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PubMed2002

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges.

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Bacteria, Anaerobic↗
PubMed2006

Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens.

Carbohydrate-binding lectins play essential roles as pattern recognition receptors in innate immunity in both vertebrates and invertebrates. The carcinolectins 5 (CL5a and CL5b, the CL5 isoforms of horseshoe crab, Carcinoscorpius rotundicauda, with apparent sizes of 36 and 40 kDa, respectively) are prominent plasma lectins that bind all representative microbes and pathogen-associated molecular pattern molecules. Different cDNA isoforms of both CL5a and CL5b were isolated, leading to our speculation on their functional divergence. Characterization of CL5 isoforms bound to microbial cell surfaces demonstrates the diversity of these lectins. The resolution patterns of the isoforms that associate with fungus differ from those that associate with bacteria, suggesting the unique roles these lectins play in the recognition and differentiation of microbes. We postulate that different populations of plasma lectins act in collaboration in frontline innate immune defense against disparate pathogens. The functional diversity of lectins in invertebrates appears to evolutionarily compensate for the lack of acquired immunity.

Amino Acid Sequence↗
PubMed1995

Intestinal microbial bile acid transformation in healthy infants.

Following the establishment of functionally active intestinal flora in three healthy Swedish children from birth up to 24 months of age, we investigated the development of different 24-carbon bile acids. The fecal bile acids were group-separated into unconjugated, glycine-conjugated, taurine-conjugated, and sulfated, so that we could follow the changes between the different fractions of conjugates. In meconium, most (55-63%) of the bile acids were conjugated with taurine; only 11-32% were conjugated with glycine. Deconjugation was the first sign of intestinal microbial activity on the bile acids. Already at 1 month of age, most of the bile acids were deconjugated; among the conjugated bile acids, the glycine-conjugated dominated over the taurine-conjugated. An unidentified conjugate of cholic and chenodeoxycholic acids (C, CDC) that separated with the sulfated bile acids was found. The unconjugated bile acids and those that arose from hydrolysis of existing conjugates were separated and identified by gas-liquid chromatography coupled to mass spectrometry (GC-MS). Twenty-nine different bile acids were identified. In meconium, 16 different bile acids were identified. C and CDC were identified in all samples. The bile acid pattern changed during the course of the study. Many of the identified bile acids were only found in one or a few of the analyzed samples, and sometimes only in samples from one child. 6 alpha-hydroxylated bile acids, probably not microbially synthesized, were present at high percentages in the children.(ABSTRACT TRUNCATED AT 250 WORDS)

Bifidobacterium↗
PubMed2004

Microbial-gut interactions in health and disease. Epithelial cell responses.

Intestinal epithelial cells are unique in that they represent the only host cells that are constantly interacting with a very large bacterial population in the lumen. The single monolayer of epithelial cells consists of subpopulations with distinct functions that include protection against luminal microorganisms. Although the microbial flora remains to be fully characterized, its normal relationship with the host intestinal epithelial cells appears to be predominantly symbiotic or commensal. The molecular complexity of the epithelial-microbial relationship has been shown in studies that have examined the establishment of the resident bacteria in germ-free mice. Recent work has also demonstrated the ability of resident bacteria to enhance epithelial protective responses. The mechanisms by which epithelial cells may avoid pro-inflammatory responses to resident microorganisms, while retaining the capacity to respond to pathogens, are also being characterized.

Enterobacteriaceae↗
PubMed2000

Detection of antibiotic resistance in vitro.

Testing the susceptibility of bacteria to antimicrobial agents is fundamental to the study of resistance. The qualitative methods that were originally devised evolved separately in different countries and as the need for more precise quantitative information became clear, different solutions to the problem emerged. Consequently, the methods now recommended vary widely between countries and achieving a consensus on standardisation has proved difficult. Sensitivity testing serves two purposes to provide meaningful results to the prescriber and to monitor changes in susceptibility of microbial populations. The diagnostic purpose is adequately served by the diversity of methods used, provided they are performed and controlled efficiently. Highly standardised methods are however needed for surveillance in the national and international context and to provide meaningful comparisons to be made between individual centres. These needs are best met by the use of agreed methods and interpretative criteria in sentinel laboratories. No sensitivity test method can provide the 'correct' answer all the time, if only because the 'correct' answer in therapeutic terms is not known with any certainty. Laboratories providing a sensitivity testing service can influence prescribing--and hence the development of resistance--in a number of different ways and the quest for standardisation at all costs should not obscure these important functions.

