Quantitative studies on competitive activities of skin bacteria growing on solid media.
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)
Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans.
FK506 and rapamycin are immunosuppressants that inhibit signalling cascades required for T-cell activation, yet both are natural products of Streptomyces that live in the soil. FK506 and rapamycin also have potent antimicrobial activity against yeast and pathogenic fungi, suggesting a natural role in inhibiting growth of competing micro-organisms. The immunosuppressive and antimicrobial activities of FK506 and rapamycin are mediated by binding to the FKBP12 prolyl isomerase and the resulting FKBP12/FK506 and FKBP12/rapamycin complexes inhibit conserved protein targets, either the phosphatase calcineurin or the TOR (target of rapamycin) kinases, respectively. Streptomyces sp., 'Streptomyces hygroscopicus subsp. ascomyceticus' and Streptomyces hygroscopicus, which produce FK506, FK520 (also known as ascomycin, a C21 ethyl derivative of FK506) and rapamycin, respectively, produced toxins that inhibited the growth of competing cells of the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans. Yeast and fungal mutants lacking FKBP12 or expressing dominant drug-resistant calcineurin or TOR mutants were resistant to FK506 and rapamycin, and to the toxins produced by Streptomyces. Streptomyces strains with mutations in the FK506 or rapamycin biosynthetic enzymes were impaired in toxin production. Finally, the toxins secreted by 'S. hygroscopicus subsp. ascomyceticus' and S. hygroscopicus promoted formation of FKBP12/calcineurin and FKBP12/TOR complexes in a two-hybrid assay and mutations that rendered calcineurin or TOR drug-resistant prevented interaction. These observations support the hypothesis that Streptomyces evolved to secrete FK506, FK520 and rapamycin as toxins to inhibit the growth of competing yeast and fungi. (+info)
Purification and properties of a basic endo-beta-1,6-glucanase (BGN16.1) from the antagonistic fungus Trichoderma harzianum.
The antagonistic fungus Trichoderma harzianum CECT 2413 produces at least two extracellular beta-1,6-glucanases, among other hydrolases acting on polysaccharides from fungal cell walls, when grown in chitin as the sole carbon source. We have previously reported on the purification and biochemical characterization of the major activity, which corresponds to an acidic enzyme named BGN16.2 [de la Cruz, J., Pintor-Toro, J.A., Benitez, T. & Llobell, A. (1995) J. Bacteriol. 177, 1864-1871]. In this paper, we report on the purification to electrophoretical homogeneity of BGN16.1, the second beta-1, 6-glucanase enzyme. BGN16.1 was purified by ammonium sulfate precipitation followed by adsorption and digestion of pustulan (a beta-1,6-glucan), chromatofocusing and gel-filtration chromatography. BGN16.1 is a non-glycosylated protein with an apparent molecular mass of 51 kDa and a basic isoelectric point (pI 7.4-7.7). The enzyme was active toward substrates containing beta-1,6-glycosidic linkages, including yeast cell walls. The Km was 0.8 mg x mL-1 with pustulan as the substrate. Reaction product analysis by HPLC clearly indicated that BGN16.1 has an endo-hydrolytic mode of action. The probable role of this enzyme in the antagonistic action of T. harzianum is also discussed. (+info)
Intestinal colonisation of gnotobiotic pigs by Salmonella organisms: interaction between isogenic and unrelated strains.
