R. Scott Pore

Protothecosis: report of a case with 20-year follow-up, and review of previously published cases

We present a Prototheca wickerhamii wound infection case that failed treatment with ketoconazole but was cured with amphotericin-B plus tetracycline. The patient was immunocompetent but had had local steroid injections. We reviewed another 159 cases from the literature. Prototheca has infected many areas of the human body, but most often skin, olecranon bursa, or wounds. Prior treatment with steroids and immune deficiencies are contributing factors. Itraconazole and fluconazole are reasonable initial treatments for patients with mild infections. For serious infections, or for infections that have failed azole treatment, amphotericin-B is the treatment of choice.

Antibiotic susceptibility testing ofCandida albicans by flow cytometry

A flow cytofluorometric susceptibility test (FCST) for in vitro antifungal drug testing ofCandida albicans was developed. Membrane damage was indicated by increased cellular fluorescence owing to propidium iodide or rose bengal uptake. Ketoconazole caused an exponential dose-response effect, best defined by Emax, at therapeutically achievable concentrations (0.02–0.2 µg/ml). This effect was not comparable to the conventional minimum inhibitory concentration (MIC) effect elicited by higher antibiotic concentrations. Amphotericin B, on the other hand, did not elicit Emax, but caused a >1 log dose-response effect which did correspond to the MIC. 5-Fluorocytosine susceptibility was also measurable. Other cytometric data indicating abnormal growth and growth inhibition, as well as conventional growth inhibition testing, confirmed that the 10-h FCST measured useful parameters of in vitro susceptibility.

Taxonomic implications of Prototheca and Chlorella cell wall polysaccharide characterization

SummaryFour polysaccharide fractions were obtained by acid and alkaline degradation of purified cell walls of Prototheca spp. and Chlorella spp. These fractions were further hydrolyzed and the component sugars identified. Five Prototheca strains and two Chlorella protothecoides strains have essentially similar polysaccharide compositions, which significantly differ from those of C. vulgaris and C. pyrenoidosa. This emphasizes the close affinity of C. protothecoides to Prototheca spp. not shared by other Chlorella spp.

Amphotericin B synergy testing by the FCST

The flow cytofluorometric susceptibility test (FCST) was incorporated into two in vitro synergy assays of amphotericin B-drug combinations. The FCST checkerboard assay and the FCST concentration-effect assay were developed to detect subtle modulation of amphotericin B fungicidal effects onCandida albicans andCryptococcus neoformans. Amphotericin B × cyclosporine A was a synergistic fungicidal combination against bothC. albicans andC. neoformans. Amphotericin B×gentamicin and amphotericin B×ketoconazole were synergistic combinations againstC. neoformans. In all cases tested, the synergy was effective when 0.50–0.62 μg amphotericin B/ml was used with>-0.50 μg of the other drug/ml. The fungicidal effect of ≥1.00 μg amphotericin B/ml overwhelmed the synergistic effects. A number of other drug combinations were additive, autonomous, or antagonistic.

Prototheca zopfii: natural, transient, occurrence in pigs and rats

Domestic swine faeces and fresh faeces from trapped barnyard rats were heavily contaminated with Prototheca zopfii, a cause of dairy cow mastitis. When the pigs and rats were maintained on Prototheca-free diets, the transient intestinal population of P. zopfii decreased precipitously and disappeared. When combined with the information that other farm animals excrete P. zopfii, it was concluded that contaminated animal feed may be the source of large numbers of P. zopfii in the farm environment. We found P. zopfii in wet spoiled feed. Rats are logical vectors for contamination of feed.

Detoxification of Chlordecone Poisoned Rats with Chlorella and Chlorella Derived Sporopollenin

Chlorella protothecoides accelerated the detoxification of chlordecone poisoned rats, decreasing the half-life of the toxin from 40 to 19 days. The ingested algae passed through the gastrointestinal tract unharmed , interrupted the enteric recirculation of the persistent insecticide, and subsequently eliminated the bound chlordecone with the feces. The detoxification was similar to that obtained with cholestyramine. Laboratory preparations were made to determine whether cell-free components retained the therapeutic properties of the whole cells. Acid and alkaline hydrolysis of the algae destroyed the cells except for the resistant cell wall components. One component was sporopollenin , a carotenoid polymer of limited natural occurrence among microorganisms and plants. Plant sporopollenin was not active, but algal cell walls and sporopollenin retained the therapeutic activity of the whole cells. The cells and cell walls have potential as detoxifying drugs for animals poisoned by chlordecone and other xenobiotic compounds with similar properties.

Synthesis and secretion fo mucous glycoprotein by the gill of Mytilus edulis I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.

Ketoconazole susceptibility of yeasts by the FCST method

The flow cytofluorometric susceptibility test (FCST) was extended fromCandida albicans to additional clinically relevant yeasts.Candida tropicalis, C. parapsilosis, C. lusitaniae, C. krusei, C. guilliermondii, Torulopsis glabrata, andRhodotorula rubra were also amenable to the FCST. Of the most frequently encountered yeasts,C. albicans andC. tropicalis exhibited the Emax response, which is believed to be an in vitro indicator of ketoconazole susceptibility with potential clinical relevance.Torulopsis glabrata, for which ketoconazole therapy is less effective, did not exhibit the Emax response; rather it exhibited an MIC effect, but usually at ketoconazole concentrations greater than those therapeutically achievable.

Prototheca associated with banana

Prototheca stagnora was found to be a habitant of older harvested banana (Musa sapientum) and plantain (M. paradisiaca) stumps while P. wickerhamii colonized fresh Musa sp. stumps and flower bract water of Heliconia sp. While Prototheca sp. were known to habituate woody plants, this is the first evidence that herbaceous plants also serve as habitats.

Microbial toxins, their functional role and phylogenetic validity

Microbially produced toxins, which appear to lack a role in microbial survival, may be antimicrobial compounds of significance to the producers. These toxin/antibiotics may act against cell metabolism shared by man or animals and other microorganisms. Protein toxin/antibiotics are produced by single species of bacteria. Those from fungi and algae are nonprotein secondary metabolites and several microorganisms may make the same or similar toxin/antibiotics.