In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

In vitro photoinactivation of bovine mastitis related pathogens.

BACKGROUND: Bovine mastitis is considered the most important disease of worldwide dairy industry. Treatment of this disease is based on the application intramammary antibiotic, which favors an increase in the number of resistant bacteria in the last decade. Photodynamic inactivation (PDI) has been investigated in different areas of Health Sciences, and has shown great potential for inactivating different pathogens, without any selection of resistant microorganisms. The objective of this study was to investigate the efficacy of PDI in the inactivation of pathogens associated with bovine mastitis. METHODS: We tested the effectiveness of PDI against antibiotic resistant strains, isolated from bovine mastitis, from the following species: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, and the alga Prototheca zopfii. Nine experimental groups were evaluated: control, no treatment; light only, irradiation of a red light-emitting diode (λ=662 (20) nm) for 180 s; exposure to 50 µM methylene blue alone for 5 min; and PDI for 5, 10, 30, 60, 120 and 180 s. RESULTS: S. dysgalactiae, S. aureus, and C. bovis were inactivated after 30s of irradiation, whereas S. agalactiae was inactivated after 120 s and P. zopfii at 180 s of irradiation. CONCLUSION: These results show that PDI can be an interesting tool for inactivating pathogens for bovine mastitis.

Biofilm formation by pathogenic Prototheca algae.

UNLABELLED: Prototheca microalgae are the only plants known to cause infections in humans and animals. The mechanisms of Prototheca infections are poorly understood, and no good treatments are available. Biofilms-surface-attached, three-dimensional microbial communities contributing to chronic infections-are formed by many pathogenic bacteria and fungi, but it is not known if Prototheca algae also have this ability. This study shows that various Prototheca species form biofilms composed of surface-attached cells in all growth phases, linked together by matrix containing DNA and polysaccharides. Biofilm formation was modulated by the presence of host plasma or milk. Compared to planktonic cells, Prototheca biofilms caused decreased release of IL-6 by mononuclear immune cells and responded differently to treatment with antimicrobials. Prototheca biofilms possibly contribute to chronic and hard-to-treat character of those algal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca algae are the only existing pathogenic plants. Almost nothing is known about mechanisms of Prototheca infections. This study identifies that, similar to pathogenic bacteria and fungi, Prototheca algae can form biofilms. These biofilms induce reduced immune cell activation relative to planktonic cells, and are also less susceptible to antimicrobials. Biofilm formation by Prototheca could be the first in vitro correlate of pathogenicity, opening a new research field for this pathogen.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.

An unusual case of granulomatous lymphadenitis due to Prototheca zopfii var. portoricensis in an immunocompetent man in China.

BACKGROUND: Protothecosis is an uncommon human infection caused by Prototheca. Prototheca spp can be considered as saprophytes, and in spite of their frequency in the environment, they are of low virulence and may cause chronic infection with low-grade inflammation in humans. At present, only three species are recognized: Prototheca wickerhamii, Prototheca zopfii and Prototheca stagnora. Of these, the former two have been associated with human disease. This study was an investigation of the clinical and microbiological features of a case of granulomatous lymphadenitis due to P. zopfii var. portoricensis in an immunocompetent man in China. METHODS: We report the case of a 39-year-old male, who presented with swollen lymph nodes, from which the organism was isolated and identified by the RapidID Yeast Plus test (Remel, Santa Fe, NM, USA) and PCR molecular analysis. The pathogenicity of the isolate was confirmed in a mouse model and antifungal drug susceptibility testing was carried out. RESULTS: The pathogen was identified as Prototheca zopfii. The DNA sequence of the 18S SSU rDNA regions of the isolate strain were 100% (1205/1205) identical with Prototheca zopfii var. portoricensis. Antifungal susceptibility tests revealed that it was sensitive to amphotericin B, but resistant to 5-flucytosine, fluconazole, ketoconazole, and itraconazole. The patient responded to treatment with intravenous itraconazole and amphotericin B. CONCLUSIONS: Based on the patient's symptoms and microscopic evaluation, cultures, and molecular analyses of the isolate, granulomatous lymphadenitis due to P. zopfii var. portoricensis was diagnosed. P. zopfii var. portoricensis as a causative agent of human lymphadenitis in an immunocompetent case has not been reported, though a few cases of protothecosis have been reported in China. The real number of protothecosis cases may be greater than that reported in the literature. Thus, clinicians should be vigilant for any unknown cause of granulomatous lymphadenitis and should undertake an intensive histopathology, mycology examination, and even molecular analysis to rule out or confirm a potential Prototheca infection.

A case of cutaneous protothecosis successfully treated with local thermal therapy as an adjunct to itraconazole therapy in an immunocompromised host.

