Hemagglutinating properties of Actinobacillus pleuropneumoniae

A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.     

Factors affecting the phospholipase activity of Candida species in vitro

The phospholipase activity of 41 isolates of oral Candida species was determined by a plate assay. Seventy nine per cent of the C. albicans isolates were phospholipase producers whereas none of the C. tropicalis, C. glabrata or C. parapsilosis isolates produced the enzyme. The degree of phospholipase activity (Pz value) of individual isolates was remarkably constant despite the large variation in activity among different isolates. Experiments with 10 phospholipase positive C. albicans isolates indicate that phospholipase production in vitro is limited to a narrow pH range (c. 3.6-4.7) and is suppressed by increasing concentrations of sucrose and galactose in the media (r = 0.9). Hence, candidal phospholipases seem to play a complex role in the aetiopathology of human candidoses.     

Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake

Fourteen obligate alkaliphilic and halotolerant bacterial isolates, exhibiting extracellular amylase activity at 55 degrees C and pH 10, were isolated from hot springs around Lake Bogoria, Kenya. From 16S rDNA sequence analysis, nine isolates shared 100% identity with Bacillus halodurans strain DSM 497T, while the rest shared 99% identity with alkaliphilic Bacillus species A-59. PCR of the intergenic spacer region between 16S and 23S rRNA genes (ISR-PCR) divided the isolates into two groups, while tDNA-PCR divided them into three groups. Bacillus halodurans DSM 497T had a different ISR pattern from the isolates, while it had a tDNA-PCR profile similar to the group that shared 99% identity with alkaliphilic Bacillus species A-59. All isolates hydrolysed soluble starch as well as amylose, amylopectin and pullulan. The amylase activity (1.2-1.8 U ml(-1)) in the culture broths had an optimum temperature of 55-65 degrees C, was stimulated by 1 mm Ca2+, and was either partially (16-30%) or completely inhibited by 1 mM EDTA. Activity staining of the cell-free culture supernatant from the isolates revealed five alkaline active amylase bands.     

Characterization of extracellular metallo- and serine-proteases of Aeromonas hydrophila strain B51

The extracellular proteases of Aeromonas hydrophila B51 were stable on heating (56 degrees C) and on storage at 4 degrees C or -20 degrees C. Inhibitor studies showed that 72% of the total activity was inhibited by EDTA (a metalloprotease inhibitor) and 26% was inhibited by phenylmethanesulphonyl fluoride (a serine protease inhibitor). Analytical isoelectric focussing revealed the presence of 33 proteins in the crude extracellular products. Using a casein overlay technique three separate zones of proteolytic activity were detected: a zone with pI 6.5-6.8, formed of two closely focussed bands (possibly isomers of the same protease) and completely inhibited by EDTA; a single band with pI 7.0, which was inhibited by EDTA; and a diffuse zone with pI 8.3-8.5, which was only partially inhibited by EDTA. It is concluded that the serine protease activity focussed in this latter zone. These results indicate the presence of at least four, and possibly five proteases. Our results differ substantially from those reported by other workers using different isolates and it is suggested that significant differences in the character of extracellular products and extracellular proteases exist between different isolates of A. hydrophila.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates

Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micra were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBMO583-like) while cluster C2 harbored two genotypes (ATCC33270-like and ICBMO36). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

Deciphering the diversity of culturable thermotolerant bacteria from Manikaran hot springs

The aim of this study was to analyze and characterize the diversity of culturable thermotolerant bacteria in Manikaran hot springs. A total of 235 isolates were obtained employing different media, and screened for temperature tolerance (40 °C–70 °C). A set of 85 isolates tolerant to 45 °C or above were placed in 42 phylogenetic clusters after amplified ribosomal DNA restriction analysis (16S rRNA-ARDRA). Sequencing of the 16S rRNA gene of 42 representative isolates followed by BLAST search revealed that the majority of isolates belonged to Firmicutes, followed by equal representation of Actinobacteria and Proteobacteria. Screening of representative isolates (42 ARDRA phylotypes) for amylase activity revealed that 26 % of the isolates were positive, while 45 % exhibited protease activity, among which one amylase and six protease producers were tolerant up to 70 °C. BIOLOG-based identification of 13 isolates exhibiting temperature tolerance up to 70 °C, using carbon utilization patterns and sensitivity to chemicals, revealed a high degree of correlation with identification based on 16S rRNA gene sequencing for all isolates, except one (M48). These promising isolates showing a range of useful metabolic attributes demand to be explored further for industrial and agricultural applications.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.     

