Cold-active chemoorganotrophic bacteria from permanently ice-covered Lake Hoare, McMurdo Dry Valleys, Antarctica

Eight strains of chemoorganotrophic bacteria were isolated from the water column of Lake Hoare, McMurdo Dry Valleys, Antarctica, using cold enrichment temperatures. The isolates were Alpha-, Beta-, and Gammaproteobacteria and Actinobacteria spp. All isolates grew at 0 degrees C, and all but one grew at subzero temperatures characteristic of the water column of Lake Hoare. Growth temperature optima varied among isolates, but the majority showed optima near 15 degrees C, indicative of cold-active phenotypes. One isolate was truly psychrophilic, growing optimally around 10 degrees C and not above 20 degrees C. Half of the isolates grew at 2% salt while the other half did not, and all but one isolate grew at 2 atm of O(2). Our isolates are the first prokaryotes from the water column of Lake Hoare to be characterized phylogenetically and physiologically and show that cold-active species of at least two major phyla of Bacteria inhabit Lake Hoare.     

Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.     

Fusarium solani species complex isolates conspecific with Fusarium solani f. sp. cucurbitae race 2 from naturally infected human and plant tissue and environmental sources are equally virulent on plants, grow at 37 degrees C and are interfertile

In a previous taxonomic study based on multilocus sequencing of Fusarium from clinical specimens and hospital environments, the most common lineage was Fusarium solani species complex group 1 (FSSC 1) which is conspecific with F. solani f. sp. cucurbitae race 2, a pathogen of cucurbit fruits. The aims of our study were to determine if clinical and environmental isolates of FSSC 1 are plant pathogens and members of the same biological species as cucurbit isolates, and to determine if all isolates can germinate, grow and sporulate at 37 degrees C. Isolates from the different sources did not differ in virulence on zucchini fruits. All FSSC 1 isolates were pathogenic and produced more rot than FSSC isolates from plant hosts other than cucurbits. Both mating types were found among isolates from each of the sources, and all isolates were sexually compatible with cucurbit isolates. All isolates germinated, grew and sporulated at 37 degrees C. This is the first report in which plant pathogenicity has been verified for a collection of human clinical isolates. Our data are consistent with the hypothesis that all FSSC 1 isolates, regardless of source, are a single biological species, equally virulent plant pathogens and tolerant of the human body temperature.     

In vitro determination of virulence factors activity associated with several Cryptococcus neoformans clinical isolates

Different phenotypic characteristics associated with virulence of the Cryptococcus neoformans species complex have shown an important role in their pathogenicity. In this study we have determined the role of phenotypically and genotypically factors of some virulence factors from clinical isolates in the two species of the complex; 35 C. neoformans and 19 Cryptococcus gattii. Growth at 37 degrees C, macroscopic and microscopic morphology, switching phenomenon, activity of 23 extracellular enzymes, variability of the colonies in agar with phloxin B; phospholipase B gene, and the mating type were determined by PCR; the molecular pattern was determined by URA5 RFLP. All isolates grew at 37 degrees C, the capsular size was greater in C. gattii (1.87 microm -/+1.47 microm) than in C. neoformans (0.83 microm -/+0.15 microm). Switching was observed mainly in isolates of C. gattii. All isolates expressed the enzyme urease, a lower activity of the proteases (Pz= 0.54), but a higher activity of the phospholipase (Pz=0.43) and phenoloxidase (Pz=0.003) was determined for C. gattii.     

Characterization of Actinomyces israelii Serotypes 1 and 2

In a previous serological study, we compared 14 isolates of Actinomyces israelii serotype 2 with 13 serotype 1 cultures. The present study reports the morphological, physiological, and biochemical characteristics of these same 27 cultures. All of the isolates exhibited similar cellular morphology, and all but one produced the typical spider type microcolony on solid media. Twelve of 13 serotype 1 isolates produced the molar tooth type macrocolony, whereas only 2 of 14 serotype 2 cultures produced this type of rough colony. All of the serotype 1 isolates fermented arabinose with the production of acid; none of the serotype 2 cultures fermented this carbohydrate. All 27 cultures produced the greatest amount of growth when cultured under anaerobic conditions and grew poorly or not at all in air. Both groups of organisms produced similar reactions on other biochemical test media; these findings suggested that A. israelii serotype 2 should not be given a species designation.     

