Growth requirements of some fungi causing maduromycosis

Three strains ofMadurella mycetomi, two ofM. grisea, and two ofRhinocladiella mansonii have been studied for possible differences in growth requirements which might be used for distinguishing these species. Under the experimental conditions, an incubation temperature of 37C suitedM. mycetomi about as well as 30C.R. mansonii grew less well at 37C than at 30C, andM. grisea did not grow at the higher temperature. M. grisea andR. mansonii further differed fromM. mycetomi in that they required thiamine for growth. The pH tolerance of all the strains was very wide. Asparagine and potassium nitrate were readily utilized by all the strains, but ammonium salts were not. Urea was poorly used byM. mycetomi; the other species did not use it. A possible relationship ofM. grisea andR. mansonii is discussed.     

Keratinolytic and keratinophilic fungi in the soils of Papua New Guinea

A study was made of soil samples collected during an expedition to the Highlands of Papua New Guinea. Fungi were isolated from the samples by the method of hair baiting (To-Ka-Va). Of the 33 species isolated, about half showed keratinolytic activity. Such activity is previously unreported for Mucor hiemalis f. hiemalis, Myrothecium roridum, Paecilomyces carneus, P. marquandii, Penicillium brevicompactum, Rhinocladiella mansonii and Verticillium lecanii. The species most active keratinolytically were Chrysosporium an. Arthroderma cuniculi, C. an. A. curreyi, C. indicum, Myceliophthora vellerea and Trichophyton ajelloi. The spectrum of fungi with keratinolytic activity isolated from the different sites differed considerably according to the frequency of use by man, heaviest use being correlated with greatest activity. The pH of the soil (varying from 5.8–7.5) had little influence on the type of such fungi isolated.     

A newHalophytophthora species,H. porrigovesica, from subtropical and tropical mangroves

A new oomycete was found from intertidal fallen leaves of mangroves in Japan and Thailand and is described here asHalophytophthora porrigovesica. This species is characterized by having an epapillate, ovate zoosporangium with a lens-shaped dehiscence plug-like material at the apex, and by forming an expanding long cylindrical vesicle prior to zoospore release. A key to 14 species and 2 varieties ofHalophytophthora including the new species is proposed. The subtropical (Iriomote is., Japan) strains and tropical (Thailand) strains were different in physiological properties and especially in the asexual reproduction. The subtropical strains showed a lower optimal temperature and wider range of suitable temperature and salinity for zoosporangium formation, whereas the tropical strains showed a higher optimal temperature and narrower range of temperature and salinity. These differences are explained as adaptations of the strains to the environmental conditions of their respective habitats. From the subtropical mangroves, six strains of the new species have been isolated only from submerged leaves ofSonneratia alba, while several strains have been isolated from tropical mangroves from the leaves of three species of mangrove trees,S. alba, Bruguiera gymnorrhiza andAvicennia alba. This indicates a change of taxon selectivity (host specificity) with the geographical distribution.     

Physiological and morphological characteristics of yeasts isolated from waste oil effluents

Pichia membranaefaciens, Cryptococcus laurentii, Rhodotorula glutinis and Candida krusei were isolated from contaminated sites. A significant variability in cell forms and in assimilation profiles was observed in the C. krusei strains. The chitin synthase activity and chitin content allowed us to differentiate three strain types. The variability of the phenotypic traits was higher in C. krusei strains isolated from heavily polluted sites. This revised version was published online in July 2006 with corrections to the Cover Date.     

Skin test-active substance prepared from culture filtrate of Fonsecaea pedrosoi

Ethanol-precipitated substance (EP) was prepared from culture filtrate of Fonsecaea pedrosoi. EP was separated into two components by passing through a Sephadex G-50 column; the faster passing component was referred to as EP-1, the slower as EP-2. EP-1 and EP-2 were evaluated as an antigen for detecting cutaneous delayed hypersensitivity in patients with chromomycosis. EP-1 elicited positive delayed skin reactions in all of 8 patients with chromomycosis, of which 7 caused by F. pedrosoi and one by Exophiala jeanselmei. Healthy subjects, patients with sporotrichosis and patients with tinea barbae failed to react to EP-1. These results indicate that EP-1 is a useful tool for detecting cutaneous delayed hypersensitivity in patients with chromomycosis caused by F. pedrosoi. It was found that precipitin test using EP-1 as an antigen had little diagnostic value in chromomycosis. EP-2 did not show antigenic activity in both skin and precipitin reactions.     

