A reemphasis-germ tubes diagnostic for Candida albicans have no constrictions

The ultrastructure of Prototheca salmonis is described, and compared with known isolates of Prototheca. Prototheca salmonis was found to be the causative agent of a disease, grossly affecting the visceral organs of Salmon parr in fresh water stocks of a fish farm. The organism was distributed widely throughout the visceral organs of the diseased fish. Prototheca salmonis, was also isolated from the infected tissues and grown in culture. The ultrastructure of the organism showed many similarities with the ultrastructure of the known isolates. The notable differences were the structure of the cell wall, the presence of glycogen, and the absence of plastid-like organelles, which were a prominent feature of the known isolates of Prototheca.     

Detection of Helicosporidium spp. in metagenomic DNA

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.     

Selective Medium for the Isolation of Prototheca

A medium was devised which permitted the selective isolation of Prototheca spp. from nature.     

Appearance of Colonies of Prototheca on CHROMagar Candida medium

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antimycotic activity of 4-thioisosteres of flavonoids towards yeast and yeast-like microorganisms

Different substituted methoxy- and hydroxy-4-thioisosteres of flavonoids were prepared and their in vitro antimycotic activity towards yeast (Candida spp., Clavispora spp., Cryptococcus spp., Filobasidiella spp., Issatchenkia spp., Pichia spp., Kluyveromyces spp., Saccharomyces spp. and Yarrowia spp.) and yeast-like (Prototheca spp.) microorganisms was tested. Further insights in the biological activities of these antioxidant, oestrogenic and antimicrobial biomimetic derivatives were obtained.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate.

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.     

The non-photosynthetic, pathogenic green alga Helicosporidium sp. has retained a modified, functional plastid genome

A fragment of the Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) plastid genome has been sequenced. The genome architecture was compared to that of both a non-photosynthetic relative (Prototheca wickerhamii) and a photosynthetic relative (Chlorella vulgaris). Comparative genomic analysis indicated that Helicosporidium and Prototheca are closely related genera. The analyses also revealed that the Helicosporidium sp. plastid genome has been rearranged. In particular, two ribosomal protein-encoding genes (rpl19 and rps23) appeared to have been transposed, or lost from the Helicosporidium sp. plastid genome. RT-PCR reactions demonstrated that the retained plastid genes were transcribed, suggesting that, despite rearrangement(s), the Helicosporidium sp. plastid genome has remained functional. The modified plastid genome architecture is a novel apomorphy that indicates that the Helicosporidia are highly derived green algae, more so than Prototheca spp. As such, they represent a promising model to study organellar genome reorganizations in parasitic protists.     

Evidence of a <Emphasis Type="Italic">Prototheca Zopfii</Emphasis> Genotype 2 Disseminated Infection in a Dog with Cutaneous Lesions

Protothecosis is a disease caused by saprophyte aerobic unicellular algae belonging to the genus Prototheca. In dogs, it mainly occurs as a disseminated form, with initial clinical manifestations often referable to the gastrointestinal tract, followed by typical ocular and neurological signs. So far, Prototheca zopfii genotype 2 infection has been reported in severe forms of disseminated protothecosis, while in dogs has never been associated with cutaneous forms. In this study, we describe a case of Prototheca zopfii genotype 2 infection in a dog characterized by nodular and ulcerative dermatitis and with evidence of dissemination. In December 2015, a 5-year-old unneutered male English Setter dog was presented with a 4-month history of footpads ulcerations and multifocal nodular lesions (3–5 cm diameter) on both front limbs. Cytological examination of the aspirated fluid collected from all nodules revealed the presence of sporangic forms compatible with Prototheca spp. organisms. Suspected Prototheca spp. colonies were isolated from the aspirated fluid and identified as Prototheca zopfii genotype 2 by molecular methods. Few days after the visit, the patient developed serious neurological and ocular signs, and the owners elected humane euthanasia. To the authors’ knowledge, this case could represent the first report of a disseminated Prototheca zopfii genotype 2 infection associated with cutaneous lesions in a dog. This study underlines the importance of considering Prototheca zopfii genotype 2 infection in the differential etiological diagnosis of nodular and ulcerative dermatitis in dogs.     

无绿藻的研究进展:从基础到临床

无绿藻是一种直径约3 ～ 30 μm的单细胞生物,广泛存在于自然界和动物体表及体内,属于条件致病性真菌.目前主要通过直接镜检、真菌培养、组织病理学检查及分子生物学等手段对无绿藻进行鉴定.现已发现无绿藻属包括五个种,其中对人有致病性的仅为中型无绿藻基因型2、小型无绿藻和P.blaschkeae,其致病机制可能与外伤和免疫力低下有关.随着研究的深入,越来越多的无绿藻病被临床确诊.根据不同的类型及其临床表现,对无绿藻病的治疗也有所区别.为了提高对无绿藻这一条件真菌及其致病性的认识,该文对其生物学特性、鉴定方法、致病性、临床表现等研究进展做一简要综述.     

Pasteurella pneumotropica and the prevalence of the AHP (Actinobacillus, Haemophilus, Pasteurella)-group in laboratory animals

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guinea pigs, rabbits and cats originating from 7 conventional laboratory animal facilities. They occurred seldom in pure culture and were found in a variety of organs. Thorough identification (41 criteria) revealed that only 83 isolates (62%) were P. pneumotropica and could be subdivided into 3 biotypes. 3 isolates were P. aerogenes, 1 was P. ureae, 11 (8%) were qualified as Actinobacillus spp. and 13 (10%) as Haemophilus spp. Close relationship of the 3 genera--the 'AHP-group' --made the differentiation difficult. 23 atypical cultures were discarded at the beginning of the study as not belonging to the 'AHP-group'. Two-thirds of isolates were associated with inflammation or suppuration; Haemophilus spp. seemed to be more pathogenic than Pasteurella and Actinobacillus species.     

