Membranes of five-fold alamethicin-resistant<Emphasis Type="Italic">Staphylococcus aureus,Enterococcus faecalis</Emphasis> and<Emphasis Type="Italic">Bacillus cereus</Emphasis> show decreased interactions with alamethicin due to changes in membrane fluidity and surface charge

The aim of this study was to understand development of resistance to alamethicin (a model barrel stave pore forming antimicrobial peptide) by investigating changes in phospholipid profile, fatty acid side chain analysis and extent of alamethicin insertion in biomimetic membrane prepared form wild type strains and five folds alamethicin resistant variants ofStaphylococcus aureus NCDC 110,Enterococcus faecalis NCDC 114 andBacillus cereus NCDC 66. The wild type strains NCDC 110, 114, 66, were sensitive to alamethicin with IC₅₀ 5.5, 3.25 and 2.0 μg/ml respectively. Wild type strains were cultured in the presence of alamethicin to select resistant variants with IC₅₀ 29.0, 17.0 and 9.5 μg/ml respectively. The phospholipid profile analysis revealed increase in amino-group containing phospholipids to amino-group lacking phospholipids ratio between wild-type and resistant variant inS. aureus and B. cereus but decreased inE. faecalis. Predominant fatty acids in all strains were composed of even number of carbons. Linoleic acid was detected only in resistant strain ofB. cereus. As indicated by saturated-to-unsaturated fatty acids ratio, the membrane fromS. aureus andE. faecalis became more rigid, whereas, inB. cereus it became more fluid. Using a colorimetricin vitro assay, a decrease in alamethicin insertion in the biomimetic membrane could be observed upon acquisition of resistance. The membranes of five-fold alamethicin-resistantS. aureus, E. faecalis andB. cereus revealed changes in membrane fluidity and surface charge upon acquisition of resistance to alamethicin.     

Thermal Resistance ofBacillus cereus spores in custard preparations

Summary The thermal resistance ofBacillus cereus spores expressed in terms ofD value (an expression of time in minutes required for the heat destruction of one log cycle of spores) in custard preparations decreased from 90°C to 100°C. With an increase in pH of custard preparations from 6.2 to 7.2, theD value ofB. cereus spores increased from 3.1 to 3.7 min at 90°C, from 2.5 to 3.0 min at 95°C and from 1.7 to 2.2 min at 100°C. However, theZ value (the change in temperature necessary to cause a tenfold change in theD value) remained unaffected by the change in pH. A decrease in the number of heatedB. cereus spores was observed with the increase in the concentration of the antimicrobial agent sodium benzoate and the antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

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Résistance thermique des spores deBacillus cereus dans les préparations de crème

Résumé La résistance thermique de spores deBacilluscereus, exprimée par la grandeur D (l'expression du temps en minutes requis pour la destuction thermique d'une décade logarithmique de spores) dans les préparations de crème, décroît de 90 °C à 100 °C. Avec une augmentation de pH des préparations de crème de 6.2 à 7.2, la valeur D pour les spores deB. cereus augmente de 3.1 à 3.7 min. à 90 °C, de 2.5 à 3.0 min. à 95 °C et de 1.7 à 2.2 min. à 100 °C. Toutefois, la grandeur Z (le changement de température requis pour engendrer un changement d'un facteur 10 dans la valeur D) n'est pas affectée par ce changement de pH. Une diminution du nombre de spores deB. cereus chauffé est observée avec l'augmentation de la concentration du benzoate de soude, un agent antimicrobien, ainsi que de l'anisole hydroxy-butylé et du toluène hydroxybutylé, deux agents antioxidants.

     

Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels

A natural lactic fermentation of mixtures of water and whole flour of either maize or high-tannin sorghum was obtained either before or after cooking to a weaning gruel: The preparations had a final pH of about 3.8 (range 3.67 to 4.00) and a ratio of lactic acid to acetic acid of 91 (w/w). The growth of added (about 10⁷ c.f.u./g gruel) Gram-negative intestinal pathogenic bacteria, enterotoxigenicEscherichia coli, Campylobacter jejuni, Shigella flexneri andSalmonella typhimurium, was strongly inhibited in the sour gruels, and the effect could primarily be explained by the low pH caused by the formation of lactic and acetic acids during the fermentation process. Of the added Gram-positive bacteria,Bacillus cereus andStaphylococcus aureus showed similar inhibited growth up to 7h after inoculation in the sour gruels. The strain ofStaphylococcus, however, showed only a continued reduction in growth in the fermented gruel samples, which had a viable lactic bacteria culture indicating the presence of a bacteriocin. This implies that a low pH (< 4.0) alone is not sufficient to sustain the inhibition of the growth ofStaphylococcus aureus. The survival studies were carried out at optimal temperatures for each respective enteropathogen.     

