Identification and characterization of antimicrobial activity in two yeast genera

A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.     

The Validity of the Methylene Blue Reduction Test in the Grading of Ice Cream

S ummary . Samples of ice cream from small manufacturers'plant were examined and graded by the Methylene Blue Reduction Test. The effect of the 17 h incubation period on bacterial populations was examined and the resultant increase in numbers was determined with a Coulter Counter. Most of the ice creams examined fell into Grades 1, 2 or 3, but only a small difference in the colony count range between Grade 1 and Grade 2 samples was observed. Sterile ice cream samples were seeded with pure cultures of selected coliforms and the effect on methylene blue grading was determined. It was found that for each grade of ice cream a far higher number of the added pure culture bacteria was required to achieve the same methylene blue reduction as bacteria of the mixed flora already present.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

A simple and rapid test for differentiation of aerobic from anaerobic bacteria

A rapid qualitative test is proposed for bacterial respiratory type based on 24 h culturing of bacteria in liquid medium supplemented with a redox indicator: methylene blue or resazurin. Five reference bacterial strains with definite respiratory type as well as nine bacterial isolates from a laboratory digester for methane fermentation were used. Results obtained showed that both indicators can be used for distinction of strict aerobes from other bacterial representatives with definite respiration. In addition, the resazurin is able to differentiate strict anaerobes from microaerophiles and other anaerobes. The main advantages of the methylene blue is that it is a cheap, easily accessible dye, wide-used in microbiological practice and the results obtained with it are more stable over time. It was also noticed that the test with both indicators gave reliable results for the bacterial respiration only when an inoculum up to 48 h old was used.     

Antibacterial acitivity of the peptide complex isolated from the preparation of leukocytic interferon

A complex of low-molecular cationic peptides having an anti-bacterial effect with respect to Gram-positive and Gram-negative bacteria was isolated from the preparation of leukocytic interferon. The antibacterial action of the peptide complex was experimentally studied in vitro. The study revealed that the degree of the antibacterial activity of the peptide complex depended on the concentration of the bacterial culture under study, the ionic power of the incubation medium and did not depend on the presence of the products of bacterial vital activity in the growth medium. The antibacterial action of the peptide complex on the test cultures of Gram-positive and Gram-negative bacteria, as well as on the cultures of bacteria isolated from patients with infectious inflammatory diseases of the organs of the urinary system, was established. These results opened prospects for the development of fundamentally new antibacterial preparation on the basis of the peptide complex obtained in our studies.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

紫云英根瘤菌及巴西固氮螺菌的氢酶表达特征

紫云英根瘤菌氢酶表达依赖于H_2并受碳底物和高O_2浓度的阻遏及cAMP的显著促进。整体细胞的吸氢活性对O_2不敏感,受碘乙酸(50mmol L~(-1))的强烈抑制。少数氧化还原电位为正值的人工电子受体可支持吸氢活性。与紫云英根瘤菌不同,巴西固氮螺菌氢酶表达并不依赖于H_2,受碳底物阻遏及cAMP促进的效应均不显著,而对O_2敏感。整体细胞吸氢活性受碘乙酸的抑制作用不明显。无论正、负值氧化还原电位人工电子受体均可支持吸氢活性。在经饥饿的静止细胞中,H_2可支持固氮活性并增强固氮酶对O_2的耐受能力。     

Effect of the Volume-to-Surface Ratio of Cultures on Escherichia coli Growth: An Experimental and Theoretical Analysis

Daidzein (4′,7-dihydroxyisoflavone), a phytoestrogen found in soybeans mainly in the form of its glycoside daidzin, is metabolized by colonic bacteria to compounds with altered estrogenic activities, which may affect human health. Antibacterial agents used for the treatment of infections can alter the composition of bacterial populations in the colon and therefore can affect daidzein metabolism. To rapidly detect the effects of different concentrations of antibiotics on daidzein metabolism by colonic bacteria of monkeys and identify the subpopulation involved in daidzein metabolism, Etest strips containing antibacterial agents from three classes (tetracyclines, fluoroquinolones, and β-lactams) were used to eliminate the colonic bacteria that were susceptible to 0–32 μg/ml of each antibacterial agent and test the surviving bacteria for their ability to metabolize daidzein. The metabolism of daidzein by the colonic microflora was measured before and after the colonic bacterial population was exposed to antibacterial agents. The metabolites were detected by high performance liquid chromatography and mass spectrometry after incubation of the cultures for various times. Exposure of colonic microflora to antibiotics had various effects on daidzein metabolism. Tetracycline completely removed the bacteria metabolizing daidzein, metabolism of daidzein was not changed in cultures of bacteria after ceftriaxone treatment, and ciprofloxacin enriched for the bacteria metabolizing daidzein. In liquid cultures treated with various concentrations of ciprofloxacin, 4 μg/ml of ciprofloxacin favored the growth of bacteria that metabolized daidzein. This is the first time in which the Etest has been used to show that, whereas some antibiotics eliminate phytoestrogen-metabolizing bacteria in colonic microflora, others enrich them by eliminating the non-metabolizing strains in the population.     