Drug Resistance, Microbial↗
PubMed2002

Introduction: innate recognition of bacteria and protozoan parasites.

Major advances have recently been achieved in the area of microbial recognition by the innate immune system. In this Forum, we discuss important issues related to innate recognition of bacteria and protozoan parasites. In particular, we highlight the structural characterization of pathogen-associated molecular patterns (PAMPs); the definition of the receptors required for recognition of PAMPs, especially the Toll-like receptors (TLRs); the signaling pathways triggered by PAMPs/PAMPs receptor interaction; and the functional consequences of these interactions for pathogenesis during microbial infection.

Animals↗
PubMed2001

Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity.

Lipoxins are eicosanoid mediators that show potent inhibitory effects on the acute inflammatory process. We show here that the induction of lipoxin A(4) (LXA(4)) accompanied the in vivo suppression of interleukin 12 (IL-12) responsiveness of murine splenic dendritic cells (DCs) after microbial stimulation with an extract of Toxoplasma gondii. This paralysis of DC function could not be triggered in mice that were deficient in a key lipoxygenase involved in LXA(4) biosynthesis. In addition, DCs pre-treated with LXA(4) became refractory to microbial stimulation for IL-12 production in vitro and mice injected with a stable LXA(4) analog showed reduced splenic DC mobilization and IL-12 responses in vivo. Together, these findings indicate that the induction of lipoxins in response to microbial stimulation can provide a potent mechanism for regulating DC function during the innate response to pathogens.

Animals↗
PubMed1999

Tetanus toxin impairs accessory and secretory functions in interferon-gamma-treated murine macrophages.

Tetanus neurotoxin (TT), a product of microbial origin, acts as a zinc endopeptidase on vesicle-associated membrane proteins (VAMP). We have demonstrated that TT displays inhibitory effects on secretory and accessory functions in the murine macrophage (Mphi) cell line GG2EE. Nitric oxide (NO) secretion was decreased when interferon (IFN)-gamma-pretreated GG2EE Mphis were coincubated with a fungal costimulus (SMP200) and TT. When heat-inactivated TT was used this effect was not evident. The TT-mediated phenomenon was dose-dependent and specific since, under the same experimental conditions, it did not affect interleukin-6 or tumor necrosis factor-alpha secretion. Furthermore, IFN-gamma-induced major histocompatibility complex class II molecule expression and GG2EE accessory function, assessed by SMP200-stimulated lymphoproliferation, were also inhibited by TT. Such inhibition was incomplete, in line with our previous results showing that TT partially cleaves VAMP proteins in murine Mφ.

Animals↗
PubMed2005

Specialized microbial databases for inductive exploration of microbial genome sequences.

BACKGROUND: The enormous amount of genome sequence data asks for user-oriented databases to manage sequences and annotations. Queries must include search tools permitting function identification through exploration of related objects. METHODS: The GenoList package for collecting and mining microbial genome databases has been rewritten using MySQL as the database management system. Functions that were not available in MySQL, such as nested subquery, have been implemented. RESULTS: Inductive reasoning in the study of genomes starts from "islands of knowledge", centered around genes with some known background. With this concept of "neighborhood" in mind, a modified version of the GenoList structure has been used for organizing sequence data from prokaryotic genomes of particular interest in China. GenoChore http://bioinfo.hku.hk/genochore.html, a set of 17 specialized end-user-oriented microbial databases (including one instance of Microsporidia, Encephalitozoon cuniculi, a member of Eukarya) has been made publicly available. These databases allow the user to browse genome sequence and annotation data using standard queries. In addition they provide a weekly update of searches against the world-wide protein sequences data libraries, allowing one to monitor annotation updates on genes of interest. Finally, they allow users to search for patterns in DNA or protein sequences, taking into account a clustering of genes into formal operons, as well as providing extra facilities to query sequences using predefined sequence patterns. CONCLUSION: This growing set of specialized microbial databases organize data created by the first Chinese bacterial genome programs (ThermaList, Thermoanaerobacter tencongensis, LeptoList, with two different genomes of Leptospira interrogans and SepiList, Staphylococcus epidermidis) associated to related organisms for comparison.