Mercury resistance determined by a self-transmissible plasmid inBacillus cereus 5

Summary Inducible mercuric reductase activity inBacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains ofB. cereus andB. thuringiensis. Transfer of mercury resistance fromB. cereus 5 toB. cereus 569 andB. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10^–5 to 10^–4.B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation ofB. cereus 569 by electroporation with pGB130 DNA isolated fromB. cereus 5 and a mercury-resistantB. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.     

The effect of fermentation on the growth and survival ofSalmonella typhimurium,Staphylococcus aureus,Bacillus cereus andPseudomonas aeruginosa in fermenting tef (Eragrostis tef)

Summary Growth ofSalmonella typhimurium, Staphylococcus aureus, Bacillus cereus andPseudomonas aeruginosa was inhibited when the pH of fermenting tef approached 5.0, 5.0, 5.5 and 5.0, respectively. However the test organisms grew in far more acidic conditions in broth than in fermenting tef and this is due to antimicrobial substance(s) being produced by some of the lactic acid bacteria. Except forBacillus cereus spores, all the test organisms were heat-inactivated during the baking process of the final tef injera.

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Effet de la fermentation sur la croissance et la survie de Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus et Pseudomonas aeruginosa dans le tef (Eragrostis tef) en fermentation

Résumé La croissance deSalmonella typhimurium, deStaphylococcus aureus, deBacillus cereus et dePseudomonas aeruginosa est inhibée lorsque les pH du tef en fermentation approchent respectivement 5:0, 5.0, 5.5 et 5.0. Toutefois, les organismes tests croissent dans des conditiones bien plus acides que dans le tef en fermentation. Ceci est dû à des substances antimicrobiennes produites par certaines bactéries lactiques. A l'exception des spores deBacillus cereus, tous les organismes tests sont inactivés par la chaleur durant le processus de cuisson.

     

Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.     

Distribution and sporulation phenology of myxomycetes in the Sonoran Desert of Arizona

All pith samples from 68 dead saguaro cacti in 3 plots and 11 isolated dead plants in Saguaro National Monument, Arizona, produced at least one species of myxomycete upon incubation at 20 or 30°C. Three species,Badhamia gracilis (Macbr.) Macbr.,Physarum straminipes Lister, andDidymium eremophilum M. Blackwell et Gilbertson, developed at high frequencies on the substrates in moist chamber culture.Perichaena corticalis (Batsch) Rost, andProtophysarum phloiogenum M. Blackwell et Alexopoulos were also present. Although previous literature reports [9] indicated that Myxomycetes grow best at low pH, these species all tolerated substrates of pH 8.7–10.4.Didymium eremophilum andP. phloiogenum had peaks in sporulation within 6 days; other species were slower. There was no difference in time of sporulation ofB. gracilis orD. eremophilum at 20 and 30°C; however, sporulation ofP. straminipes was significantly later at 30°C. Reduced spore germination and slower buildup of critically sized amoebal populations ofP. straminipes at 30°C may be a factor.     

Lytic effect ofVibrio cholerae elastase on Gram-positive and-negative bacteria

Elastase ofVibrio cholerae caused the lysis of freshly grown cells of Gram-negative (Pseudomonas aeruginosa, Proteus vulgaris, Salmonella paratyphi A andKlebsiella pneumoniae) bacteria. Gram-positive (Staphylococcus aureus andS. epidermidis) organisms were resistant to this enzyme. Heat killed and lyophilized Gram-positive and-negative bacteria (exceptS. aureus andS. epidermidis) showed higher sensitivity to elastase. Both Gram-negative and-positive bacteria were lyzed maximally by elastase at pH 8.0. At this pH, lytic activity of elastase was maximum in Tris-HCl and glycine-NaOH buffers followed by Tris-maleate and cacodylate buffers.     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Screening ofBifidobacterium strains for bacteriocin production

Summary Thirteen strains of bifidobacteria (Bifidobacterium) were examined for bacteriocin production. One strain among these produced bacteriocin which is active against lactic streptococci strains and against other species ofBifidobacterium, Clostridium andLactobacillus, but not against Gram-negative bacteria such asPseudomonas, Klebsiella, Serratia andEscherichia coli.Bacteriocin activity was inhibited by proteolytic enzymes, but not by heating 15 min or30 min at 100°C, and it was resistant for 15 min at 121°C and active at pH between 2 to 10.     

