Characterization of bacteroides melaninogenicus.
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Fifty-eight human isolates of Bacteroides melaninogenicus, 42 from a variety of clinical infections and the rest from normal flora, were studied for pigment production and ultraviolet light fluorescence and by forty biochemical and other tests, including end-product analysis by gas-liquid chromatography. In a number of instances, tests were repeated several times and the results were reproducible. Agar plate dilution susceptibility tests were also performed to 12 antimicrobial agents. These 58 strains could be reliably placed into three groups, corresponding to the three subspecies described, based on seven characteristics. These included acid production in peptone-yeast-glucose medium, production of n-butyric acid from peptone-yeast-glucose medium, esculin hydrolysis, starch hydrolysis, indole production, effect on milk, and lipase production. Production of hydrogen gas in peptone-yeast-fructose medium may be another distinguishing characteristic. In general there was not much difference in the susceptibility of the three groups to the various antimicrobial agents tested. Two strains had a minimal inhibitory concentration of penicillin G of 16 and 32 U/ml, respectively. Three strains did not produce a black pigment in spite of prolonged incubation on blood-containing media. (+info)
Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.
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As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. (+info)
Evaluation of the pathotec Rapid I-D system for identification of Enterobacteriaceae.
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The PathoTec Rapid I-D System for identifying Enterobacteriaceae was evaluated with 471 cultures. In 4,910 individual test comparisons, 95.5% of the results agreed, with results of only two test strips, those for esculin hydrolysis and urease production, agreeing with conventional tests in less than 94% of the trials. The PathoTec system exhibited 94.3% accuracy in identifying these cultures in a double-blind study with conventional media and procedures as the alternate system. Two newly developed test strips, for 0-nitrophenyl-beta-D-galactopyranoside and ornithine decarboxylase, were found to be highly reliable. (+info)
A novel beta-glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose.
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A regulon from Streptococcus mutans that plays a role in the utilization of beta-glucosides has been isolated, sequenced and subjected to sequence analysis. This regulon encodes a beta-glucoside-specific Enzyme II (EII) component (bglP) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a phospho-beta-glucosidase (bglA) which is responsible for the breakdown of the phospho-beta-glucosides within the cell. Both the bglP and bglA gene products have significant similarity with proteins that have similar functions from Clostridium longisporum, Listeria monocytogenes, Erwinia chrysanthemi, Escherichia coli, Klebsellia oxytoca and Bacillus subtilis. The potential functions of the BglP and BglA proteins are supported by phenotypic data from both S. mutans and E. coli. A chromosomal deletion in S. mutans spanning the bglP and bglA genes resulted in a strain that was unable to hydrolyse the beta-glucoside aesculin in the presence of glucose. When glucose was removed from the medium, the deletion strain regained the ability to break down aesculin. These data suggest that S. mutans possesses an alternative mechanism from the one described in this report for breaking down beta-glucosides. This second mechanism was repressed by glucose while the regulon described here was not. Complementation studies in E. coli CC118 also suggest a potential role for this regulon in the utilization of other beta-glucosides. When a plasmid containing the 8 kb beta-glucoside-specific regulon was transformed into E. coli CC118, the transformed strain was able to break down the beta-glucoside arbutin. (+info)
Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. coli subsp. nov., isolated from humans and food.
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Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculin-positive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056T = NCDO 599T) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 = NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species. (+info)
Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl-/GABA (hGAT1) cotransporters.
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1. Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na(+)/glucose (hSGLT1) and Na(+)/Cl(-)/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. 2. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. 3. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7x12A. Important hydrogen bond interactions occur at five positions bordering this surface. 4. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site approximately 8A from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). 5. Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. (+info)
Comparison of three procedures for biochemical testing of anaerobic bacteria.
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The Analytab Products, Inc. (API), anaerobic multitest microsystem (MICRO) was compared with the Center for Disease Control conventional (CONV) thioglycolate (supplemented with hemin and vitamin K1) system and with pre-reduced anaerobically sterilized (PRAS) media as recommended by the Virginia Polytechnic Institute. Growth from a solid medium was suspended to produce standard inocula. Substrates included 16 carbohydrates, indole, urea, gelatin, and esculin. API strips were inoculated in air and incubated in GasPak (BBL) jars. MICRO tests were read at 1 and 2 days. CONV tests at 1, 2, and 7 days, and PRAS tests at 3 weeks. One hundred thirty well-characterized strains of anaerobes (76 gram-negative rods, 16 cocci, 26 gram-positive nonsporeforming rods, and 12 clostridia), including 48 reference strains, were studied. Of 2,600 tests performed, 2,085 (80.2%) showed agreement with all three methods. There was 90.9% agreement between the MICRO and CONV, 84.9% between the MICRO and PRAS, and 84.6% between the CONV and PRAS tests. All MICRO tests were reliable except for indole, which was not sensitive enough, and gelatin, which was very insensitive. The MICRO system permits performance of biochemical tests at the workbench in the average clinical laboratory without the need for expensive equipment and time-consuming procedures. (+info)
Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests.
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A simplified method for speciation of group D streptococci is described. A total of 4,156 streptococcal isolates from human clinical material was tested for ability to hydrolyze esculin in the presence of 40% bile, ferment pyruvate, hydrolyze arginine, and grow in media containing 40% bile or 6.5% NaCl. Streptococci which hydrolyzed esculin in 40% bile, but which did not hydrolyze arginine, were also tested for their ability to ferment raffinose or sorbose. Sixty percent (2,503) of the isolates hydrolyzed esculin in the presence of 40% bile and were thus presumptively identified as group D. By application of the other criteria, 84% of these were speciated as Streptococcus faecalis, 7% were speciated as S. faecium, 6% were speciated as S. bovis, 2% were speciated as S. avium, and 1% were not identified. This scheme was shown to be both reliable and practical for use in the diagnostic laboratory. S. avium and S. bovis isolates were characterized, and 18 S. bovis isolates from patients with bacterial endocarditis were compared physiologically with 151 isolates of this species from other sources. (+info)