Cinco cepas de Prototheca de origen humano

Resumen Se han aislado cinco cepas dePrototheca de muestras de origen humano: raspado vulvar, tinea corporis, secreción de oído, plieques de la mano, onicopatía inespecífica. Se estudian los caracteres morfológicos (propiedades asimilativas, termofilia) de estas cepas. Dos de ellas pertenecen a la especiePrototheca zopfii. Dos a la especieP. wickerhamii y la quinta es unaP. trispora. El autor hace resalter la frecuencia cada vez mayor con que se viene aislando este género de algas incoloras en el material patológico y la necesidad de su reconocimiento ya que son fácilmente confundibles con las levaduras.

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FivePrototheca strains of human origin are isolated. Morphological and physiological characters are studied. Two strains belong to the speciesPrototheca zophii, two to the speciesP. wickerhamii and the other one toProtheca trispora. The author emphasizes the frequency, with which this genus of chloric algae are lately reported, and the importance of their identification easily confused with that of the yeasts.Ultimamente hemos venido encontrando cada vez con mayor frecuencia en el material clínico, ejemplares del género Prototheca. La ubicación taxonómica de este género es aún dudosa; se le ha considerado un grupo intermedio entre algas incoloras y hongos (Tubaki, 1959). Nuestras 5 cepas han sido encontradas en un lapso de cuatro años, de un total de 320 muestras.

     

Investigacion preliminar de la accion inhibitoria in vitro de los antibioticos frente a las algas del genero Prototheca

Resumen Se investigó la posible acción inhibitoria in vitro de antibióticos en disco sobre algas microscópicas, potencialmente patógenas para el hombre, del Género Prototheca y la especie P. wickerhamii.Se utilizó el método de disco-placa en medio de Agar de Saboureaud.Resultaron como inhibidores in vitro: Amikacina, Colistina, Dibekacina, Framicetina, Gentamicina, Kanamicina, Lividomicina, Neomicina, Polimixina, Paromomicina, Ribostamicina, Sisomicina y Tobramicina.Se destaca el interés del estudio de sensibilidad a estas drogas ante un caso de Protothecosis humana.

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We investigated the possible inhibitory action in vitro by antibiotic containing discs against microscopic alga that are potential human pathogens of the genus Prototheca especially P. wickerhamii.The results indicated that inhibitors in vitro included amicacin, colistin, dibecacin, framicetin, gentamycin, kanamycin, lividomycin, neomycin, polymyxin, paromomycin, ribostamycin, sisomycin and tobramycin.We emphasize the interest in the effectiveness at these drugs in a case of human protothecosis.

     

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.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

Enzymatic profile of Prototheca species

The enzymatic profiles of algae of genus Prototheca were studied using 19 substrates included in the API ZYM system. Chymotrypsin and -glucuronidase were not detected. Of the enzymes detected the major percentages were alkaline phosphatase, esterase (C4), lipase esterase (C8), acid phosphatase and phosphoamidase.     

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.     

Prototheca stagnora, an encapsulated organism

The type species of Prototheca stagnora, SEC-L-1690 (ATCC 16528) (2) was found to have a large capsule surrounding the cells, in India ink preparations. Cryptococcus neoformans and P. stagnora are very similar organisms in their morphology and they should be differentiated by their reproduction process or by their biochemical and physiological characteristics.     

Emergence of Fungal-Like Organisms: Prototheca

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

     

Selective Medium for the Isolation of Prototheca

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

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.     

First isolation of Prototheca species in Spain

One-hundred forty samples of waste water were studied in search of algae of the genus Prototheca, that are potentially pathogenic to man. The samples were taken from collector outlets opening onto the Guadalquivir River.Nineteen cultures of Prototheca were isolated and identified as P. wickerhamii 9, P. zopffi 8 and P. stagnore 2.This is the first report of these algae in Spain and it is to be anticipated that these opportunist human pathogens will eventually be found to cause disease in the general population.     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.