Agar Medium (agar + medium)

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Selected Abstracts

Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Nicolai Müller
Summary Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 108 cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22,28 h at 28°C. Cells were small, 0.5 × 5 ,m in size, Gram-positive, and formed terminal oval spores. At 20°C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO2. At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture. [source]

Occurrence and diversity of nitrogen-fixing Sphingomonas bacteria associated with rice plants grown in Brazil

Sandy Sampaio Videira
Abstract So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted to three strains of Sphingomonas azotifigens. In this work, a group of 46 Sphingomonas -like isolates, which originated from two rice varieties grown in two soils in Brazil, were characterized based on morphological, physiological and genetic analyses. The PCR genus specifically applied indicated that all 46 isolates belonged to the Sphingomonas genus and confirmed the results based on the yellow pigment of the colonies grown on potato agar medium and the BIOLOG data. It was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the acetylene reduction method and confirmed by nifH gene detection. The genetic diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis) showed that the isolates formed two distinct groups at a similarity value of 60%. Furthermore, five clusters at 60% similarity were observed with the 16S,23S intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the 16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing isolates formed clusters apart from that of the S. azotifigens. This is the first report on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice grown in Brazil. [source]

Importance of Cryphonectria parasitica stromata production and intermediate-pigmented isolates in spread of Cryphonectria hypovirus 1 on grafted American chestnut trees

E. P. Hogan
Summary Large, surviving American chestnut trees, grafted in 1980, were inoculated in 1982 and 1983 with four ,white' European (French and Italian) hypovirulent strains of Cryphonectria parasitica infected with Cryphonectria hypovirus 1 (CHV1). Spread of Italian CHV1-Euro7, indicated by nucleotide sequence analysis of CHV1 isolates, and a high level of blight control, occurred on these trees for over 20 years. However, the means by which CHV1 spreads and the possible role of stromata production in that spread are unknown. In this study, 249 C. parasitica isolates were recovered from stromata excised from natural cankers on the grafted trees and plated on an agar medium; 5.2% of the stromata yielded white phenotype isolates, 9.2% yielded intermediate-pigmented isolates (30,70% pigmentation) and the remainder were normal-pigmented isolates. For comparison, cankers artificially established on blight-free, forest-clear-cut American chestnut trees, following inoculation with three Italian white hypovirulent strains, were evaluated in a similar manner. Of 241 C. parasitica isolates recovered from stromata, 66.4% had a white colony phenotype, 19.1% had an intermediate-pigmented phenotype and the remainder were normal-pigmented isolates. For single conidia collected from stromata and plated, nearly equal frequencies of only white and intermediate-pigmented colony phenotypes were obtained. Following dsRNA extraction and electrophoresis, 21 of 33 intermediate-pigmented isolates were positive for CHV1. Some normal pigmented isolates also were positive for CHV1. Single-sporing a CHV1-positve, normal-pigmented, natural-canker, stroma isolate (Str 1) from the grafts resulted in several deeply red-orange pigmented (JR) isolates as well as some white isolates. The dsRNA in the JR isolate was extracted and cDNAs made by reverse transcriptase-polymerase chain reaction (RT-PCR) for part of a region (p29) in ORF A. Nucleotide sequencing indicated 100% identity to CHV1 present in the inoculated Italian white strain, Ep 47. The results indicate that stromata production on the grafted trees may contribute to CHV1 spread, and the presence of CHV1 in intermediate-pigmented isolates and some normal pigmented isolates indicates these isolates, often overlooked, may be important in CHV1 spread and the high level of blight control on the grafted trees. [source]

Methods for inoculum production and inoculation of Cistella japonica, the causal agent of resinous stem canker in Chamaecyparis obtusa

T. Yamanobe
Summary The ascomycete Cistella japonica was cultured on potato dextrose agar medium (PDA) for inoculation into Chamaecyparis obtusa, enabling the development of an inoculation method suitable for use in a breeding programme aimed at selecting for families of Ch. obtusa resistant to resinous stem canker. Using PDA to generate the inoculum resolved the problems encountered with the previously used mixed medium of rice bran and wheat bran, including unfavourable characteristics, uncertain growth of Ci. japonica mycelia, and a complex culturing operation. The inoculation test induced resin exudation similar to that observed in natural infections, and reproduced clonal differences with regard to damage severity. [source]

