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PubMed2000

Detection and substrate selectivity of new microbial D-amino acid oxidases.

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.

Journal Article↗

PubMed2004

Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds.

OBJECTIVE: To evaluate sensitivity of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis (MAP) in large dairy herds and assess the use of the method for estimation of MAP prevalence. ANIMALS: 1,740 lactating cows from 29 dairy herds in California. PROCEDURE: Serum from each cow was tested by use of a commercial ELISA kit. Individual fecal samples were cultured and used to create pooled fecal samples (10 randomly selected fecal samples/pool; 6 pooled samples/herd). Sensitivity of MAP detection was compared between Herrold's egg yolk (HEY) agar and a new liquid culture method. Bayesian methods were used to estimate true prevalence of MAP-infected cows and herd sensitivity. RESULTS: Estimated sensitivity for pooled fecal samples among all herds was 0.69 (25 culture-positive pools/36 pools that were MAP positive). Sensitivity increased as the number of culture-positive samples in a pool increased. The HEY agar method detected more infected cows than the liquid culture method but had lower sensitivity for pooled fecal samples. Prevalence of MAP-infected cows was estimated to be 4% (95% probability interval, 2% to 6%) on the basis of culture of pooled fecal samples. Herd-level sensitivity estimate ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity for culture of pooled fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Use of pooled fecal samples from 10 cows was a cost-effective tool for herd screening and may provide a good estimate of the percentage of MAP-infected cows in dairy herds with a low prevalence of MAP.

Animals↗

PubMed1978

Gas liquid chromatography for detection of bacteriuria: examination for volatile acidic and neutral compounds.

Direct injection of clinically infected urines on porous-polymer columns was investigated to determine which microbial metabolites were consistently detectable, and whether their presence could be used as a reliable index of infection. Chromosorb 101 was found to be the most suitable porous polymer for the detection of microbial metabolites; greater sensitivity of detection was achieved by partial purification of the urine before injection. Acetic acid was the only compound found consistently and it enabled 10(6) microorganisms per ml to be detected in urine. However, as urinary tract infection is diagnosed by the presence of 10(5) organisms or more per ml, our method is insufficiently sensitive for the detection of bacteriuria. Escherichia coli, Enterobacter cloacae, Klebsiella spp, Proteus spp, Stapyhlococcus albus, and Streptocococcus faecalis were detectable by our method but Pseudomonas aeruginosa and Candida albicans were not.

Acetates↗

PubMed2004

Multipathogen oligonucleotide microarray for environmental and biodefense applications.

Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

Bacteria↗

PubMed2006

Estimation of microbial growth using population measurements subject to a detection limit.

In this paper we develop a maximum likelihood estimation procedure for determining the mean and variance in microbial population size from microbial population measurements subject to a detection limit. Existing estimation methods generally set non-detectable measurements equal to the detection limit and are highly biased. Because changes in the mean and variance in the microbial population size are typical in industrial processes we also outline statistical tests for detecting such changes when measurements are subject to a detection limit, which is critical for process control. In an industrial process there may also potentially be variability in the microbial growth rate due to variation in the microbial strain, environment, and food characteristics. Accordingly, we also present a maximum likelihood procedure for estimating microbial growth model parameters and their variance components from microbial population measurements subject to a detection limit. Such information can be used to generate the mean and variance through time of the microbial population size, which is vital for the application of predictive microbiological models to risk assessment and food product shelf-life estimation.

Bacteria↗

PubMed2000

Microbial contamination of in-use lubricants for non-touch urethral catheters in intermittent self-catheterization.

There are no reports on microbial contamination of repeatedly used lubricant (84-87% glycerin containing 0.02% benzalkonium chloride) for non-touch urethral catheters in intermittent self-catheterization. In this work, we evaluated microbial contamination of in-use lubricant and its prevention. Between September and December, 1996, microbial contamination and water activity of in-use lubricants in sheathed and lubricated non-touch catheters connected to a tube used by 46 outpatients at our hospital was examined. Microbial contamination was detected at 5 to 2.6 x 10(8) colony-forming units (CFU)/ml in 14 (30.4%) of 46 samples. With higher water activity of the lubricant (a higher dilution rate of the lubricant with water), a higher concentration of microbial contamination was observed. In 3 (21.4%) of the 14 patients using contaminated lubricant, urine samples showed the same microbial species as those detected in the lubricant. To prevent microbial contamination of the lubricant, dilution of the lubricant with water, as a result of repeated use, should be avoided.

Bacteria↗

PubMed1992

The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes.

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.

Acrylic Resins↗

PubMed1983

The use of a B subtilis in a pre-incubation assay for the detection of dna-modifying agents.

A microbial bioassay for the detection of agents which induce increased lethal damage in DNA repair deficient strains of B. subtilis was modified and evaluated using six established mutagens. This modified procedure used a liquid pre-incubation technique with the plate incorporation assay allowing detection of mutagenic compounds which require metabolic activation and are sparingly soluble in water.

Bacillus subtilis↗

PubMed2006

Bran fermentation as a means to enhance technological properties and bioactivity of rye.