Characterization of clinical isolates of pathogenic <Emphasis Type="Italic">Nocardia</Emphasis> strains and related actinomycetes in Thailand from 1996 to 2003

In Thailand from 1996 to 2003, 171 strains of pathogenic aerobic actinomycetes from clinical specimens were isolated. Of those strains, 134 were mycolic acid containing actinomycetes, including 96 strains of Nocardia species. Others included 10 strains of Gordonia, 14 strains of Rhodococcus, and 22 strains of Mycobacterium. One strain each of the genera Tsukamurella and Corynebacterium were also isolated. Also identified were 27 strains of non-mycolic acid containing actinomycetes. Our identification studies of 96 strains of Nocardia species showed that significant pathogens in Thailand were N. beijingensis (18 strains), N. cyriacigeorgica (13 strains), and N. farcinica (34 strains); the most prevalent species was N. farcinica (35.4%). We also isolated four strains of N. asiatica, five strains of N. asteroides sensu stricto, four strains of N. nova, seven strains of N. otitidiscaviarum, eight strains of N. transvalensis, and two strains of N. pseudobrasiliensis.     

Physiological studies in the black yeasts

Summary A collection of 159 strains of black yeasts obtained from a number of culture collections and a wide variety of substrata was studied. Morphologically, four species were present:Phialophora jeanselmei with a mycelium that produced radulaspores and also phialides;Sclerophoma pithyophila, which produces pycnidial initials and sometimes pycnidia in culture; and two species ofAureobasidium, A. pullulans with wide mycelium and large radulaspores, andA. mansonii, which produces narrow mycelium and small radulaspores.Physiologically, these species form two distinct groups:A. mansonii andP. jeanselmei, which grow slowly; andA. pullulans andS. pithyophila, which grow more rapidly. Except for rate of growth, there was little difference between these species in their abilities to use the carbon and nitrogen sources tested. None produced significant capabilities for fermentation in the sugars and at the pH level tested. All varied in their response to a technique designed to measure amounts of growth by turbidometry.The black yeasts, especiallyA. pullulans, are a highly polymorphic group of organisms capable of growing in a wide variety of locations. For the most part they are capable of using the simpler organic compounds found in nature, which indicates they apparently act largely on terminal or subterminal products of decay in the final stages of the mineralization of organic compounds.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences

The nuclear small subunit rRNA genes of authentic strains of the black yeastsExophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaeomuriformis, Capronia mansonii, Nadsoniella nigra var.hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var.jeanselmei andE. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA ofExophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences fromNeurospora crassa andAureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated betweenExophiala dermatitidis, Wangiella dermatitidis andSarcinomyces phaeomuriformis. This monophyletic clade also contains the teleomorph speciesCapronia mansonii thus confirming the concept of a teleomorph connection of the genusExophiala to a member of the Herpotrichiellaceae. However,Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph ofCapronia mansonii as it was postulated.     

Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius

Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.     

New clade of silicified bolidophytes that belong to Triparma (Bolidophyceae,Stramenopiles)

The small phytoplankton genus Triparma belongs to the class Bolidophyceae and contains two distinct forms: silicified species and naked flagellated species (formerly Bolidomonas). Recent studies showed that four silicified species/strains (Triparma laevis f. inornata, T. laevis f. longispina, T. strigata, and T. aff. verrucosa) belong to a single clade that is paraphyletic, because it also contains an unclassified flagellated strain, and is sister to a flagellated species, T. eleuthera. In this study, we isolated and characterized two new strains of silicified species to test the phylogenetic unity of silicified bolidophytes. The isolates were identified as T. retinervis strains because they possessed fine areolation on the cell wall. 18S rDNA and rbcL phylogenetic analyses demonstrated that T. retinervis formed a new silicified clade that is sister to the flagellated species T. pacifica. This reveals that there are at least two distinct clades including both silicified and flagellated Triparma species.     