Effects of Arene-type Hydrocarbon Air Pollutants on Bacillus megaterium

Effects of certain common carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbon organic air pollutants on Bacillus megaterium cultures were noted. Depending on the medium used, either growth suppression or induction of atypical cell forms was observed in cultures grown in the presence of a carcinogen. By contrast, no such alterations were apparent in cultures grown in media supplemented with a noncarcinogen. Both carcinogenic and noncarcinogenic hydrocarbons exerted an enhancing influence, of varying degree, on lipogenesis, glycolysis, and methylene blue reductase activity. A higher than normal level of these reactions, however, was associated with cultures exposed to a carcinogen. In addition, infrared examination of lipids revealed unique spectral characteristics for materials extracted from carcinogen-treated cultures. No difference was noted between materials derived from noncarcinogen-treated cultures and from control cultures.     

Pseudo-Second-Order Kinetic Model for Biosorption of Methylene Blue onto Tamarind Fruit Shell: Comparison of Linear and Nonlinear Methods

In this study, the sorption of methylene blue, a basic dye, onto tamarind fruit shell was studied by performing batch kinetic sorption experiments. The equilibrium kinetic data were analyzed using the pseudo-second-order kinetic model. A comparison between linear least squares method and nonlinear regression method of estimating the kinetic parameters was examined. Four pseudo-second-order kinetic linear equations were discussed. The coefficient of determination (r ²), and the chi-square (χ²) test were employed as error analysis methods to determine the best-fitting equation. Kinetic parameters obtained from four kinetic linear equations using the linear method differed but they were the same when nonlinear method was used. Present investigation showed that by linear method a Type 1 expression very well represent the kinetic uptake of methylene blue onto tamarind fruit shell. Linear method was found to check only the hypothesis instead of verifying the kinetic model. Nonlinear regression method was found to be the more appropriate method to determine the rate kinetic parameters.     

Preliminary characterization of some Streptomyces species isolated from a composting process and their antimicrobial potential

The aim of this study was to screen Streptomycetes isolates with antimicrobial and antiviral activity, in a search for new metabolites. The isolates were obtained from a composting process, and identified based on morphological characteristics and molecular biological methods. The antimicrobial activity was determined using the double-layer agar method against 53 test organisms (bacteria, yeasts, and filamentous fungi). All isolates were grown in submerged culture, in mineral salts-starch-casein (SC) broth and ISP2 media, and the filtrate cultures were used in the assays for antibacterial and antiviral activity. Bovine Herpes virus (BoHV-I) was used for the antiviral activity. The morphological and molecular characteristics confirmed that all 25 isolates belonged to the genus Streptomyces. In the assay for antimicrobial activity, 80% of the Streptomyces isolates were able to inhibit at least one of the test organisms. Of these, 80% were active against bacteria and 45% against fungi. Eight of the isolates showed a broad spectrum of inhibitory activity; of these, the isolate Streptomyces spp. 1S was able to inhibit 46 of the test organisms, and, most importantly, the 16 Gram-negative strains were inhibited. Of the 25 isolates, 44.4% of the isolates were able to grow and produce bioactive metabolites when grown in submerged culture. Four extracts showed a cytopathic effect in 10 CCID₅₀ MDBK cell, even though no viricidal effect was observed. The results obtained with these isolates indicated good biotechnological potential of these Streptomyces strains.     