Interfertility between North American and European strains of Phlebiopsis gigantea

R. Grillo
Summary Thirteen homokaryotic strains of Phlebiopsis gigantea from Canada, six strains from the US and 10 strains from Europe were paired in all possible combinations in order to determine the degree of interfertility between them. The diagnosis of interfertility was based on the production of heterokaryotic fruit bodies in the pairings. Among the resulting 406 pairings, 253 (62%) fruited. Within the strains originating from Canada, USA and Europe, 64, 80 and 64% of the pairings fruited, respectively. The fruiting frequency in pairings between the Canadian and US strains was 65%, between the Canadian and European strains 55%, and between the US and European strains 67%. True hybridization between the European and North American P. gigantea was shown by analysing the single-spore progeny using DNA fingerprinting. In spite of the relatively low interfertility in pairings within and between continents, no clear indication of the existence of intersterility groups was found. The low interfertility is probably due to the ageing of the pure cultures and to deficient fruiting ability of certain heterokaryons on agar medium. The results strongly suggest that although the North American and European strains of P. gigantea show some differentiation they can be regarded as belonging to the same biological species. Résumé Treize souches homocaryotiques de Phlebiopsis gigantea du Canada, 6 des Etats-Unis et 10 d'Europe ont été confrontées par paires selon toutes les combinaisons possibles pour déterminer leur degré d'interfertilité. Le diagnostic d'interfertilité est basé sur la production de fructifications hétérocaryotiques dans les confrontations. Parmi les 406 paires, 253 (62%) ont fructifié. Ce pourcentage est de 64, 80 et 64% respectivement pour les souches du Canada, des Etats-Unis ou d'Europe entre elles. La fréquence de fructification pour les confrontations entre souches du Canada et des Etats-Unis est de 65%, entre souches européennes et canadiennes de 55% et entre souches des USA et d'Europe de 67%. La réalité de l'hybridation entre souches européennes et nord américaines a été démontrée par analyse de la descendance de spores par empreinte génétique. Malgré la relativement faible interfertilité dans les confrontations intra ou inter continents, l'existence de groupes d'interstérilité n'a pu être montrée. La faible interfertilité est probablement due au vieillissement des cultures pures et à une faible capacitéà fructifier sur milieu gélosé de certains hétérocaryons. Les résultats suggèrent fortement que malgré une certaine différenciation entre souches nord américaines et européennes de P. gigantea, celles-ci peuvent être considérées comme appartenant à la même espèce. Zusammenfassung Zur Bestimmung der Interfertilität wurden 13 homokaryotische Isolate von Phlebiopsis gigantea aus Kanada, 6 aus den USA und 10 aus Europa in allen möglichen Kombinationen miteinander gekreuzt. Der Erfolg der Kreuzungen wurde anhand der Bildung von heterokaryotischen Fruchtkörpern beurteilt. Bei insgesamt 406 Kreuzungen wurden in 253 Fällen (62%) Fruchtkörper gebildet. Unter den Stämmen aus Kanada, den USA und Europa kam es in 64, 80 und 64% der Fälle zur Fruchtkörperbildung. Bei Paarungen zwischen Isolaten aus Kanada und den USA fruktifizierten 65%, bei Paarungen zwischen Isolaten aus Kanada und Europa waren dies 55%, Paarungen zwischen Isolaten aus den USA und Europa fruktifizierten zu 67%. Die echte Hybridisierung zwischen europäischen und nordamerikanischen Isolaten von P. gigantea wurde durch Analysen der Einzelsporisolate aus den Fruchtkörpern durch DNA-Fingerprinting nachgewiesen. Trotz der relativ geringen Interfertilität innerhalb und zwischen den Kontinenten wurden keine klaren Hinweise auf die Existenz von Intersterilitätsgruppen gefunden. Die geringe Interfertilität ist möglicherweise durch eine Degeneration der Kulturen in vitro und durch die fehlende Fähigkeit mancher Heterokaryen zur Fruchtkörperbildung auf Agarmedien erklärbar. Die Ergebnisse zeigen, dass Isolate von P. gigantea aus Nordamerika und Europa trotz einer gewissen geographischen Differenzierung zu einer einzigen biologischen Art gehören. [source]

