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PubMed2004

16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure.

A laboratory-scale continuously stirred anaerobic thermophilic batch digester was inoculated with cattle manure. Bacterial and archaeal communities, as well as digester performances, were analysed during reactor start-up for about 20 days. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used for overall detection and for study of the dynamics of microbial populations. Dominant bacteria and archaea 16S rDNAs were sequenced from the sample on day 12. Ten bacteria and 3 archaea OTUs (operational taxonomic units) were identified from the 52 clones sequenced. Sequences corresponding to the dominant bacterial SSCP peak were phylogenetically close to the 16S rDNA sequence of Bacillus thermoterrestris, whereas sequences corresponding to the two dominant archaeal SSCP peaks were phylogenetically close to the 16S rDNA sequence of Methanoculleus thermophilicus and Methanosarcina thermophila.

Anaerobiosis↗

PubMed2005

Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines.

The Toll-like receptor (TLR) family of cell surface molecules represent a major component of the pattern recognition system, which enables both vertebrates and invertebrates to detect invading microorganisms and mount an anti-microbial response. The TLR repertoire of mouse and man has been intensively studied and in this manuscript we report the identification of ESTs with homology to chTLR5 and chTLR7, and independently confirm the identification of chTLR 1/6/10 and 3 in the EST databases. We have determined the mRNA expression patterns for seven chicken TLRs (chTLR) in a wide range of chicken tissues, isolated immune cell types and cultured cells. Some of the chTLR were expressed in most tissues (chTLR1/6/10, chTLR3, chTLR4 and chTLR5), whereas others exhibited more restricted expression patterns (chTLR2 type 1, type 2 and chTLR7). Similarly distinct patterns of chTLR expression were seen with innate and adaptive immune cell types isolated from peripheral blood or spleen and with cultured cells of somatic or immunological origin. An understanding of the TLR repertoire for different tissues, immune cell subsets and cultured cell types allows more refined interpretation of immune induction in response to chicken pathogens.

Animals↗

PubMed1998

Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects.

Polyhdroxyalkanoates (PHAs), stored as bacterial reserve materials for carbon and energy, are biodegradable substitutes to fossil fuel plastics that can be produced from renewable raw materials. PHAs can be produced under controlled conditions by biotechnological processes. By varying the producing strains, substrates and cosubstrates, a number of polyesters can be synthesized which differ in monomer composition. By this means, PHAs with tailored interesting physical features can be produced. All of them are completely degradable to carbon dioxide and water through natural microbiological mineralization. Consequently, neither their production nor their use or degradation have a negative ecological impact. After a historical review, possibilities for the synthesis of novel PHAs applying different micro-organisms are discussed, and pathways of PHA synthesis and degradation are shown in detail for important PHA producers. This is followed by a discussion of the physiological role of the accumulation product in different micro-organisms. Detection, analysis, and extraction methods of PHAs from microbial biomass are shown, in addition to methods for polyester characterization. Strategies for PHA production under discontinuous and continuous regimes are discussed in detail in addition to the use of different cheap carbon sources from the point of view of different PHA producing strains. An outlook on PHA production by transgenic plants closes the review.

Bacteria↗

PubMed1998

Reactive iron species in biological fluids activate the iron-sulphur cluster of aconitase.

Low molecular mass iron (LMrFe) can appear in plasma when the transferrin becomes fully iron loaded. Such iron poses a risk factor for oxidative damage, and for microbial virulence. A previous novel approach to the detection and measurement of LMrFe in plasma was the use of the iron-binding properties of the glycopeptide antitumour antibiotic bleomycin and its ability to degrade DNA in the presence of oxygen, bound iron, and an iron reducing agent. Since bleomycin is a non-physiological ligand with iron-binding and redox cycling properties, it has been suggested that it may not be a valid biological model for detecting and measuring LMrFe. To address these concerns we have developed a biological approach to the detection and measurement of LMrFe based on the activation of iron-requiring aconitase. Parallel measurements, in a variety of clinical conditions in which there was a complete saturation of the plasma transferrin, showed that the bleomycin assay and the aconitase assay can give similar results for LMrFe.

Aconitate Hydratase↗

PubMed1998

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157.

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter) was not suitable for detection of epidemiological relatedness. All but one E. Coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5'-GAG GGT GGC GGT TCT-3' and primer 1247 (5'-AAGAGCCCGT-3'). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.

Adolescent↗

PubMed2006

Microbial etiology of acute gastroenteritis in hospitalized children in Taiwan.