Biomineralization Potential of Bacillus subtilis,Rummeliibacillus Stabekisii and Staphylococcus Epidermidis Strains In Vitro Isolated from Speleothems,Khasi Hill Caves,Meghalaya, India

Microorganisms were isolated and identified from speleothems at Khasi hill caves, Meghalaya. The aim was to understand their biomineralization potential. Analyses of the speleothems from Krem Soitan, Krem Mawpun, and Krem Lawbah using scanning electron microscope (SEM) showed evidences for microbe–mineral interactions. SEM showed microbial reticulate and beaded filaments, cells, fiber calcites, and clusters of coccoid-like structures. A total of 113 bacterial strains were isolated and identified by a combination of conventional and molecular based tools. 105 strains that were sequenced belonged to the genus: Bacillus, Rummeliibacillus, Staphylococcus, and Brevibacterium. The BLASTn sequence search of 16S rRNA sequences with the National Centre for Biotechnology Information database to establish the identity of the strains yielded similarity scores of ≥99% with the respective organisms. The strains were identified as Bacillus simplex, Bacillus gaemokensis, Bacillus subtilis, Bacillus thuringiensis, Bacillus albus, Bacillus cereus, Bacillus anthracis, Bacillus weihenstephanensis, Rummeliibacillus stabekisii, Bacillus wiedmannii, Staphylococcus epidermidis, Rummeliibacillus pycnus, Kurthia zopfii, and Brevibacterium frigoritolerans. These strains were tested for biomineralization on B-4 medium. Five strains (B. subtilis, R. stabekisii, Staphylococcus epiderdimis, B. cereus, and B. wiedmannii) had the capability to precipitate biominerals in vitro. B. subtilis, R. stabekisii, and S. epidermidis precipitated 0.24, 0.36, and 0.35 g/L of biominerals at 22°C at the end of the four week experiment period. These strains increased the pH of the medium from 7 to 8.95. The precipitated biominerals were imaged using an ultra-high resolution field emission SEM. X-ray diffraction of the biomineral precipitated by R. stabekisii showed that it was composed of vaterite and jungite. Whereas S. epidermidis showed that it was composed of calcite, vaterite, and jungite. B. subtilis produced small, circular calcite crystals. This is the first comprehensive report on the possible evidences about the role of R. stabekisii and S. epidermidis in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains have biomineralization potential. Further evidences of the coexistence of exopolysaccharides, whisker fiber calcites, microbial filaments, and coccoid-like forms point to biogenic inputs in the cave mineral formations.     

Molecular identification of endophytic fungi from medicinal plant <Emphasis Type="Italic">Huperzia serrata</Emphasis> based on rDNA ITS analysis

Fifty-two endophytic fungi strains with different colony morphologies were isolated from stems, leaves and roots of Huperzia serrata (Thunb. ex Murray) Trevis. collected from Bawangling Reserve of Hainan Province in southern China. They were identified mainly based on rDNA ITS sequences and phylogenetic analysis. The results showed that all strains belonged to four classes, i.e. Sordariomycetes (92.31%), Dothideomycetes (3.85%), Pezizomycetes (1.92%) and Agaricomycetes (1.92%). Forty-seven strains were identified at the genus level, including Glomerella (Colletotrichum), Hypocrea (Trichoderma), Pleurostoma, Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium, Cazia and Phellinus. As to the other five strains, three were identified at the order level and two at the family level, indicating that a great diversity of fungi taxa exists in H. serrata. Most isolated strains belonged to the genus of Glomerella (Colletotrichum) and Hypoxylon, twenty-one from Glomerella and its anamorph Colletotrichum (42.3% of total isolated strains) and ten from Hypoxylon (19.2% of total isolated strains). Pleurostoma, Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium, Cazia and Phellinus were reported as endophytic fungi isolated from H. serrata for the first time.     

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants

We have used the toxic non-metabolizabie glucose/ mannose analogue 2-deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, meli-biose, galactose or fructose. We found that the frequency at which 2-deoxygiucose-resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxygiucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of III^Man, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of III^Man_L, a low-molecular-weight form of III^Man_L (35200), class 2 was composed of strains exhibiting a reduced level of III^Man_L, class 3 was composed of strains devoid of both forms of III^Man (III^Man_L as well as III^Man_H, the high-molecular-weight form of III^Man (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2-deoxygiucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of III^Man but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.     