Interactions between Tricholomopsis rutilans and ectomycorrhizal fungi in paired culture and in association with seedlings of lodgepole pine and Sitka-spruce

E. A. Murphy
Interactions between two isolates (Avondhu and CBS) of Tricholomopsis rutilans and ectomycorrhizal fungi (Hebeloma crustuliniforme, Laccaria laccata and Paxillus involutus) were examined on agar medium in the presence or absence of woodchips. The CBS isolate showed more competitiveness than the Avondhu isolate, when paired with ectomycorrhizal fungi. There was an inhibition of the growth of mycelium of the ectomycorrhizal fungi ranging from overgrowth to avoidance. The ectomycorrhizal fungi exhibited hyphal abnormalities such as discoloration, excessive vacuolation and curling. Formation of mycorrhizas by H. crustuliniforme in Pinus contorta and Picea abies was unaffected by the presence of the CBS isolate, whereas a reduction occurred in the presence of the Avondhu isolate. In non-mycorrhizal seedlings of both conifers, the Avondhu isolate reduced root and shoot dry mass and number of root laterals and caused a lower number of short roots in lodgepole pine. The significance of these interactions between T. rutilans and ectomycorrhizal fungi in paired culture and during symbiosis is discussed. Interactions entre Tricholomopsis rutilans et des champignons ectomycorhiziens en confrontation culturale et en association avec des semis de Pinus contorta et de Picea sitchensis Les interactions entre deux isolats de Tricholomopsis rutilans (Avondhu et CBS), et les champignons ectomycorhiziens Hebeloma crustuliniforme, Laccaria laccata et Paxillus involutus, ont étéétudiées sur milieu gélosé en présence ou absence de copeaux de bois. L'isolat CBS s'est montré plus compétitif que l'isolat Avondhu dans les confrontations avec les ectomycorhiziens. Il y avait une inhibition de la croissance mycélienne des ectomycorhiziens allant de l'envahissement à l'évitement. Les hyphes des ectomycorhiziens présentaient des anomalies telles que la coloration, la vacuolisation excessive et l'enroulement. La formation de mycorhizes par H. crustuliniforme chez Pinus contorta et Picea abies n'était pas affectée en présence de l'isolat CBS mais une réduction avait lieu en présence de l'isolat Avondhu. Chez les plants non mycorhizés des deux résineux, l'isolat Avondhu réduisait le poids sec des racines et des pousses et le nombre de racines latérales; chez le pin il réduisait le nombre de racines courtes. La signification de ces interactions entre T. rutilans et les champignons ectomycorhiziens en culture et durant la symbiose est discutée. Interaktionen zwischen Tricholomopsis rutilans und Ektomykorrhizapilzen in Dualkulturen sowie in Assoziation mit Sämlingen von Pinus contorta und Picea sitchensis Die Interaktionen zwischen zwei Isolaten von Tricholomopsis rutilans (Avondhu und CBS) und Ektomykorrhizapilzen (Hebeloma crustuliniforme, Laccaria laccata und Paxillus involutus) wurden auf einem Agar-Medium mit und ohne Zusatz von Holzfragmenten untersucht. Das CBS-Isolat war gegen die Ektomykorrhizapilze konkurrenzfähiger als das Avondhu-Isolat. Die Wirkung auf das Wachstum der Ektomykorrhizapilze reichte vom Überwachsen bis zur Ausbildung einer Hemmzone. Die Hyphen der Ektomykorrhizapilze verfärbten sich, waren stark vakuolisiert und gekräuselt. Die Mykorrhizabildung durch H. crustuliniforme wurde bei Pinus contorta und Picea sitchensis durch das CBS-Isolat nicht beeinflusst, unter dem Einfluss des Avondhu-Isolates war sie jedoch reduziert. Bei nicht mykorrhizierten Sämlingen beider Koniferen verursachte das Avondhu-Isolat eine Reduktion der Wurzel-und Triebtrockenmasse sowie der Anzahl der Seitenwurzeln; bei P. contorta verringerte es die Anzahl der Kurzwurzeln. Die ökologische Bedeutung dieser Befunde wird diskutiert. [source]