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

栗疫病菌不同毒力菌株胞外酶活性和草酸产量的比较

对栗疫病菌不同毒力菌株产生胞外酶的种类、活性和草酸产量以及草酸对多聚半乳糖醛酸酶水解聚果胶酸钙的影响进行了研究。所有供试菌株均未能检测到淀粉酶活性。栗疫病菌在培养中可分泌漆酶,多聚半乳糖醛酸酶、蛋白酶、纤维素酶和脂酶,但不同毒力菌株产生这些酶的能力不同。总的来说,强毒力菌株均可分泌这些酶,且活性强,但弱毒力菌株的酶活性较弱或不分泌这些酶。菌丝产量和草酸产量分析表明,强毒力菌株的草酸产量明显高于弱毒力菌株。菌丝产量与草酸产量没有相关性。在没有草酸盐存在的条件下,多聚半乳糖醛酸酶不能降解聚果胶酸钙。     

Gamma-proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov. infect protozoa and require activated charcoal for growth in laboratory media

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at S?o Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus Rickettsiella, which cause disease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' disease and Pontiac fever. The S?o Gemil strains grew only on media containing charcoal, as is also true of the species of the genus LEGIONELLA: Unlike the vast majority of Legionella isolates, the new isolates did not require L-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for alpha-ketoglutarate. Strains SGT-39(T) and SGT-56 grew consistently between 30 and 43 degrees C, while strains SGT-108(T) and SGT-109 grew between 30 and 40 degrees C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39(T) and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108(T) and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39(T), for which we propose the name Aquicella lusitana, while strain SGT-108(T) represents a second species of the same genus, for which we propose the name Aquicella siphonis.     

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.     

Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage.

Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal fungi and 27 isolates of 15 species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 mm in diameter, were removed from the cultures and stored in sterile distilled water in test tubes at 5 degrees C. Sixty-four, 61, and 41 isolates of the symbiotic fungi were viable after 1, 2, and 3 years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, four fungi (three symbionts and one pathogen) were tested and found to have retained their original growth rates and root-infecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 degrees C and subcultured every 4 to 6 months, grew slower and did not infect as many feeder roots of pine as the water-stored isolates.     

Cold-Active Chemoorganotrophic Bacteria from Permanently Ice-Covered Lake Hoare, McMurdo Dry Valleys, Antarctica

Eight strains of chemoorganotrophic bacteria were isolated from the water column of Lake Hoare, McMurdo Dry Valleys, Antarctica, using cold enrichment temperatures. The isolates were Alpha-, Beta-, and Gammaproteobacteria and Actinobacteria spp. All isolates grew at 0°C, and all but one grew at subzero temperatures characteristic of the water column of Lake Hoare. Growth temperature optima varied among isolates, but the majority showed optima near 15°C, indicative of cold-active phenotypes. One isolate was truly psychrophilic, growing optimally around 10°C and not above 20°C. Half of the isolates grew at 2% salt while the other half did not, and all but one isolate grew at 2 atm of O₂. Our isolates are the first prokaryotes from the water column of Lake Hoare to be characterized phylogenetically and physiologically and show that cold-active species of at least two major phyla of Bacteria inhabit Lake Hoare.     