Response surface methodology was applied to study the effects of fermentation on the levels of phytochemicals (folates, phenolic compounds, alkylresorcinols) and on the solubilization of pentosans in rye bran from native and peeled grains. Furthermore, the microbial composition of the brans before and after fermentation was studied. Peeling reduced the microbial load and lower microbial counts were detected in the fermentation experiments carried out with the bran from peeled grains. High temperature and long fermentation time favoured the growth of indigenous lactic acid bacteria (LAB), and a diverse microbial community was detected. The brans contained low levels of aerobic spore-forming bacteria, but their number was not increased during the fermentations. Fermentation of both brans increased the levels of folates, easily extractable total phenolics and free ferulic acid. During fermentation of bran from native grains, the levels of alkylresorcinols slightly increased but during fermentation of bran from peeled grains they decreased. Significant increase in soluble pentosans was established in both types of rye bran fermentations. Enhanced bioactivity and solubilization of pentosans with limited microbial growth were obtained after 12-14 h fermentation at 25 degrees C. The results suggest that fermentation is a potential bioprocessing technology for improved technological properties and bioactivity of rye bran.

Bread↗

PubMed2006

Assessment of various diagnostic methods of ureaplasma respiratory tract infections in newborns.

We compared three methods used microbial culturing for detection of ureaplasmas in endotracheal aspirate from 500 prematurely born neonates with respiratory disturbances: BioMerieux test, PCR and microbial culturing. Ureaplasmas were detected in respiratory tracts of 79 (16%) newborns. Correlation of the results of culture with those obtained with the BioMerieux kit, culture with PCR and BioMerieux kit with PCR was 97%, 89% and 90%, respectively. Sensitivity and specificity of PCR in comparison with culture was 86% and 98%, respectively, and of the BioMerieux kit 96% and 98%. PCR can be recommended in rapid diagnostics of respiratory infections in newborns suffering from respiratory disorders. It allows the detection of ureaplasmas in case of parallel infections and identification of their species.

DNA Primers↗

PubMed2002

Biofilm formation in drinking water affected by low concentrations of phosphorus.

Abstract: There are geographical regions where microbial growth in drinking waters is limited by phosphorus instead of organic carbon. In these drinking waters even a low amount of phosphorus can strongly enhance microbial growth. The formation of biofilm can be limited by low availability of phosphorus in drinking waters with low content of phosphorus. The formation of biofilms on polyvinyl chloride plates was studied in laboratory experiments with water containing 48 microg/L assimilable organic carbon and 0.19 microg/L microbially available phosphorus. We found that low additions of phosphate (1-5 microg/L PO4(3-)-P) to water increased microbial growth in the water and in the biofilm. The effect of phosphorus on microbial growth could be detected by determining either the microbial cell production or the content of ATP in biofilms. Also, in steady-state biofilms, microbial concentrations were higher with phosphorus addition as enumerated by heterotrophic plate counts on R2A-agar and acridine orange direct counting. This work confirms the earlier findings of the importance of phosphorus for microbial growth in humic-rich drinking waters.

Bacterial Physiological Phenomena↗

PubMed2000

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes.

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

Bacteria↗

PubMed2006

mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray.

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.

Ecosystem↗

PubMed2005

Phylogenetic detection of conserved gene clusters in microbial genomes.

BACKGROUND: Microbial genomes contain an abundance of genes with conserved proximity forming clusters on the chromosome. However, the conservation can be a result of many factors such as vertical inheritance, or functional selection. Thus, identification of conserved gene clusters that are under functional selection provides an effective channel for gene annotation, microarray screening, and pathway reconstruction. The problem of devising a robust method to identify these conserved gene clusters and to evaluate the significance of the conservation in multiple genomes has a number of implications for comparative, evolutionary and functional genomics as well as synthetic biology. RESULTS: In this paper we describe a new method for detecting conserved gene clusters that incorporates the information captured by a genome phylogenetic tree. We show that our method can overcome the common problem of overestimation of significance due to the bias in the genome database and thereby achieve better accuracy when detecting functionally connected gene clusters. Our results can be accessed at database GeneChords http://genomics10.bu.edu/GeneChords. CONCLUSION: The methodology described in this paper gives a scalable framework for discovering conserved gene clusters in microbial genomes. It serves as a platform for many other functional genomic analyses in microorganisms, such as operon prediction, regulatory site prediction, functional annotation of genes, evolutionary origin and development of gene clusters.

Algorithms↗

PubMed2002

Bioreporters in microbial ecology.

Bioreporters are effective research tools for gaining an understanding of a microbe's perception of the world. Fitted with a fusion of an environmentally responsive promoter to a suitable reporter gene, a bacterial or fungal bioreporter is able to communicate its metabolic or transcriptional behavior in a habitat, and furnish us with information on the chemical, physical or biological properties of its immediate surroundings. This review details recent developments in the use of such bioreporters in microbial ecology. Emphasis is placed on reporter genes that allow detection in individual microbial cells, as they provide a high-resolution description of the habitat under investigation. In an outlook on the future of bioreporter technology, this review stresses the need to interpret the activity of a bioreporter within the context of its biology.

Bacteria↗

PubMed2005

Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix-dominated freshwater lakes.

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.

Cyanobacteria↗

PubMed1994

Adenosine triphosphate bioluminescence as a method to determine microbial levels in scald and chill tanks at a poultry abattoir.

According to Hazard Analysis of Critical Control Points (HACCP) programs developed for the poultry industry, poultry processing waters should be actively monitored to minimize cross-contamination between chicken carcasses. In order to monitor HACCP programs, a test is required that provides results on a real time basis. A modified adenosine triphosphate (ATP) bioluminescence test has been developed that can assess microbial levels in poultry processing waters within 15 min. A study was conducted to determine the effectiveness of this test for examining scald, prechill, and chill tank waters. The results showed that the modified ATP bioluminescence method gave results comparable to plate counts. The microbial levels were dependent on the tank and the time of sampling. The highest microbial levels were detected in the scald tank. In all three tanks, the microbial levels increased over time during the day.

Abattoirs↗

PubMed2005

Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

Culture Media↗