BACKGROUND/PURPOSE: Viral infections are the most common causes of acute infectious diarrhea in the pediatric population. To explore any possible microbial etiologies of acute gastroenteritis in children, we detected stool viral antigen including rotavirus, adenovirus, norovirus and astrovirus. We also studied the possible precipitating factors. METHODS: During a period of 1 year (from October 2003 to September 2004), children from birth to 15 years old admitted to the pediatric ward were prospectively surveyed. Stool specimens were collected within 48 hours after admission and then frozen at -40 degrees C until analysis. Enzyme immunoassay was used to detect rotavirus, astrovirus, norovirus and adenovirus. Bacterial culture was performed at the same time. RESULTS: During the study period, 82 stool samples were collected due to acute gastroenteritis and fit the definition of a diarrhea episode. Forty-two (51.2%) patients with viral infection, 11 (13.4%) with bacterial infection, and six (7.3%) with mixed viral and bacterial infection were detected. The most prevalent virus was rotavirus (35.4%), followed by norovirus (29.3%). The most prevalent cause of bacterial infection was Salmonella (19.5%). With regard to clinical severity, rotavirus resulted in longer hospital stay, higher rate of vomiting, stool occult blood, leukocytosis, lower rate in stool pus cell, and C-reactive protein elevation more than 5 mg/dL as compared with norovirus. Only the difference in hospital stay reached significant statistical difference. CONCLUSION: Norovirus is an important cause of acute gastroenteritis in children, although rotavirus is still the leading cause of pediatric acute gastroenteritis.

Acute Disease↗

PubMed2003

cDNA cloning, purification, properties, and function of a beta-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella.

Microorganisms possess distinctive biochemical or molecular patterns on their cell surfaces, such as those formed by the lipopolysaccharides, lipoteichoic acids, and/or peptidoglycans of bacteria and the beta-1,3-glucans of fungi. Pattern recognition proteins that bind to these surface moieties have been implicated in the activation of the innate immune response in insects and other invertebrates. We report the purification and cloning of a cDNA for a 53-kDa beta-1,3-glucan recognition protein (betaGRP) from the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). BetaGRP cDNA contains an open reading frame that encodes 488 amino acids, of which the first 17 residues comprise the secretion signal peptide. The calculated molecular mass of the 471-residue mature protein is 53,311 Da. The protein consists of a carboxyl-terminal domain that is similar to other recognition proteins from invertebrates, beta-1,3-glucanases from bacteria, and a beta-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of betaGRP shares sequence similarity with other invertebrate recognition molecules and the beta-1,3-glucanase from S. purpuratus. Affinity purification of a 53-kDa protein and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the betaGRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that betaGRP is constitutively expressed in all life-stages, with no detectable induction following exposure of wandering larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb betaGRP transcript is transcribed within the fat body. Recombinant betaGRP retains beta-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, causes aggregation of microorganisms, and activates the prophenoloxidase cascade in the presence of soluble beta-1,3-glucan. These data support the hypothesis that the 53-kDa betaGRP functions to recognize pathogen surface molecules as nonself and subsequently activates insect innate immune responses.

Amino Acid Sequence↗

PubMed2002

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay, the micronucleus assay and the HPRT gene mutation assay.

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.

Air Microbiology↗

PubMed2002

Immune escape through C-type lectins on dendritic cells.

Dendritic cells (DCs) detect different pathogens and elicit tailored anti-microbial immune responses. They express C-type lectins that recognise carbohydrate profiles on microorganisms, resulting in internalisation, processing and presentation. Intracellular sequences of distinct DC-specific lectins point to differences in intracellular routing that influence antigen presentation. Moreover, putative signalling motifs hint to the activation of DCs on carbohydrate recognition. Recent evidence shows that not only pathogens, but also tumour antigens, exploit C-type lectins to escape intracellular degradation resulting in abortive immunity. More insight into ligand specificity, intracellular targeting and signalling will reveal the pathways by which pathogens modulate immunity through C-type lectins.

Animals↗

PubMed2001

Study of the degradation of the herbicides 2,4-D and MCPA at different depths in contaminated agricultural soil.