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs

Aims:  To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs.
Methods and Results:  Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property.
Conclusions:  HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants.
Significance and Impact of the Study:  Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.     

The ultrastructure of Prototheca wickerhamii

Two clinical isolates of Prototheca wickerhamii were freeze-dried, fixed and studied by electron microscopy and were also examined growing in culture medium by phase contrast light microscopy. Resting spores placed on fresh medium developed cytoplasmic extensions which sequestrated before proliferation occurred. In the presence of adequate nutrients vegetative spores grew and subdivided to form up to 12 endospores within large sporangia which ruptured to release free spores every 5–6 hours. These proliferating or vegetative spores contained much more endoplasmic reticulum and mitochondria than the resting spores which contained more lipid, and often starch granules as well, but relatively few membranous organelles.     

Phenotypic and genetic diversity among intestinal spirochaetes (genus Brachyspira) in free-living wild mallards (Anas platyrhynchos) sampled in southern Sweden

Brachyspira spp. are anaerobic intestinal spirochaetes that colonize vertebrates. Some species cause enteric diseases in pigs, chickens and possibly in humans, whereas others display a commensual relationship with their hosts. The aims were to investigate the prevalence among colonized free-living wild mallards (Anas platyrhynchos) of three enteropathogenic Brachyspira spp., and to describe the biodiversity of Brachyspira spp. isolates. Isolates from 150 birds were screened by PCR for 3 pathogenic Brachyspira spp., and 35 isolates from 20 mallards, 4 pigs and 1 chicken were subjected to phenotypic tests, 9 diagnostic PCRs, sequencing of the 16S rRNA and NADH oxidase (nox) genes, phylogenetic analysis and nox gene restriction enzyme analysis in silico. Of the 150 birds, 47%, 33% and 11% were positive by PCR for Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira hyodysenteriae, respectively. Thirty-one characterized isolates were provisionally identified as B. intermedia, Brachyspira alvinipulli, "Brachyspira pulli", or B. pilosicoli, whereas 4 were of indeterminate species affiliation. Many isolates were phylogenetically related to isolates from livestock. Isolates identified by PCR as B. pilosicoli displayed particularly high biodiversity. Up to five different Brachyspira genotypes were found from the same bird. Sequencing of amplicons from isolates that displayed ambiguous results as judged from PCR and phenotyping showed that several diagnostic PCRs were non-specific. Nox gene restriction enzyme analysis in silico correctly identified 2 of 34 characterized isolates. A culture technique based on filtration that produced uncontaminated spirochaete isolates was described. The results show that mallard intestines support a high degree of biodiversity among Brachyspira spp.     

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.     

Paenibacillus isolates possess diverse dextran-degrading enzymes

AIMS: To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced. METHODS AND RESULTS: Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70 degrees C were closely aligned to Paenibacillus spp. The three isolates purified at 60 degrees C had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50 degrees C produced dextranolytic activity in the spent media with maximal activity at 40 or 45 degrees C under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70 degrees C. CONCLUSIONS: The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.     

Isolation, identification and analysis of antibacterial activity of soil streptomycetes isolates from north Jordan

A total of 90 different Streptomyces isolates were recovered from 36 soil samples and assessed for their antibacterial activity. Nine isolates were identified by the absence of an aerial mycelium. The rest were grouped into six colour series, namely grey, white, yellow, green, red and polymorphic colours (pink, orange or violet) with total numbers of 29, 18, 14, 8, 3 and 9, respectively. The isolates (68%) showed a reverse side culture pigmentation, 30% produced melanin and 25% produced other soluble pigments. Isolates (48%) were characterized by flexuous spore chains, 21% with spiral and 10% for each of the rectus and retinaculum apertum arrangement. The antibiotic activity against a wide range of bacteria was exhibited by 54% of the isolates which were effective against Bacillus subtilis (57%), Staphylococcus aureus (47%), Escherichia coli (24%), Klebsiella spp (16%), and Shigella spp (12%). The lowest activity (8%) was exhibited against Pseudomonas spp and Salmonella spp. The antibacterial activity of the isolates was divided into four groups according to the diameter of the inhibition zone produced. Groups 3 and 4 with larger inhibition zones indicated their potential as a possible source of novel antibiotics.     

Identification of the Prototheca Species by Immunofluorescence

Studies were carried out to develop fluorescent antibody reagents for the identification of the Prototheca species and for their differentiation from morphologically similar fungi of various genera in formalin-fixed tissues. Antisera against representative isolates of P. filamenta, P. moriformis, P. stagnora, P. wickerhamii, and P. zopfii were produced in rabbits. Antiglobulins, labeled with fluorescein-isothiocyanate that intensely stained most cells of the homologous species, were selected for use as potential diagnostic reagents. By adsorbing the conjugates with selected heterologous cross-staining protothecae, reagents that were both sensitive and specific were obtained. Evaluation of the adsorbed conjugates with sections of tissue infected with protothecae, sections of tissue infected with morphologically similar fungi, and cultures of protothecae showed that these reagents are useful for the rapid and reliable identification of the Prototheca species.     

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains.

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.