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

E. van Rensburg
Abstract Aims:, To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results:,Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, ,18 and ,75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain,heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions:,Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study:, Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen. [source]

Inhibition of human breast cancer cell (MBA-MD-231) invasion by the Ea4-peptide of rainbow trout pro-IGF-I

Sineenat Siri
Abstract It was shown previously that Ea4-peptide of trout pro-IGF-I exerted mitogenic activity in non-transformed cells and inhibited colony formation in a soft agar medium of established human cancer cells. Here we report that the same peptide inhibits the invasion of human breast cancer cells (MDA-MB-231) through a matrigel membrane in a dose-dependent manner. The expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI1) genes in MDA-MB-231 cells were downregulated by treatment with rtEa4-peptide. The inhibition of expression of these genes in response to rtEa4-peptide treatment was reduced to the control level when inhibitors for c-Jun N-terminal kinase 1/2 (JNK1/2), mitogen activated protein kinase kinase 1/2 (Mek1/2), p38 mitogen activated protein kinase (p38 MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphokinase C (PKC) were used. These results suggest that inhibition of invasion of MDA-MB-231 cells by rtEa4-peptide may be mediated via the suppression of uPA, tPA, and PAI1 gene activities through signal transduction pathways. J. Cell. Biochem. 99: 1363,1373, 2006. © 2006 Wiley-Liss, Inc. [source]


ABSTRACT Eighty-two whole black olive samples gathered from six different olive oil processing facilities were surveyed to determine levels of aflatoxins using immunoaffinity column extraction and reversed-phase high-performance liquid chromatography. Two different analytical procedures adopted for the analysis of aflatoxins were investigated for their suitability by spiking the blank olive samples with five different known levels of aflatoxins to determine which one had higher recovery rates. Although some of the olive samples had been exposed to adverse conditions, such as rain and high temperatures, none were found to contain aflatoxins at the determined detection limit. Although the samples were kept in high relative humidity (75%) and high temperature (30C) for 3 months and were tested at 1-month intervals, no aflatoxins were detected. In addition, the olives were inoculated on a potato dextrose agar medium and incubated for 7 days at 25C to characterize the microflora. Because there is no evidence of aflatoxins in fresh whole olives, the next step of processing the contaminated olives into olive oils and testing them for the aflatoxins was not pursued. [source]


ABSTRACT The selectivities to pathogenic Klebsiella strains of different isolation media were compared by known standard strains. The modified MacConkey-inositol-carbenicillin (MCIC) medium (Named MCIAC, MacConkey-inositol-adonitol-carbenicillin) supplemented with adonitol gave no false-negative colonies, and exhibited higher selectivity. MCIC and Simmons citrate agar with inositol (SCAI) media gave two false-negative colonies, respectively. These three media all gave two false-positive colonies, respectively. Salmonella Shigella medium gave four false-negative colonies and five false-positive colonies. Violet red bile glucose agar medium gave the most false-positive colonies, although it gave no false-negative colonies. One hundred samples of powdered milk were examined by MCIAC, MCIC and SCAI plates. The typical positive colonies were further identified using Vitek GNI Auto Microbic system and API 20E system. The results showed that the specificity of the MCIAC medium was higher than MCIC and SCAI media. PRACTICAL APPLICATIONS MacConkey-inositol-carbenicillin (MCIC) is the most commonly used selective medium for the detection of Klebsiella. But some inositol-nonfermenting Klebsiella strains would be missed when selected by this medium. We improved the MCIC medium by supplementing with 1% adonitol. The new modified medium (MacConkey-inositol-adonitol-carbenicillin, MCIAC) had advantages over other selective Klebsiella media in having a higher selectivity and an incubation time of only 16,24 h. MCIAC could be routinely used for pathogenic Klebsiella selection of powdered milk and other food samples. [source]

Analysis of Mycelial Growth Rates and RAPD-PCR Profiles in a Population of Gaeumannomyces graminis var. tritici Originating from Wheat Plants Grown from Fungicide-treated Seed

