. gingivalis samples. For samples positive by both reference methods, probe sensitivity was 82% in 27 B. forsythus samples and 73% in 15 P. gingivalis samples. B. forsythus was detected more frequently by culture compared with IFM; the reverse was observed for P. gingivalis. The rapid DNA probe assay for B. forsythus and P. gingivalis was comparable to cultivable and IF analyses.

Adult↗

PubMed2004

Real-time PCR in the microbiology laboratory.

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

Animals↗

PubMed1998

Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution.

BACKGROUND: Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS: The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS: The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMedDate not supplied

Delvotest SP for detection of cloxacillin and sulfamethoxazole in milk: IDF interlaboratory study. International Federation Dairy.

Under the auspices of the International Dairy Federation's Group E503, a collaborative study of the Delvotest SP multiplate microbial inhibitor test was performed to gain information about the detection limits of 2 antimicrobial agents (a beta-lactam and a sulfa compound), the variation of test results between users and 2 batches of the test, and the reasons for deviating results. Lyophilized milk samples spiked with various concentrations of cloxacillin or sulfamethoxazole were analyzed. Each substance/concentration combination was tested with each of 2 test batches 14 or 15 times per participating laboratory. Test results were to be read by more than one person and reported on separate forms. Results were obtained from 29 laboratories, which included 5 with no experience and 11 with limited experience with this test. Detection limits for cloxacillin (22.5 or 30 micrograms/kg, depending on batch) and sulfamethoxazole (45 micrograms/kg) were established from dose-response curves. A small difference in cloxacillin detection levels between the 2 test batches was observed. Analyses of samples gave almost unanimous results (> or = 95%). Of deviating results, defined as anomalous results (1.4% of readings), half could be attributed to human errors and half to procedural errors.

Animals↗

PubMed2001

A real-time approach to detect seal defects in food packages using ultrasonic imaging.

The microbial integrity of many types of flexible food packages depends on a zero defect level in the fused seam seal. Human inspection for defects in these seals is marginal at best, and secondary incubation protocols are often used to spot packages with compromised integrity before releasing product for sale. A new type of inspection method has been developed and is being evaluated for robustness. The purpose of the study was to evaluate a new raster scanning geometry to simulate continuous motion, online ultrasonic inspection of the seal region in flexible food package seals. A principal engineering tradeoff of scanning inspection systems is between increased line speed that results from decreased spatial sampling (less acquired data to process) and decreased image quality. The previously developed pulse-echo Backscattered Amplitude Integral (BAI) mode imaging technique is used to form ultrasound images using the new scanning geometry. At an ultrasonic frequency of 22.9 MHz, 38- and 50-microm-diameter air-filled channel defects in all-plastic transparent trilaminate are evaluated. The contrast-to-noise ratio (CNR) of the processed BAI-mode image is used to quantify image quality as a function of spatial sampling. Results show seal defects (38- and 50-microm diameter) are still detectable for undersampled conditions, although image quality degrades as spatial sampling decreases. Further, it is concluded that the raster scanning geometry is feasible for online inspection.

Food Contamination↗

PubMed1969

Marine microorganisms associated with the food of young salmon.

The biomass of microorganisms in the digestive tracts of young salmon in the sea of Saanich Inlet was examined by (i) the direct microscopic method, (ii) the plate count method and (iii) the physiological method (microbial biomass assumed from the release of carbon dioxide). By all methods, most microbial biomass in foods was shown to decrease during its stay in the digestive organ. This observation was supported by the detection of many bacteria at different stages of digestion. The microbial biomass was quantitatively influenced by the composition of food ingested and by the sampling time.

Journal Article↗