Algorithms↗
PubMed1998

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology.

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M(r) of 25-30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

Acid Phosphatase↗
PubMed2005

Soil health -- a new challenge for microbiologists and chemists.

Soil health refers to the biological, chemical, and physical features of soil that are essential to long-term, sustainable agricultural productivity with minimal environmental impact. Thus, soil health provides an overall picture of soil functionality. Although it cannot be measured directly, soil health can be inferred by measuring specific soil properties (e.g. organic matter content) and by observing soil status (e.g. fertility). There is also increased interest in studying soil microorganisms in their particular environments, as microbial diversity is intimately related to soil structure and function. One of the key objectives in determining soil health is to acquire indicators that can be used to evaluate the soil's current status and hence to develop sustainable agricultural systems. In this regard, significant progress has been made over the last few years in the development of specific biomarkers and macromolecular probes, enabling rapid and reliable measurements of soil microbial communities. In addition, modern molecular biological techniques, such as fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE), and terminal restriction fragment length polymorphism (T-RFLP), have facilitated the analysis of microbial biodiversity and activity, whereas the application of modern analytical techniques, such as nuclear magnetic resonance (NMR) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), have provided data on soil chemistry. The combination of these two approaches offers promise in determining soil health status.

Bacteria↗
PubMed1998

Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes.

Secretory leukocyte protease inhibitor (SLPI) is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte-derived proteinases, has anti-HIV-1, antibacterial, and anti-fungal properties, and interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages. We now report that at both the mRNA and the protein level, SLPI shows inducible expression in a nonglandular epithelium. A weak expression of SLPI was found in the stratum granulosum of adult normal human epidermis; however, in lesional psoriatic epidermis and in migrating keratinocytes of healing wounds, a strong cytoplasmic staining was seen in the suprabasal keratinocytes. Remarkably, in the dermis adjacent to SLPI-expressing keratinocytes, SLPI was found extracellularly associated with elastin fibers, whereas the dermis in normal skin was negative. In cell culture, SLPI was hardly expressed in monolayers of proliferating keratinocytes. Differentiating cultures with a phenotype of normal skin expressed low levels of SLPI, whereas cultures with a regenerative/psoriatic phenotype expressed high levels. Functional studies with recombinant SLPI indicated that its antibacterial spectrum and potency are distinct from other anti-microbial peptides such as lysozyme and defensins. In view of the multiple functions of SLPI and the inducibility, we propose that it acts as an important first line defence mechanism in cutaneous injury.

Adult↗
PubMed1997

Atopic versus infectious diseases in childhood: a question of balance?

There is an increasing interest in the concept that respiratory tract infections during early childhood may in some circumstances confer protection against sensitisation to aeroallergens, via "bystander" stimulation of Th-1 associated immune functions in the regional lymph nodes draining the airway mucosa. We hypothesise below that this phenomenon may be but one component of a broader process operative during early postnatal life, in which generalised contact with the microbial environment plays an obligatory role in stimulating the functional maturation of the Th-1 arm of the immune response. We argue further that one of the most potent sources of such stimulation is provided by the normal commensal flora of the gastrointestinal tract, which is establish during early infancy.

Cytokines↗
PubMed2000

Epithelial structural proteins of the skin and oral cavity: function in health and disease.

Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.

Animals↗
PubMedDate not supplied

Effect of oral administration of a variety of bacteria on depressed macrophage functions in tumour-bearing rats.