The effect of carbon dioxide on reduction of methylene blue by microorganisms

Summary and Conclusions The experiments ofHes which indicate that CO₂ is essential for the reduction of methylene blue by microorganisms presumably because it is required for the functioning of the cellular hydrogen transport mechanisms, have been repeated and results have been obtained that seemingly differ from those ofHes. We could not demonstrate any effect of CO₂ on the rate of methylene blue reduction by various bacteria in the presence of utilizable compounds such as glucose, sucrose, lactate and pyruvate. UsingE. coli and other bacteria we found, however, that the time required for the reduction of methylene blue was greatly increased by CO₂ removal when no substrates were added. This effect of CO₂ on the endogenous metabolism was observed only when the cells were depleted of endogenous reserves and of CO₂ by aeration or dilution.     

Bacteriocinogenic activity of strains of the genus Erwinia

Antibacterial activity of 272 Erwinia strains was studied. It was found that 182 or 66.9 per cent of the strains were capable of producing spontaneously antibacterial substances belonging to the class of bacteriocins; 125 bacteriocynogenic strains were divided into 25 groups on the basis of their antibacterial spectrum similarity; 57 bacteriocynogenic strains were not included into any of these groups because of their significant heterogenicity with respect to the feature studied. It was shown that most of the strains inhibited viability of the bacteria of both its own and other species. Investigation of the antagonistic activity of the Erwinia strains with broad antibacterial spectra with respect to E. coli indicative for colicins gave negative results. The study of the Erwinia strains sensitivity to the antibacterial effect of the bacteriocynogenic cultures showed that 210 out of 272 cultures were sensitive to separate bacteriocins.     

Identification of Mycoplasmatales: Characterization Procedures

A large collection of avian Mycoplasma cultures was used in studies to improve and develop biological and biochemical characterization techniques. Differential patterns among 11 avian serotypes were shown by carbohydrate fermentation, tetrazolium- and methylene blue-reduction reactions, breakdown of arginine, and the formation of film on egg yolk-agar. Some cultures fermented as many as 14 carbohydrates. Polyhydric alcohols and pentoses were among the compounds fermented. An improved procedure for determining methylene blue reduction by Mycoplasma was developed. These simple, rapid procedures are reproducible and should be useful in grouping Mycoplasma isolates prior to definitive identification by serological or other means.     

Antibacterial peptides designed as analogs or hybrids of cecropins and melittin.

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.     

Growth stimulation produced by methylene blue treatment in sweet potato

Methylene blue as an alternative treatment to gamma rays to stimulate growth in sweet potato tissue cultures, was applied in two different ways: – pre-incubation of nodal explants with methylene blue for 1 h using urea as a permeabilizer; – methylene blue directly incorporated into the culture medium. Both treatments stimulated growth, but the better performance being obtained with the second treatment, which had no toxic effect. The activity and electrophoresis pattern of peroxidase after treatment ofIpomoea batatas plantlets with methylene blue or gamma rays did not show similar results for the two treatments. Peroxidase activity was greater in leaves of gamma ray treated plants compared to the non-treated control. The results obtained with the Methylene blue treatment did not significantly change the peroxidase activity relative to the control. This revised version was published online in June 2006 with corrections to the Cover Date.     

The synthesis and antibacterial activity of totarol derivatives. Part 1: modifications of ring-C and pro-drugs.

A series of analogues of, and potential pro-drugs derived from, the potent antibacterial diterpene totarol (1) were synthesized in order to elucidate the minimum structural requirements for antibacterial activity and to seek compounds with good bioavailability in vivo. These analogues varied in the structural features of their aromatic rings and the prodrugs were O-glycosylated derivatives. They were tested in vitro against three gram-positive bacteria: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA); and against the gram-negative multi-drug-resistant Klebsiella pneumoniae. None of the analogues was more potent than totarol itself, which is effective against these gram-positive bacteria at MIC values of 7 microM. The results were evaluated in terms of a structure-activity relationship and this showed that a phenolic moiety was essential for potent antibacterial activity. Amongst the pro-drugs, totaryl alpha-D-mannopyranoside (22) proved the most active in vitro (MIC 18 microM). The in vivo antibacterial activities of compounds 1, 22 and totarol beta-lactoside (23) were assessed in a mouse model of infection, but they were found to be ineffective. Compounds 1 and 22 were shown to be cytotoxic towards proliferating human cell cultures, CH 2983, HeLa, and MG 63, but only at concentrations of > 30 microM.     

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.     

Factors affecting the antibacterial activity of the ruminal bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h(-1)) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2-1.2 h(-1)) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h(-1)) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h(-1)) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml(-1)) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application.