Seasonal variation in allelopathic potential of<Emphasis Type="Italic">Artemisia princeps</Emphasis> var.<Emphasis Type="Italic">orientalis</Emphasis> on plants and microbes

We investigated seasonal variations in allelopathic potential ofArtemisia princeps var.orientalis. Aqueous and meth-anol extracts and volatile substances were prepared in the laboratory from samples collected monthly (April through October). Their impacts were then assessed on the germination and seedling growth ofLactuca sativa andAchyranthes japonica. The allelopathic potential varied with the time of sample collection and the concentration tested. For example, germination ofL. sativa was not inhibited by the aqueous extract but seedling growth (shoots and roots) was, with its seasonal effect being significant. ForA. japonica, seed germination was not inhibited at lower concentrations (except for August samples). However, at higher concentrations and in certain months (especially July), germination was more negatively affected. The degree of seedling growth inhibition also differed by month and by extract concentration, with roots being impacted more than shoots. Volatile substances also had a time-dependent influence on the germination and seedling elongation ofA. japonica. In a separate experiment, the ethyl-acetate and water fractions of a crude methanol extract were prepared monthly fromA. princeps var.orientalis. Here, we examined their antimicrobial activities against three gram-positive bacteria (Bacillus cereus, Bacillus subtilis, andStaphylococcus aureus), two gramnegative bacteria (Escherichia coli andPseudomonas fluorescens), and one lactic acid bacterium,Lactobacillus plantar urn. The ethyl-acetate fraction that was sampled in September was remarkably potent againstB. cereus andB. subtilis, whereas the water fraction collected in August and September showed great antimicrobial activity against the grampositive and -negative bacteria. In contrast,L. plantarum was not inhibited by the water fraction, regardless of the sampling month. Likewise, the ethyl-acetate and water fractions collected in April and October had the lowest levels of antimicrobial activity.     

Photobiological activity of exogenous and endogenous porphyrin derivatives in Escherichia coli and Enterococcus hirae cells

Photodynamic treatment, the combined application of a photosensitiser and visible light, represents a new and promising approach for the inactivation of microorganisms. The photosensitising potentials of exogenous zinc-phthalocyanine-tetrasulphonate (ZnPsTS), tetraphenylporphyrins (TPPs) and endogenous porphyrin derivatives were tested and compared on Gram-negative and Gram-positive bacteria, Escherichia coli B. and Enterococcus hirae, respectively. The synthesis of endogenous porphyrins was induced by 5-aminolevulinic acid (δ-ALA). The porphyrin- or δ-ALA-treated cells were irradiated with white light. The photosensitising efficiency of endogenous derivatives on both types of bacteria is ZnPcTS < TP(4-OGluOH)₃P < TP(4-OGluOH)₄P. However, neither exogenous derivatives exhibit appreciable photosensitising activity for disinfection application. ALA-induced photodynamic treatment showed good potential for the inactivation of Escherichia coli cells, but not towards Enterococcus hirae cells. The failure of photosensitisation of the Enterococcus hirae strain selected indicates that apart from the Gram-positive character, other structural elements of the membrane can influence the result of photodynamic treatments. Received: 13 October 1999 / Accepted: 1 January 2001     

Survival and activity ofStreptococcus faecalis andEscherichia coli in tropical freshwater

The survival ofStreptococcus faecalis andEscherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities ofS. faecalis andE. coli decreased less than 1 log unit after 105 hours as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 hours,E. coli was more active thanS. faecalis as measured by nucleic acid composition. In this tropical rain forest watershed,E. coli andS. faecalis survived and remained active for more than 5 days; consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

Autecological investigations on marine diatoms. 4:Biddulphia aurita (Lyngb.) Brebisson et Godey—A succession of spring diatoms

Comparison of generation times ofBiddulphia aurita (Lyngb.) Brebisson and Godey with other diatoms indicated that the species can reach its best competitive position by growth from –1.5°C to about 6°C. The diatom demonstrates its optimum temperature at about 12°C. Yet, flowerings will occur at the lower temperatures only. Its presence in the early spring plankton is facilitated by its non-adhrence behaviour, that proved to be temperature dependent. At 0°C almost no adherence occurred, whereas at high temperaturesB. aurita adhered completely to the bottom of the glas vessels or pipettes. The importance ofB. aurita in the benthic community is discussed. The mutual differences in growth ofB. aurita, Chaetoceros debilis andThalassiosira nordenskioeldii resulted in a succession comparable with that found in nature.     

