Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis.
(17/1602)
Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. (+info)
Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.
(18/1602)
The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora. (+info)
Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.
(19/1602)
Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes. (+info)
Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.
(20/1602)
We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone. (+info)
Phylogenetic analysis of genus Marinilabilia and related bacteria based on the amino acid sequences of gyrB and emended description of Marinilabilia salmonicolor with Marinilabilia agarovorans as its subjective synonym.
(21/1602)
The detailed phylogenetic relationships for genus Marinilabilia and related taxa were analysed by using DNA gyrase B subunit gene (gyrB) sequences. Anaerobic bacteria in the Cytophaga-Flavobacterium-Bacteroides phylum, namely genera Marinilabilia, Bacteroides, Rikenella, Prevotella and Porphyromonas and Cytophaga fermentans, were clustered in the same branch and the facultative anaerobes Marinilabilia and Cytophaga fermentans formed a subcluster in the branch of the anaerobic bacteria. Phylogenetic analysis using 16S rDNA sequences gave a similar result but with a lower bootstrap value for each cluster. The gyrB sequences of Marinilabilia salmonicolor and Marinilabilia agarovorans were the same, and the relatedness of their chromosomal DNA, as determined by DNA-DNA hybridization, was greater than 70%. These genetic aspects led to the conclusion that M. salmonicolor IFO 15948T and M. agarovorans IFO 14957T belong to a single species. Since M. salmonicolor was described first, as Cytophaga salmonicolor, M. salmonicolor is a senior subjective synonym of M. agarovorans. Therefore, the name M. salmonicolor should be retained and strain IFO 14957T should be reclassified as M. salmonicolor. However, the agar-degrading ability of strain IFO 14957T is a prominent biochemical characteristic. It is therefore proposed that strain IFO 14957T should be renamed M. salmonicolor biovar agarovorans. (+info)
The faecal flora of children in the United Kingdom.
(22/1602)
The faecal flora of 55 children (aged 8 days to 8 years) and 16 adults was determined. All the children were artificially fed from birth. The faecal flora of the youngest age group was generally less complex and less predictable than that of adults. Some bacterial groups commonly found in adult stools, for example bacilli, lactobacilli and yeasts, were rarely found in the youngest infants. Most of the changes towards the adult pattern took place between 4 and 12 months. The faecal flora of children aged 1-4 years generally resembled that of adults, although lactobacilli were still infrequently isolated. (+info)
Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron.
(23/1602)
Results from previous studies had suggested that Bacteroides thetaiotaomicron utilizes starch by binding the polysaccharide to the bacterial surface and subsequently degrading the polymer by using cell-associated enzymes. Most of the starch-degrading activity was localized to the periplasm, but a portion appeared to be membrane associated. This raised the possibility that some breakdown might occur in the outer membrane prior to exposure of the polysaccharide to the periplasmic polysaccharide-degrading enzymes. In this study, we show that SusG, an outer membrane protein which has been shown genetically to be essential for starch utilization, has enzymatic activity. Results of protease accessibility experiments support the hypothesis that SusG is exposed on the cell surface. Results of [(14)C]starch binding assays, however, show that SusG plays a negligible role in binding of starch to the cell surface. Consistent with this, SusG has a relatively high K(m) for starch and by itself is not sufficient to allow cells to grow on starch or to bind starch. Hence, the main role of SusG is to hydrolyze starch, but the binding of starch to the cell surface is evidently mediated by other proteins presumably interacting with SusG. (+info)
Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes.
(24/1602)
Two strains of Listeria monocytogenes, one that formed smooth colonies on agar surfaces and a varient of it that formed rough colonies, colonized the gastrointestinal tracts of germfree mice. Within 24 h after mice were inoculated orally with about 100 bacteria, the population levels per gram (wet weight) of tissue of both strains were 10(5) to 10(7) in the stomach and ileum and 10(8) to 10(9) in the cecum and colon, respectively. As detected in Gram-stained histological sections, in such gnotobiotes, the bacteria colonized the lumen in all areas of the tract and much of the mucus layer on the epithelial surface in the proximal colon. The strain that formed smooth colonies did not colonize the tracts of specific-pathogen-free mice, but did colonize, to the same levels as in germfree mice, the stomachs and bowels of ex-germfree mice previously associated with two members of the indigenous flora (Bacteroides and Clostridium). In the latter animals, however, the listeria did not form layers on the colonic epithelium as efficiently as they did in monoassociated gnotobiotes. (+info)