Physiological characteristics of 13 common fungal species in bathrooms

To clarify the factors affecting fungal contamination in bathrooms, the growth of 13 common fungal species (13 isolates) in bathrooms was studied under various environmental conditions. Most of the fungi examined grew on media of 0.01% and 0.05% sodium fatty acid and on media of 0.01% anion surfactant. On media of non-ion surfactant, however, growth varied from species to species. Fungi found commonly in bathrooms can be divided into two groups. The first group, including six species — Cladophialophora boppii, Exophiala spinifera, E. salmonis, Phialophora europaea, Phoma herbarum, and Scolecobasidium constrictum — grew on media of 0.01%, 0.05%, and 0.25% non-ion surfactant, with the latter five species also growing on alkali medium. Most of them did not grow at 33°C, or on media with 10% NaCl, however. Fungi of these six species, identified using DNA and morphological analysis, were common in bathrooms, but not in other indoor environments, for example, in house dust or on windows. The second group contained seven species including Aureobasidium sp., Cladosporium cladosporioides, and Fusarium sp., which were common both in house dust and in bathrooms; they did not grow on media of 0.05% or 0.25% non-ion surfactant, but most grew comparatively fast on normal medium (1/4 PDA), and were able to grow on media with 10% NaCl and also at 33°C. The characteristic fungi found in bathrooms were able to exploit surfactant but were unable to grow well under comparatively dry or high-temperature conditions.     

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.     

The frequency and characteristics of highly thermophilic bacteria in cool soil environments

Following enrichment at 70 degrees C and 80 degrees C, five highly thermophilic aerobic eubacteria have been isolated from cool soil environments. These organisms show a temperature range for growth of 40-80 degrees C and have optimal and very high growth rates around 70 degrees C with generation times less than 30 min. All isolates are narrow rods, which stain Gram-negative, but have a Gram-positive cell wall structure and only one of five isolates is a spore former. All cultures contain a small proportion of previously unreported extremely long flexuous rods, which can be seen to divide eventually. Biochemical testing of five strains reveals a significant ability to utilize alkanes and some aromatic hydrocarbons. Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA the five strains were differentiated into three categories, which paralleled the biochemical results. 16S rDNA sequences showed high similarity with thermophilic Bacillus species now reclassified as Geobacillus. These bacteria are present in high numbers in apparently all soils and the question is raised of how these organisms, which are apparently unable to grow at the temperatures experienced in these cool soils, are so prominent.     

Characterization of Sphingomonas isolates from Finnish and Swedish drinking water distribution systems

Sphingomonas species were commonly isolated from biofilms in drinking water distribution systems in Finland (three water meters) and Sweden (five water taps in different buildings). The Sphingomonas isolates (n = 38) were characterized by chemotaxonomic, physiological and phylogenetic methods. Fifteen isolates were designated to species Sphingomonas aromaticivorans, seven isolates to S. subterranea, two isolates to S. xenophaga and one isolate to S. stygia. Thirteen isolates represented one or more new species of Sphingomonas. Thirty-three isolates out of 38 grew at 5 degrees C on trypticase soy broth agar (TSBA) and may therefore proliferate in the Nordic drinking water pipeline where the temperature typically ranges from 2 to 12 degrees C. Thirty-three isolates out of 38 grew at 37 degrees C on TSBA and 15 isolates also grew on blood agar at 37 degrees C. Considering the potentially pathogenic features of sphingomonas, their presence in drinking water distribution systems may not be desirable.     

Systematics of Catenulifera (anamorphic Hyaloscyphaceae) with an assessment of the phylogenetic position of Phialophora hyalina

