Diversity of the bacterial and fungal microflora from the midgut and cuticle of phlebotomine sand flies collected in North-Western Iran.

BACKGROUND: Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies. METHODOLOGY/PRINCIPAL FINDINGS: FIVE SAND FLY SPECIES: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates. CONCLUSION/SIGNIFICANCE: Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.

Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes.

The antifungal activity of onion (Allium cepa L.) on two important dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations >3.12% (v/v). The fungus T. mentagrophytes was more affected by the onion as compared to T. rubrum at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for T. rubrum compared with T. mentagrophytes. The hyphae of T. rubrum were found to be mainly affected by converting to resistant forms, i.e., chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of T. rubrum and T. mentagrophytes caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (-SH) groups present in essential compartments of the fungal cells was postulated.