Z. Weber
Abstract Linear mycelial growth rates of 70 isolates of Gaeumannomyces graminis var. tritici on agar medium amended or unamended with the fungicide silthiofam were not correlated. Mycelial growth rate was not influenced by the fungicide applied to the seed of the plants from, which the isolates originated. DNA polymorphism determined by randomly amplified polymorphic DNA (RAPD) polymerase chain reaction was used to assess genetic variation among isolates. Thirty RAPD markers generated with five arbitrary 10-mer primers revealed DNA polymorphism suitable for assessing variability in this fungal population. Cluster analysis of RAPD data identified two groups at the 54% similarity level. There was a significant relationship between the presence of 11 markers and sensitivity to silthiofam. [source]

Modulation of water activity on fungicide effect on Aspergillus niger growth in Sabouraud dextrose agar medium,

X. Ni
Abstract Aims:, To examine whether water activity (aw) in combination with low concentration of fungicides can be used to effectively control Aspergillus niger van Tieghem growth in cultural medium, the Sabouraud dextrose agar (SDA). The data would be used as baseline information for reducing A. niger contamination in insect artificial diets. Methods and Results:,Aspergillus niger was isolated from an insect artificial diet. Four concentration levels (i.e. 0, 1, 10 and 20 ,mol) of two fungicides (i.e. amphotericin B and itraconazole) were tested against A. niger under four aw levels (i.e. 0·994, 0·961, 0·921 and 0·859) adjusted by including 0, 12·5, 25 and 38% of glycerol in the medium mixture. Aspergillus niger growth was significantly reduced at low fungicide concentration (1 ,mol), and at reduced aw. The spore germination was prevented with either higher fungicide concentration (>10 ,mol), or low aw in the medium (aw < 0·921). The two ecological determinants (fungicides and aw) showed a significant impact on A. niger survival in the medium (P < 0·0001). Itraconazole is more effective than amphotericin B in controlling A. niger contamination in the agar medium. Conclusion:, Adjustment of aw (with 12·5% of glycerol) in combination with 1 ,mol of itraconazole can effectively prevent A. niger growth in the SDA cultural medium. Significance and Impact of the Study:,Aspergillus niger contaminations have frequently affected the quality of insects produced from mass rearing facilities. Low aw in combination with low fungicide concentration has the potential to become one of the most cost-effective management strategies to prevent A. niger contamination in insect artificial diets. The effect of fungicides and low aw in artificial diets on insect biology needs to be further examined. [source]

Bifidobacterium -selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium

S.N. Thitaram
Abstract Aims:, To determine the efficacy and selectivity of an acidified, antibiotic-selective, oligosaccharide-containing media for enumerating Bifidobacterium spp. from chicken caeca samples. Methods and Results:, Transoligosaccharide propionate agar medium (TOS) modified by addition of mupirocin (50 ,g ml,1) and glacial acetic acid (1%, v/v), did not inhibit the growth of bifidobacteria compared with the control media yet inhibited the growth of Lactobacillus acidophilus, Lactobacillus gallinarum, Lactobacillus helveticus and Streptococcus gordonii. Conclusions:, Addition of mupirocin (50 ,g ml,1) and glacial acetic acid (1%, v/v) to TOS (TOS-AM50), is an effective selective medium for isolation and enumeration of Bifidobacterium spp. from chicken caeca samples. Significance and Impact of the Study:, The development of an intestinal bifidobacteria-selective media contributes to the study of probiotics and prebiotics in poultry and potentially other species. [source]

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

G. Gutiérrez-Sánchez
Abstract Aims:, To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. Methods and Results:,Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0,1 and 0,1·36 g l,1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l,1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0·90 g l,1 of caffeine and 0·14 g l,1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9·87 g l,1) with a specific growth rate (,) of 0·073 h,1 and caffeine degradation rate of 0·033 g l,1 h,1, was observed under these conditions. Conclusions:, Caffeine degradation as well as biomass production were characterized. Significance and Impact of the Study:, These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry. [source]

A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cells

Mari Nomoto
ABSTRACT The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc2155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes. [source]

Susceptibility of oral obligate anaerobes to telithromycin, moxifloxacin and a number of commonly used antibacterials

