Quantitative studies on competitive activities of skin bacteria growing on solid media.
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Earlier quantitative investigations of antagonism between skin bacteria were based on the use of liquid cultures, but a more realistic model has now been devised, based on the use of the surfaces of solid media. Pure or mixed inocula were spread evenly over suitable agar media in Petri dishes marked out with a standard grid. Growth curves were constructed from viable counts of the surface bacteria after they had been removed from excised squares of the agar media and dispersed. The method was highly reproducible, and competitive interactions were revealed more clearly than in studies with liquid media. An antibiotic-producing strain of Staphylococcus epidermidis (S6+) readily suppressed strains of Micrococcus, Corynebacterium and Streptococcus species. However, a Staphylococcus aureus strain which was less sensitive to the antibiotic effect of S6+ interacted in a complex manner, depending on the absolute and relative size of the S6+ inoculum. (+info)
Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic.
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Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range). (+info)
A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade.
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Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection. (+info)
Antimicrobial activity of human cervical mucus.
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The antibacterial activity of human cervical mucus (CM) was examined on standardized microbial colonized agar plates (agar diffusion test). In parallel, the lysozyme content of CM was determined by means of a turbidimetric test system in aliquots of the same CM specimens. Suspensions of living lyophilized Micrococcus lysedeikticus were used as bacterial substrate. Testing was performed in a total of 133 CM samples, obtained at mid-cycle from sexually active women from unselected infertile couples with a median age of 30 (range 21-42) years. All mucus specimens showed considerable antibacterial activity with clearly visible circular inhibition zones around the CM-filled holes in the colonized agar plates. Related to the effect of hen's egg white (HEW)-lysozyme on the same plates, the median activity of the CM specimens in the agar diffusion test was equivalent to 33.0 (range 6.4-391.4) microg/ml HEW-lysozyme. However, there was a wide inter-individual range of antibacterial effects of cervical secretions. The cervical index did not significantly influence the outcome of either test. The pH of the endocervical CM also was not correlated with the antibacterial effect. Sexual activity leading to the presence of spermatozoa in CM considerably increased its antibacterial effect. The activity was markedly higher in samples obtained within hours after intercourse compared with those taken after sexual abstinence of >/=5 days (P < 0.05). In microbially colonized CM specimens compared to sterile CM, all obtained under hormonally standardized conditions, the antibacterial activity in the agar plate test was significantly lower (P < 0.05). The results of this pilot study demonstrate the considerable antibacterial activity of human CM. (+info)
Characterization of Micrococcus antarcticus sp. nov., a psychrophilic bacterium from Antarctica.
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A Gram-positive, cold-adapted, aerobic, spherical actinobacterium (strain T2T) with a quite low cardinal growth temperature was isolated from Chinese Great-Wall station in Antarctica. Sequence comparisons of the 16S rDNA indicated the isolate to be a phylogenetic member of the genus Micrococcus, family Micrococcaceae, in which it represents a novel lineage. The phylogenetic distinctness of the isolate with respect to the type strains Micrococcus luteus and Micrococcus lylae was supported by DNA-DNA similarity values of less than 40%. Chemotaxonomic properties supported the placement of the isolate in the genus Micrococcus. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine. The predominant menaquinones are MK-8 and MK-8(H2). The G + C content of the DNA of the isolate is 66.4 mol%. Genotypic, morphological and physiological characteristics were used to describe a new species of Micrococcus, for which the name Micrococcus antarcticus is proposed. The type strain is T2T (= AS 1.2372T). (+info)
Kinetic studies on the phosphorolysis of polynucleotides by polynucleotide phosphorylase.
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The kinetics of the phosphorolysis of polynucleotide (as differentiated from oligonucleotide) by polynucleotide phosphorylase of Micrococcus luteus has been investigated. Double reciprocal plots of initial velocity against either inorganic phosphate or polynucleotide concentration are linear, and furthermore, the affinity of the enzyme for either substrate is unaffected by the presence of the other. dADP, an analogue of ADP product, is a competitive inhibitor with respect to Pi and polynucleotidy. (Ap)tA-cyclic-p is a competitive inhibitor with respect to Pi. The results are almost identical with both primer-independent (Form-I) and primer-dependent (Form-T) enzymes, although the various kinetic constants differ. On the vasis of these data a rapid equilibrium random Bi Bi mechanism is proposed. The demonstration of two different inhibitor constants for dADP and the difference between the Michaelis and the inhibitor constant for polyadenylic acid in polynucleotide phosphorolysis indicate at least two binding sites for polyadenylic acid and dADP on M. luteus polynucleotide phosphorylase. Its is suggested that in the phosphorolysis of long chain polymers the second binding site permits the polynucleotide to snap right back into position after removal of I mononucleotide unit and thus leads to the observed processive degradation. A general discussion of oligonucleotide and polynucleotide phosphorolysis and the differences between Form-I and Form-T enzymes in de novo synthesis and degradation of polynucleotides is presented. (+info)
Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms.
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An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: D-glucose or D-mannose for ConA and N-acetyl-D-glucosamine or N-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix. (+info)
Preparation and antibacterial activity upon Micrococcus luteus of derivatives of iturin A, mycosubtilin and bacillomycin L, antibiotics from Bacillus subtilis.
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Methylated and acetylated derivatives of iturin A and mycosubtilin and methylated derivatives of bacillomycin L were prepared and their antibacterial activity on Micrococcus luteus was compared with the activity of the original substance. the results obtained show the importance of polar groups for the antibiotic activity of the substances of iturin group. (+info)