Catenulifera, typified by C. rhodogena (=Scopulariopsis rhodogena), was established to accommodate the anamorphs of Hyphodiscus (Ascomycota, Helotiales) and to delimit these taxa from members of Phialophora section Catenulatae. Catenulifera rhodogena has been inferred as monophyletic, but its relationship to ascomycetes with poorly differentiated phialidic anamorphs remains enigmatic. To test the hypothesis that C. rhodogena is closely related to morphologically similar species of Phialophora and to further explore the systematics of Catenulifera, we analyzed nuclear rDNA and β-tubulin gene sequences of isolates identified as C. rhodogena, Hyphodiscus hymeniophilus, P. brachyconia, P. brevicollaris and P. hyalina. ITS-LSU and ITS-LSU-β-tubulin phylogenies positioned all isolates except P. hyalina in a single, well-supported clade that consisted of three subclades. Subclade 1 included fungicolous isolates of C. rhodogena and H. hymeniophilus that did not fluoresce when exposed to long-wave UV light. Subclade 2 contained fungicolous and lignicolous strains of C. rhodogena that produced fluorescent colonies and possessed a 366bp indel in the LSU rRNA gene. Neither lineage encompassed the ex-type strains of Cistella rubescens (=H. hymeniophilus) or S. rhodogena, but the former isolate was inferred as sister to Subclade 2. Subclade 3 included all isolates of P. brachyconia, a species recognized here as C. brachyconia comb. nov. A fourth isolate of P. brachyconia that was extralimital to Subclade 3 is described as C. luxurians sp. nov. The positions of C. brevicollaris comb. nov., a species based on P. brevicollaris, and C. luxurians were not resolved in the ITS-LSU phylogeny. P. hyalina is not closely related to Catenulifera.     

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.     

Distribution and phylogeny of hexachlorocyclohexane-degrading bacteria in soils from Spain

Hexachlorocyclohexane (HCH)-degrading bacteria are believed to mediate natural attenuation of HCH contamination and have potential for active bioremediation processes. This study addressed the very limited understanding of the distribution, diversity and substrate specificity of such bacteria from 13 soil samples, varying in levels of HCH contamination, from four sites in Spain. Hexachlorocyclohexane removal occurred in 16 of 36 enrichment cultures. Hexachlorocyclohexane-degrading populations were clearly associated with HCH-contaminated soils, and populations growing on the delta-HCH isomer were only found in soil contaminated with delta-HCH. beta-Hexachlorocyclohexane was persistent in enrichment cultures, and there was no evidence for populations growing on beta-HCH. From alpha- and gamma-HCH enrichment cultures, nine HCH-degrading isolates were obtained, which were all Sphingomonas spp. Attempts to isolate organisms from delta-HCH enrichment cultures failed. None of the isolates grew on HCH as a sole organic substrate in pure culture. All isolates degraded alpha- and gamma-HCH, and most degraded beta-HCH. delta-Hexachlorocyclohexane inhibited growth of most isolates, but could be degraded by cell suspensions of at least four strains. Denaturing gradient gel electrophoresis indicated that the isolates represented predominant populations in the enrichment cultures, but additional predominant populations, including some Pseudomonas spp., could not be isolated.     

Proteolytic anaerobic bacteria from lake sediments of Antarctica

Amongst twenty five proteolytic bacteria isolated from lake sediment samples of Antarctica, six isolates were selected based on SDS PAGE protein profile and zone of hydrolysis on casein agar at 10 degrees C. Most of the cultures were rod shaped and motile with two showing terminal bulging spores. Isolates grew between 5 degrees C to 37 degrees C and protease was induced in the late log, stationary or death phase. Isolate SPA-3 grew maximally at 10 degrees C and SPA-6 at 37 degrees C while others preferred 20 degrees C-30 degrees C for growth. The growth and protease production on casein, skimmed milk, bovine serum albumin and gelatin varied with the isolates. Acetate was the dominant volatile fatty acid (24-66% of total VFA) produced during hydrolysis of protein substrate.     

Effect of pH on Growth and Survival of Three Avian and One Saprophytic Mycoplasma Species

The ability of Mycoplasma meleagridis, M. laidlawii, an unnamed, nonpathogenic avian species, and three isolates of M. gallisepticum to grow and survive in liquid media of various pH values was investigated. All species grew over a pH range that was generally about two units. The most significant finding was that cultures initiated in media within a pH range of approximately 8.5 to 8.9 remained viable for 45 to 60 days at 37 C, whereas cultures initiated at lower values survived for shorter periods.