Characterization of a novel South American population of the astaxanthin producing yeast <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> (<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>)

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.     

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of moderately halophilic bacteria isolated from the Weihai Solar Saltern (China)

A total of 45 moderately halophilic bacteria was isolated from sediment and saline water collected from the Weihai Solar Saltern (China). The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The halophilic strains were tested for their antimicrobial activity. Cytotoxicity assay was performed to determine which of the halophilic strains could inhibit proliferation of human hepatocellular carcinoma Bel 7402 cells. Our results showed that all of the isolated 45 strains displayed moderately halophilic characteristics. Phylogenetic analysis indicated that 17 of the isolated strains were related to the phylum Firmicutes and belonged to four genera, Bacillus, Halobacillus, Planococcus and Salinicoccus. The other strains identified as genus of Halomonas belonged to phylum γ-Proteobacteria. Most of the halophilic bacterial strains showed potent activities against Gram-positive bacteria, human pathogenic fungi and plant pathogenic fungi. In addition, the crude extracts from 14 halophilic bacterial strains showed cytotoxic activity against tumor cells Bel 7402, and five of them showed remarkable activities with IC₅₀ less than 40 μg ml⁻¹. Our results suggest that the moderately halophilic bacteria may be developed as promising sources for the discovery of novel bioactive substances.     

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.     

Virulence genes,antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods

Aim: To determine the virulence genes, antibiotic resistance and plasmid profiles of 16 Enterococcus faecium and 68 Enterococcus faecalis strains isolated from various naturally fermented foods. Methods and Results: The presence of virulence genes (agg₂, gelE, cylM, cylB, cylA, espfs, espfm, efaAfs, efaAfm, cpd, cop, ccf, cad) and also the genes vanA and vanB were investigated by polymerase chain reaction (PCR). Antibiotic resistance of the isolates was determined by disc diffusion method. Most of the tested isolates were positive for virulence genes and resistant to some antibiotics. One of the Ent. faecalis strains isolated from a cheese sample carried the vanA gene and was intermediately resistant to vancomycin. The strains usually contained large plasmids, which might harbour acquired antibiotic resistance. Conclusion: The study showed that Ent. faecium and Ent. faecalis strains isolated from naturally fermented Turkish foods may be potential risk factors for consumer health in terms of virulence genes and acquired antibiotic resistance. Significance and Impact of the Study: The results indicate the importance of enterococcal contamination in terms of the safety of some fermented Turkish foods.     

Mating behavior of a Northern Italian population of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> associated with maize

Fusarium verticillioides, the most common causal organism of Fusarium stalk and ear rot of maize in Northern Italy, produces important mycotoxins such as fumonisins. Reproductive biology of F. verticillioides has been widely studied in numerous maize growing areas, but up to now no information is available on the mating behavior and genetic structure of this plant pathogen in Italy. Mating type and female fertility distribution and effective population number, N _e , were assessed for a population of 181 F. verticillioides strains isolated from three fields located in Lombardia region (Northern Italy) during 2007-2008 maize growing season. The ratio of MAT-1:MAT-2 was significantly different from the theoretical 1:1 ratio expected in an idealized population in which individuals mate at random. The frequency of hermaphroditic strains was 20 % of the total population. N _e for mating type was 89 % of the count (total population) and the N _e for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited N _e in the examined population. Under equilibrium cycle, assuming that female fertility has been lost due to selection and mutation rate during asexual reproduction, sexual reproduction needed to occur only once per 40 to 118 asexual generations to maintain this level of sexual fertility.     

Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments

Summary A total of 94 actinomycete strains were isolated from the marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others were Micromonospora spp. Fifty-one percent of the actinomycete strains showed activity against the pathogenic Vibrio spp. strains. Thirty-eight percent of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar plates. Seven strains of Streptomyces were found to produce siderophores and to inhibit the growth of Vibrio spp. in vitro. Two of them belonged to the Cinerogriseus group, the most frequently isolated group of Streptomyces. The results showed that streptomycetes could be a promising source for biocontrol agents in aquaculture.