Two phenoxyacid herbicides (2,4-D and MCPA) and their six corresponding phenols were determined in soil by using gas chomatography with electron impact mass spectrometry (GC/MS) for confirmation/quantitation. An automatic extraction (leaching), preconcentration, and cleanup (sorption) module was developed to extract the eight compounds from soil. The average recovery of all species, spiked to soil at microg/kg-mg/kg levels, was 95% (average standard deviation +/- 5%). A plot of agricultural clayey soil (approximately 12 m2) was contaminated with both herbicides (approximately 96 g/m3, depth 10 cm, density 1.23 g/cm3) and irrigated with (17 mm) at variable time intervals. Both herbicides and their corresponding phenol compounds were monitored at different soil depths over a 50 day period. The degradation of both herbicides in the surface layer (t(1/2) approximately 5 days) is a result of photodecomposition and microbial action; in the deeper layers, the degradation products occur in lower proportions by effect of leaching and are also the result of microbial action. The six phenol metabolites are only detected in the surface layer as they form preferentially by photodecomposition. The main metabolites (viz. 2,4-DCP for 2,4-D and 4-C-2-MP for MCPA) are formed within 24 h after the soil is contaminated; their concentration peaks are at day 8 in the absence of irrigation.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2001

Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.

Base Sequence↗

PubMed2003

Dendritic cells and host resistance to infection.

Host defence against infection requires an integrated response of both the innate and adaptive arms of the immune system. Emerging data indicate that dendritic cells contribute an essential part to the initiation and regulation of adaptive immunity. Dendritic cells guard the sites of pathogen entry to the host and are uniquely suited to detect and capture invading microbes. Upon recognition of microbial structures and appropriate activation, a maturation programme is triggered and dendritic cells migrate to lymphoid organs to stimulate a primary cell-mediated immune response. Moreover, dendritic cells play a critical role in shaping the emerging response, thereby controlling the course of infection. They can discriminate between various types of microorganisms and are capable of producing different cytokines in response to different microbial stimuli. On the other hand, pathogens developed numerous strategies to evade and subvert dendritic cell functions. Elucidating the interactions of dendritic cells with microbial pathogens may lead to novel strategies for combating infectious diseases by dendritic cell-based vaccination and immunotherapy. This review highlights recent advances in our knowledge of the unique role of dendritic cells in counteracting microbial infections.

Animals↗

PubMed2001

Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis: is Propionibacterium acnes involved in granuloma formation?

The etiopathogenesis of primary biliary cirrhosis (PBC) remains speculative. Epithelioid granulomas are often found in the vicinity of damaged interlobular bile ducts in PBC, raising the possibility of a reaction to microbial materials. In this study, we tried to detect and identify bacterial DNA within granulomatous lesions in PBC. Using liver sections from 9 patients with PBC and 13 control livers, granuloma in portal tracts, portal tracts without granuloma, and adjacent hepatic parenchyma were selectively microdissected from sections, and then DNA was extracted from them. First, part of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from DNA samples extracted from 5 PBC and 6 control livers, and their amplicons were sequenced for the identification of bacterial species. Several indigenous bacteria were identified. Among them, Propionibacterium acnes (P. acnes) was detected as a major clone in 20% to 50% of sequenced clones from granuloma of PBC, but the detection rate of P. acnes was 0% to 20% in those cloned from adjacent hepatic parenchyma of PBC. Then, a P. acnes-specific PCR was performed using all microdissected samples. Distinct PCR products were identified in epithelioid granuloma in all 9 PBC cases. The result that P. acnes DNA is present as a major clone in granulomas of PBC, suggest that P. acnes is involved in the pathogenesis of granuloma in PBC.

DNA, Bacterial↗

PubMedDate not supplied

[Persistent Lactobacillus casei subspecies rhamnosus bacteremia in a 14 year old girl with acute myeloid leukemia. A case report].

The present clinical observation is related to a 14-year-old girl suffering from acute myeloid leukemia. The clinical course was complicated by episodes of severe enterocolitis, E. coli- septicemia, pancreatitis and pneumonia. In the course of continued cytostatic and antibiotic treatment a persistent asymptomatic Lactobacillus casei subsp. rhamnosus-bacteremia became detectable by a total of 18 blood cultures. Microbial cultures of the faeces revealed colony-forming unites of this germ in orders of 10(9)/g. Antibiotic eradication attempts according to the resistogram were not successful. The Lactobacillus-bacteremia disappeared only after 13 months when the cytostatic therapy was terminated. An adjuvant influence of the Lactobacillus infection on the outcome of the underlying disease cannot be excluded.

Adolescent↗

PubMed2001

Amniotic fluid concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during pregnancy and labor.