The effect of intestinal colonisation by a Salmonella strain on the establishment in the gut of an isogenic mutant administered orally 24 h after the first strain was studied in gnotobiotic pigs. Irrespective of the clinical outcome of the infection, the extensive colonisation of one Salmonella strain prevented a similar degree of colonisation by an otherwise isogenic antibiotic resistant strain; in some cases the second strain was hardly detectable. The poor colonisation of the challenge Salmonella strains was generally reflected in very low counts of organisms in the tissues. Colonisation by a strain of Escherichia coli reduced the rate of establishment of an isogenic E. coli, strain but did not prevent colonisation by an S. Typhimurium strain. S. Typhimurium with mutations in the tsr (serine chemotaxis receptor protein) or oxrA (transcriptional regulator of anaerobic metabolism) genes did not inhibit colonisation. Mutations in cya (adenylate cyclase), tar and trg (chemotaxis receptor proteins for aspartate and ribose respectively) genes were less inhibitory, while motB (non-motile) and cheR (impaired motility) mutants were fully inhibitory. (+info)
A new alkaline pH-adjusted medium enhances detection of beta-hemolytic streptococci by minimizing bacterial interference due to Streptococcus salivarius.
A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing Streptococcus salivarius against beta-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of beta-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of S. salivarius bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of beta-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The beta-hemolytic streptococcal isolates recovered from the samples included 314 group A (S. pyogenes), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded S. pyogenes, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of S. pyogenes isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of S. pyogenes isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07; P < 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient S. pyogenes strains which might otherwise go undetected on SBA and other isolation media. (+info)
Selected bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio proteolyticus CW8T2.
In this study Vibrio proteolyticus CW8T2 has been identified as a virulent pathogen for Artemia spp. Its infection route has been visualized with transmission electron microscopy. The pathogen affected microvilli and gut epithelial cells, disrupted epithelial cell junctions, and reached the body cavity, where it devastated cells and tissues. In vivo antagonism tests showed that preemptive colonization of the culture water with nine selected bacterial strains protected Artemia juveniles against the pathogenic effects. Two categories of the selected strains could be distinguished: (i) strains providing total protection, as no mortality occurred 2 days after the experimental infection with V. proteolyticus CW8T2, with strain LVS8 as a representative, and (ii) strains providing partial protection, as significant but not total mortality was observed, with strain LVS2 as a representative. The growth of V. proteolyticus CW8T2 in the culture medium was slowed down in the presence of strains LVS2 and LVS8, but growth suppression was distinctly higher with LVS8 than with LVS2. It was striking that the strains that gave only partial protection against the pathogen in the in vivo antagonism test showed also a restricted capability to colonize the Artemia compared to the strains providing total protection. The in vivo antagonism tests and the filtrate experiments showed that probably no extracellular bacterial compounds were involved in the protective action but that the living cells were required to protect Artemia against V. proteolyticus CW8T2. (+info)
Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 cells.
To gain further insight into the mechanism by which lactobacilli develop antimicrobial activity, we have examined how Lactobacillus acidophilus LB inhibits the promoted cellular injuries and intracellular lifestyle of Salmonella enterica serovar Typhimurium SL1344 infecting the cultured, fully differentiated human intestinal cell line Caco-2/TC-7. We showed that the spent culture supernatant of strain LB (LB-SCS) decreases the number of apical serovar Typhimurium-induced F-actin rearrangements in infected cells. LB-SCS treatment efficiently decreased transcellular passage of S. enterica serovar Typhimurium. Moreover, LB-SCS treatment inhibited intracellular growth of serovar Typhimurium, since treated intracellular bacteria displayed a small, rounded morphology resembling that of resting bacteria. We also showed that LB-SCS treatment inhibits adhesion-dependent serovar Typhimurium-induced interleukin-8 production. (+info)
The role of normal flora in Giardia lamblia infections in mice.
The presence of normal bacterial flora in the intestinal tract is thought to protect against colonization by pathogens. Only a few specific examples of this protection have been demonstrated for bacterial pathogens and protozoan infections. Mice from one commercial breeding farm were found to be less susceptible to infection with Giardia lamblia than were isogenic mice from another facility. When mice were housed together, resistance to infection was readily transferred to normally susceptible mice. After resistant mice were treated with neomycin, differences in susceptibility to infection were shown to be due to differences in the resident flora present in these mice. These results suggest the possible use of probiotic therapy for prevention of G. lamblia infections and may help explain some of the variability of outcomes seen in G. lamblia infections in humans. (+info)