Characteristics of the bacteriocin produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> CTC 204 and the effect of this compound on the mesophilic bacteria associated with raw beef

Summary >Screening for the bacteriocin production of strains of lactic acid bacteria from various meat and meat products resulted in the detection of a bacteriocin-producing Lactococcus lactis subsp. cremoris CTC 204, isolated from chicken. The bacteriocin inhibited not only closely related lactic acid bacteria (Lactobacillus helveticus), but also pathogenic microorganisms (Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens). It was inactivated by α-chymotrypsin, ficin, papain, and pronase E, but not by lipase or pepsin. This compound was heat stable even at autoclaving temperature (121°C for 10min) and was produced during refrigerated storage. It was also active over a wide pH range (2–10), but the highest activity was observed in the lower pH range. The results indicated that dipping raw beef in the bacteriocin produced by strain CTC 204 could contribute to the extension of the shelf life of refrigerated bovine meat.     

Effect of sorbic acid and potassium sorbate on growth ofBacillus cereus andBacillus subtilis in rice filling of Karelian pasty

Summary The Karelian pasty is a perishable bakery product: the pH of the rice filling is approximately 6.60 and the a_W=0.98. Contamination after baking was shown to be the most important reason for microbial spoilage. The rice fillings of the pasties were artificially contaminated by spores ofBacillus cereus andB. subtilis, and growth at room temperature was followed.B. cereus was totally inhibited by an addition of 0.20% sorbic acid or 0.40% potassium sorbate.B. subtilis was totally inhibited by an addition of 0.10% sorbic acid or 0.26% potassium sorbate. An addition of 0.10% sorbic acid to the filling before baking is recommended. This level of addition does not alter the organoleptic quality of the pasty but is a considerable safety factor for Karelian pasty production.     

In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains

Kephir is a fermented carbonated refreshing milk, with a slightly acidic aromatic taste and creamy foam composition which contains lactobacilli, leuconostocci, acetic acid bacteria, lactostreptococci and yeasts. Recent studies have demonstrated its antibacterial, immunostimulating, antitumoral and cholesterol-lowering activities.

Purpose

The purpose of this study was to investigate the antimicrobial activity of kephir against Bacillus subtilis spp. spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. The kephir fermented for 24 h and 48 h, as well and after 7 days preservation at 4–8 °C was tested by in vitro disk diffusion method. The intensity of the antimicrobial activity was interpreted by comparison with two antibiotics, i.e. ampicillin and neomycin.

Results

The antimicrobial activity of 24 h as well as 48 fermented kephir, fresh or after 7 days preservation at 4–8 °C was similar and observed against B. subtilis, S. aureus, E. coli, E. faecalis and S. enteritidis. For E. coli, E. faecalis and S. enteritidis the antimicrobial activity was superior to both tested antibiotics and for B. subtilis and S. aureus to one antibiotic. The tested products exhibited no activity against P. aeruginosa and C. albicans.

Conclusion

Kephir is exhibiting large spectrum and strong antibacterial activity probably due to the complex viable probiotic strains association producing antimicrobial substances.     

Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Absence of a role for lytic microorganisms in the decline of bacteria andSaccharomyces introduced into soil

The populations ofKlebsieila pneumoniae, Escherichia coli, Enterobacter aerogenes, andPseudomonas sp. fell following their addition to soil, but species lysing these gram-negative bacteria were not detected. The numbers ofStaphylococcus aureus andMicrococcus flavus fell by more than four orders of magnitude and ofSaccharomyces cerevisiae by more than two orders after their addition to soil. Organisms lysing these gram-positive bacteria were present in soil, but their numbers did not increase as a result of the additions. Lytic activity againstS. aureus was detected in soil filtrates, but this activity was not enhanced by inoculation of soil with the bacterium. Addition of cycloheximide to soil suspensions delayed the fall in abundance ofM. flavus but did not suppress the lytic populations. We conclude that lysis is not responsible for the decline of bacteria orS. cerevisiae added to soil.