I. Tomás
Introduction:, Obligate anaerobes are closely involved in the pathogenesis of oral and focal infections. The objective of this study was to evaluate the susceptibility profiles of obligate anaerobes of oral origin to telithromycin (TLM), moxifloxacin (MXF), and other antibiotics that are commonly used in dentistry. Methods:, The study sample comprised 172 obligate anaerobes isolated from the saliva of 43 adult volunteers. The minimum inhibitory concentrations (MICs) were determined by the agar dilution technique in Brucella agar medium supplemented with vitamin K, haemin and 5% (volume/volume) laked sheep blood, and incubated under anaerobic conditions. The Clinical and Laboratory Standards Institute methodology was followed and its criteria were used for the qualitative interpretation of the results. The antibiotics evaluated were: amoxicillin (AMX), amoxicillin-clavulanic acid (AMX-CLA), clindamycin (CM), metronidazole (MTZ), azithromycin (AZM), TLM and MXF. Results:, Resistance to AMX (MIC90 16 mg/l) was observed in 45.3% of the obligate anaerobes and resistance to CM (MIC90 16 mg/l) was found in 18.6%. All the isolates were sensitive to MTZ (MIC90 = 1 mg/l) and 98.8% were sensitive to AMX-CLA (MIC90 = 2 mg/l). The MIC90 values for AZM, TLM and MXF were 16, 8 and 2 mg/l, respectively. Conclusion:, Pathogenic, opportunistic and non-pathogenic obligate anaerobes showed high percentages of resistance to AMX and CM, and high MIC values for AZM in the absence of recently administered antibiotics. MXF showed a higher activity than TLM, similar to that detected for AMX-CLA and MTZ. In consequence, MXF could represent a possible alternative antimicrobial against obligate anaerobes of oral origin, particularly in those patients with allergy, intolerance or lack of response to AMX-CLA or MTZ. [source]

Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor

P. E. Courty
Summary ,,In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. ,,The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases (lcc) sensu stricto (lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases (lcc10 and lcc11) that are not present in C. cinerea. The fet3 -like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/sidA gene for siderophore biosynthesis. ,,Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase,polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas (lcc3 and lcc8), in fruiting bodies (lcc7) or in the free-living mycelium grown on agar medium (lcc9 and lcc10), suggesting a specific function of these MCOs. ,,The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi. [source]

A high throughput membrane BIO-PCR technique for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola

N. W. Schaad
Molecular-based methods such as PCR have greatly improved detection of bacteria in environmental samples. However, the sensitivity of PCR is not high when compared to agar plating assays, and inhibitors from plants are often a problem. Pre-enriching bacteria on agar media (BIO-PCR) can increase the sensitivity of PCR by more than 100% and reduce the effects of inhibitors. To further increase the sensitivity and also reduce the labour needed for BIO-PCR, a high throughput 96-well membrane BIO-PCR technique is described for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola (PSP) (syn. P. phaseolicola) in washings of seeds and leaves of Phaseolus vulgaris, using available classical PCR primers and newly designed real-time primers and probe. The primers and probe, designed from a tox-argK chromosomal cluster of the PSP-specific phaseolotoxin gene, were confirmed to be specific to PSP. Samples (1·2 mL) were filtered under vacuum in 96-well membrane plates. After incubating on soft agar medium for 48,52 h, each well is washed with 200 µL of sterile water and used immediately for nested (two-step) PCR or real-time PCR or stored at ,20°C. Results of assaying spiked seed washings showed that classical PCR was unable to detect PSP at mean concentrations of 40 colony forming units (cfu) mL,1. BIO-PCR detected PSP in five out of six samples at 40 mean cfu mL,1 but none at mean concentrations of 4·2 and 0·4 mean cfu mL,1. In contrast, membrane BIO-PCR detected the bacterium in all six samples tested containing as few as 0·4 mean cfu mL,1. The sensitivity of detection from leaf washings was lower but the results were similar, classical and BIO-PCR were negative from all three levels of inoculum while membrane BIO-PCR detected three out of three samples at 80 mean cfu mL,1 and one out of three at 40 mean cfu mL,1. [source]

A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus

M. Fatmi
Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10,14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors. [source]

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus

Anne Beauvais
Summary Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, ,1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens. [source]