We report a case of human protothecosis in an immunocompromised host which was caused by Prototheca wickerhamii and was successfully treated with thermal adjunct therapy combined with systemic itraconazole therapy. A 78-year-old man taking 30 mg prednisolone daily had a 1-week history of erythematous plaques on the dorsal aspect of his right hand and forearm after sustaining a small traumatic injury. Histopathology of the lesions revealed granulomatous inflammatory changes with numerous microorganisms that had multiple septations in their cytoplasm. On the basis of mycological features and the results of the sugar assimilation test, the etiologic agent was identified as Prototheca wickerhamii. Although the lesion showed no response to the systemic itraconazole therapy and topical ketoconazole treatment, a complete resolution was achieved by the use of thermal therapy as an adjunct to systemic itraconazole.

In vitro antimycotic activity of some plant extracts towards yeast and yeast-like strains.

As part of screening aimed at the selection of novel antimycotic compounds of vegetable origin, leaf extracts of Camellia sinensis L., Cupressus sempervirens L. and Pistacia lentiscus L. and the seed extract of Glycine soja Sieb. et Zucc. were tested against yeast and yeast-like species implicated in human mycoses. Of the extracts only those of C. sinensis (obtained from a commercial preparation of green tea) exhibited broad activity towards Candida glabrata, Clavispora lusitatiae, Cryptococcus laurentii, Filobasidiella neoformans, Issatchenkia orientalis, Saccharomyces cerevisiae and Prototheca wickerhamii strains. MICs ranging from 300 to 4800 microg extract/mL (corresponding to 130-2010 microg/mL total polyphenols) were observed. Concentrations of the C. sinensis extract over 25 000 microg/mL caused a rapid decrease of viable cells of Fil. neoformans and its activity was dose-dependent. Tests carried out using the pure polyphenols present in C. sinensis extract composition, showed that only epicatechin-3-O-gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) possess antimycotic activity.

Protothecosis in patients with cancer: case series and literature review.

OBJECTIVE: To review our recent experience with protothecosis in patients with cancer at The University of Texas MD Anderson Cancer Center, and compare these cases with others reported in the literature. METHODS: We report on three patients with protothecosis and cancer who were seen at The University of Texas MD Anderson Cancer Center from January 1979 to May 2002, and reviewed all cases of protothecosis in patients with cancer reported in the literature since 1966. RESULTS: Overall, 13 cases of protothecosis complicating cancer were evaluated. The median age of the patients was 41 years (range, 7-73 years). Seven patients (54%) had an underlying hematologic malignancy, and one infection occurred after bone marrow transplantation. Neutropenia was uncommon in these patients (14%). Prototheca wickerhamii was the most common Prototheca species identified as the causative agent of infection. Skin infection was the most common presentation of protothecosis, occurring in five patients (38%), followed by disseminated disease in three patients (23%), algaemia in three patients (23%), pulmonary infection in one patient (8%), and olecranon bursitis in one patient (8%). Information on the use of antifungal therapy was available for ten patients. Seven of the ten patients received amphotericin B, while three received triazoles (fluconazole in two, itraconazole in one). Breakthrough protothecosis occurred during the administration of systemic antifungal therapy with itraconazole in one patient. All seven patients who received amphotericin B showed a response, as did one of the three patients given triazoles. Seven (58%) of the patients died during the study period, only one (17%) of protothecosis. CONCLUSIONS: Protothecosis is an uncommon infection in cancer patients, implying that Prototheca spp. have a low pathogenic potential in this population. Pulmonary involvement in particular is uncommon in these patients. Amphotericin B appears to be the most effective antifungal agent; the role of triazoles in treating protothecosis is uncertain, but they may be less effective.

Actividad inhibitoria "in vitro" de algunos antisépticos frente a cultivos de Prototheca wickerhamii / \"In vitro\" inhibitory activity of some antiseptic drugs against Prototheca wickerhamii strains

Se estudió la acción inhibidora "in vitro" de los siguientes antisépticos: etil mercurio tiosalicilato de sodio, azida sódica, borato de sodio, yoduro de potasio, fenol y cloro, frente a cultivos de 5 cepas de Prototheca wickerhamii. La acción "in vitro" de estas drogas fue medida mediante el halo de inhibición que produjeron en los cultivos de Prototheca en medio de agar-miel de Sabouraud incubados a 28ºC. Los resultados obtenidos permitieron establecer que todas las cepas eran uniformemente susceptibles al etilmercurio tiosalicilato de sodio y a la azida sódica, la mayor parte fue inhibida por el yoduro de potasio, en tanto que, el borato de sodio, el fenol y el cloro resultaron inactivos en las diluciones utilizadas

Progressive cutaneous protothecosis.

The diagnosis of severe progressive cutaneous protothecosis in a 34-year-old woman was made by skin biopsy and culture. Analysis of host defense mechanisms revealed a persistent defect in the ability of the patient's PMN to kill the infecting organism. Specific IgG and IgE antibody was demonstrated. Serum levels of complement and immunoglobulins were normal or elevated. The patient was not anergic and peripheral blood lymphocytes responded to nonspecific mitogens. Treatment with amphotericin B and tetracycline resulted in resolution of skin lesions and negative cultures.