In consideration of the well documented influence of normal microbial flora on the level of activation of macrophages, we evaluated the effects of oral administration of bacteria on rats with depressed macrophage functions. An oral, killed polyvalent vaccine (Diplococcus pneumoniae types I, II and III, Streptococcus haemolyticus, Staphylococcus aureus and Haemophilus influenzae), the live lyophilized Streptococcus faecium and spores of Bacillus subtilis, respectively, were administered orally to immuno-depressed rats. Results demonstrate the restoration of phagocytosis, intracellular killing and the chemotactic activity of macrophages. These experimental observations suggest that bacterial flora associated with mucosae can influence the level of activation of peritoneal macrophages.

Administration, Oral↗
PubMed2003

The microflora recovered from the outer-surfaces of the Frialit-2 implanto-prosthetic connector.

The aim of the present investigation was to examine the outer-surface microbiota of the prosthetic connector of Frialit-2 implants, and to compare the microbial findings with the peri-implant parameters 2 years after functional loading. In 16 implant-treated patients (55.8 +/- 9.5 years) the outer-surface micro-organisms of the prosthetic connectors were determined in 32 Frialit-2 implants. The functional loading time of the prosthetic suprastructures was 24.1 +/- 13.8 months on average. After removal of the implant-supported restoration, microbial samples were obtained from the outer-surfaces of the Frialit-2 prosthetic connector. The microbial plaque samples were specified on CDC-blood agar as percentages of the total cultivable flora. Actinobacillus actinomycetemcomitans was semiquantitively determined on TSBV-agar in CFU/ml. The microbial plaque samples were dominated by Actinomyces israelii (68.8%), Eubacterium lentum (56.3%) and Veillonella parvula (43.8%) with proportions ranking between 3.9% (V. parvula) and 11.1% (A. israelii). The most frequently detected gram-negative microorganisms were Fusobacterium nucleatum (87.5%), Porphyromonas gingivalis (81.3%), and Peptostreptococcus micros (68.8%) with enhanced proportions for P. gingivalis (11.4%) and P. micros (11.4%). No statistical significant correlation could be established between the microbiota present on the outer-surfaces of the F2-connector and the peri-implant parameters examined. The outer-surface microflora recovered from the implanto-prosthetic-connector of Frialit-2 implants reveals a colonization with gram-positive bacteria and potentially harmful gram-negative micro-organisms that were frequently detected, but present at low levels. After 2 years of restorative loading, the outer-surface microbial colonization is compatible with peri-implant soft tissue health.

Actinomyces↗
PubMed2001

Altered macrophage-like functions of preadipocytes in inflammation and genetic obesity.

We recently demonstrated that preadipocytes exhibit functional features of macrophages, such as phagocytosis and anti-microbial activity, suggesting that preadipose cells could play a role in the inflammatory process or immune response. The aim of this study was to compare these functions of both macrophages and cells from stroma-vascular fraction (SVF) of the adipose tissue in two different situations, obesity and inflammation, characterized by alterations in immune responsiveness. We demonstrated that ob/ob mice exhibited strong decrease in antimicrobial activity of both macrophages and SVF. This defect is compensated in SVF, at least in part, by an enhancement of phagocytosis that does not seem to be due to an increased macrophage number. In vitro leptin treatment of SVF and macrophages from obese mice did not restore their immune defects. Thioglycollate treatment of lean and obese mice induced an inflammatory process that led to an increase in macrophage activity in both strains. This stimulation also observed in SVF from lean mice is not present in obese ones. This work demonstrated that SVF immune functions could be modified in different pathological situations such as inflammation and obesity and sustained the new physiological role of preadipocytes in these processes.

Adipocytes↗
PubMed1982

Isolation of single atrial and ventricular cells from the human heart.

The single isolated heart cell has recently emerged as a model for the study of the structure and function of cardiac cells. Heart muscle cells of adult animals of various species have been successfully isolated by enzymatic digestion of intact cardiac tissue. In this paper a dissociation method that yields living cells from atrial and ventricular tissue of young and adult humans is detailed. The cells retain the morphologic features of cells in intact cardiac tissue, and they generate action potentials and contractions in response to electrical stimulation. The study of isolated human heart cells should make a valuable contribution to knowledge of the normal and diseased heart.

Adult↗