OBJECTIVE: This study was undertaken to determine whether obstetric factors affect the concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 in the amniotic fluid. STUDY DESIGN: We prospectively collected amniotic fluid samples from 109 women at various stages of pregnancy and labor and determined matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 concentrations by means of enzyme-linked immunosorbent sandwich assay systems. With multiple regression analysis we evaluated relationships between amniotic fluid matrix metalloproteinase 9 concentration and tissue inhibitor of metalloproteinase 1 concentration and the following factors: gestational age, presence of labor, cervical dilatation, membrane status, presence of clinical chorioamnionitis, and microbial colonization of the amniotic fluid. RESULTS: The detectable presence of amniotic fluid matrix metalloproteinase 9 was independently associated with intra-amniotic infection, labor, cervical dilatation, and spontaneous rupture of membranes. Chorioamnionitis and amniotic fluid matrix metalloproteinase 9 concentrations were correlated with tissue inhibitor of metalloproteinase 1 levels. CONCLUSIONS: Intra-amniotic infection, advanced labor, and rupture of membranes before the onset of labor were independently associated with the presence of matrix metalloproteinase 9 in the amniotic fluid. Both pathologic and physiologic processes appear to produce shifts in the balance between degradation and synthesis of the extracellular matrix.

Amniotic Fluid↗

PubMed2004

ORFcurator: molecular curation of genes and gene clusters in prokaryotic organisms.

UNLABELLED: The ability to detect clusters of functionally related genes in multiple microbial genomes has enormous potential for enhancing studies on gene function and microbial evolution. The staggering amount of new genome sequence data presents a largely untapped resource for gene cluster discovery. To date, gene cluster analysis has not been fully automated, and one must rely on manual, tedious and time-consuming manipulation of sequences. To facilitate accurate and rapid identification of conserved gene clusters, we developed a database-driven web application, called ORFcurator. We used ORFcurator to find clusters containing any genes similar to those of the 14-gene Widespread Colonization Island of Actinobacillus actinomycetemcomitans. From 126 genomes, ORFcurator identified all 73 clusters previously determined by manual searching. AVAILABILITY: ORFcurator and all associated scripts are freely available as supplementary information. SUPPLEMENTARY INFORMATION: http://www.genomecurator.org/ORFcurator/

Algorithms↗

PubMed2006

Measuring multiple fluxes through plant metabolic networks.

Fluxes through metabolic networks are crucial for cell function, and a knowledge of these fluxes is essential for understanding and manipulating metabolic phenotypes. Labeling provides the key to flux measurement, and in network flux analysis the measurement of multiple fluxes allows a flux map to be superimposed on the metabolic network. The principles and practice of two complementary methods, dynamic and steady-state labeling, are described, emphasizing best practice and illustrating their contribution to network flux analysis with examples taken from the plant and microbial literature. The principal analytical methods for the detection of stable isotopes are also described, as well as the procedures for obtaining flux maps from labeling data. A series of boxes summarizing the key concepts of network flux analysis is provided for convenience.

Chromatography, Gas↗

PubMed1998

Post-transfusion hepatitis: current risks and causes.

Viral hepatitis which follows transfusions (post-transfusion hepatitis) may be due to those transfusions, i.e., transfusion-transmitted hepatitis (TTH), or may be incident to the reason for the transfusion and, thus, may be transfusion-associated, but not transfusion-transmitted. The current risks of TTH, today, are extremely small, but are still due, primarily, to the hepatitis B virus (HBV) and the hepatitis C virus (HCV), the latter, formerly being known as "non-A, non-B hepatitis." The residual, now, of TTH which is non-A, non-B, and non-C is extremely small and may be due to a variety of agents. Using volunteer (unpaid), repeat, blood donors, who are carefully screened for hepatitis risk factors and then tested for evidence of HBV infection, the risk of HBV being transmitted by a transfusion today is in the order of 1 per 63,000 units of blood. For transfusion-transmitted HCV, with the same repeat, volunteer (unpaid) donors, careful screening and a sensitive assay for anti-HCV, the risk is in the order of 1 in 125,000 units. These risks of HBV and HCV due to transfusions are so small that other means of acquiring these viruses should be sought when patients develop hepatitis following blood transfusions. However, efforts to further reduce the current risks of HBV and HCV transmission by transfusions should continue; these include restricting transfusions to those which are necessary or appropriate, utilizing alternatives to transfusion, employing novel assays to detect viral nucleic acids, and, finally, implementing various microbial inactivation techniques on blood, blood components and plasma derivatives.

Blood Donors↗