Structural and functional interactions between extraradical mycelia of ectomycorrhizal Pisolithus isolates

Extraradical mycelia from different ectomycorrhizal (ECM) roots coexist and interact under the forest floor. We investigated structural connections of conspecific mycelia and translocation of carbon and phosphorus between the same or different genets. Paired ECM Pinus thunbergii seedlings colonized by the same or different Pisolithus isolates were grown side by side in a rhizobox as their mycelia contacted each other. (14)CO(2) or (33)P-phosphoric acid was fed to leaves or a spot on the mycelium in one of the paired seedlings. Time-course distributions of (14)C and (33)P were visualized using a digital autoradiographic technique with imaging plates. Hyphal connections were observed between mycelia of the same Pisolithus isolate near the contact site, but hyphae did not connect between different isolates. (14)C and (33)P were translocated between mycelia of the same isolate. In (33)P-fed mycelia, accumulation of (33)P from the feeding spot toward the host ECM roots was observed. No (14)C and (33)P translocation occurred between mycelia of different isolates. These results provide direct evidence that contact and hyphal connection between mycelia of the same ECM isolate can cause nutrient translocation. The ecological significance of contact between extraradical mycelia is discussed.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

Production of extracellular proteins,cellulases and antifungal metabolites by Chaetomium globosum Kunze ex. Fr.

Chaetomium globosum has been well-known potential antagonist of several seed and soilborne fungus. Eight isolates of C. globosum were obtained from different sources and were identified by morphological characters. C. globosum isolates examined for the presence of extra cellular proteins, cellulases and antifungal metabolites in culture filtrate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), thin-layer chromatography and high-performance liquid chromatography. Variation in the mycelial protein of C. globosum isolates was noted in the SDS-PAGE analysis. Different C. globosum isolates that showed more number of bands in protein profile was further screened for the production of cellulases in culture filtrate. Cellulase activity of C. globosum isolates revealed that maximum activity was observed in the isolate Cg-6 after 11?days of incubation, while Cg-2 had least activity. C. globosum isolates were tested for antibiotic production, among which three isolates viz. Cg-6, Cg-7 and Cg-5 were found to produce the antibiotic Chaetoglobosin A in the culture filtrate. The antibiotic Chaetoglobosin A appeared blue colour under UV spectrum with a wavelength of 250?nm.     

Heat-labile and heat-stable haemolysins of Campylobacter jejuni

Abstract During studies on the virulence mechanisms of Campylobacter jejuni clinical isolates it became apparent that some strains produced one or more haemolysins and some did not. There was no great difference between Group C (cholera-like) strains and Group D (dysentery-like) strains. The protein haemolysin(s) showed a spectrum of activity against erythrocytes from different animals; with maximum activity against rabbit and minimal activity against chicken erythrocytes. The results suggested a two-stage activation mechanism for haemolysis which involved a multi-hit lytic activity. It was concluded that the C. jejuni haemolysins were not identical to those described in other organisms and they may be involved in iron acquisition in vivo.     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

In vitro susceptibilities of Candida spp. to Panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434

Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo-beta-1,3-glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time-kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC(90) values of 1 microg/ml. It displayed significant activity against C. albicans and C. parapsilosis with MIC(90) values of 4 and 2 microg/ml, respectively. For C. krusei, the MIC(90) value was 8 microg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4xMIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

Antifungal Susceptibility, Enzymatic Activity, PCR-Fingerprinting and ITS Sequencing of Environmental Cryptococcus laurentii Isolates from Uberaba, Minas Gerais, Brazil

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.     

Genetic and Phenotypic Diversity among Botrytis cinerea Isolates in Iran

Forty-four Botrytis cinerea isolates from different hosts and geographical regions were studied for colony morphology, mycelial growth rate at different temperatures, pathogenicity and molecular diversity. Botrytis cinerea isolates had temperature optima of 20–25°C and isolates showed variation in growth rate at different temperatures. Two morphological types were identified among tested isolates: mycelial and sclerotial. The pathogenicity of isolates was tested on grapevine leaves, and it was revealed that nine of 44 isolates were non-pathogenic and among them seven were of mycelial type. There was no correlation between mycelium growth rate and pathogenicity. Genetic diversity was investigated using nine arbitrary decaprimers. No relationship was found between molecular clusters and geographical region or sampling time; whereas isolates from the same plant host tended to cluster with each other. Seven of nine non-pathogenic isolates were separated from pathogenic ones.