Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4,6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35°C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4°C were also detected.Two Nosema species have been related to pathology in the honeybee: Nosema apis (18) a parasite of Apis mellifera, the western honeybee, and Nosema ceranae (4), a parasite of Apis cerana, the eastern honeybee. Currently, however, N. ceranae is considered an emergent and important parasite of Apis mellifera (4).Over the last few years, an increase in infections by this microsporidian has been detected in several European countries, together with an increase in honeybee colony deaths and a consequent decrease in the production of honey (9). However, it is not clear if N. ceranae infection may be the only factor related to this disorder, since this pathogen has also been found in healthy colonies (14). In Spain, Higes et al. (7,9) have demonstrated the presence of this parasite in honeybee samples from colonies with clear signs of population depletion, relating the colony collapse disorder to N. ceranae. The presence of this microsporidian is not exclusive to Europe, since it has also been described in bee samples collected about a decade ago in the United States (3).The pathology produced by N. ceranae in A. mellifera bees may be higher than that produced by N. apis, showing a rapid autoinfective capacity of the spores to spread the infection among epithelial cells, producing high mortality (6). On the other hand, reduced longevity of caged N. ceranae-infected worker bees compared to bees infected by N. apis has also been found (15).To date, continuous cultures of N. ceranae are not available and there is no effective treatment. For this reason, it is important to study the effects of different exposure conditions, such as time, temperature, and desiccation, on the viability of spores kept in the laboratory for use in the search for new treatments and for development of culture protocols. In addition, as different levels of thermotolerance in the environment and different epidemiological patterns have been described for these microsporidia, available data on resistance of N. apis spores cannot be extrapolated to N. ceranae.Spores. Spores from N. apis and N. ceranae were provided by the experimental apiary of the Regional Apicultural Center in Marchamalo, Spain. Recently collected spores were purified as described previously (8). The spore concentration was determined by counting with a hematocytometer chamber. Two stocks of N. ceranae spores were used (S1 and S2). All studies were carried out at a concentration of 5 ϫ 10 5 spores/ml. The Nosema species was determined by multiplex PCR, which amplifies the 16S rRNA locus (12).Spore treatments. The effects of time, temperature, and desiccation on viability were measured at different points. All ...

In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme ␤-1,3-D-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, ␤-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated. Candidemia is a frequent disease among immunosuppressed patients that is caused by opportunistic fungal pathogens from the genus Candida. Candida albicans is the most abundant species found in invasive candidiasis, although an increase in the abundance of other non-albicans Candida species has been described in the last years (1, 2). Echinocandin administration constitutes the main treatment for this disease. Currently, three echinocandins drugs, caspofungin (CAS), micafungin, and anidulafungin, are available for clinical practice. These antifungals are fungicidal against most Candida species and are effective against Candida isolates that are resistant to other antifungals (3). Echinocandins are lipopeptides that inhibit the activity of ␤-1,3-Dglucan synthase, which is encoded by FKS genes (4).Resistance to echinocandins has been described at a low frequency. The main resistance mechanism is associated with mutations in two regions of the FKS gene, denoted hot spot (HS) regions. These mutations result in proteins with reduced affinity for the antifungal (2, 5-7). However, in addition, there are other situations in which yeasts can grow in the presence of the antifungal. In particular, paradoxical growth (PG) (also known as the Eagle ef...

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

eWe have morphologically characterized Candida tropicalis isolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ␤-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization of Galleria mellonella larvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.A mphotericin B (AmB) is a widely used antifungal drug that presents strong killing activity against fungi after binding to ergosterol. Classically, it has been described that AmB induced cell death by forming pores at the level of the cell membrane (1, 2). However, recent findings suggest that AmB elicits its fungicidal effect through multiple mechanisms. In this sense, AmB induces ergosterol sequestration, which causes alterations in the cell membrane that result in killing of the cells (3, 4). In addition, AmB induces a significant accumulation of reactive oxygen species (ROS), which has been correlated with cell damage, apoptosis induction, and death (5-9).Resistance to AmB is a complex process, and multiple mechanisms have been described to be responsible for it. Lack of ergosterol at the cell membrane has been frequently correlated with reduced susceptibility to AmB (10-14). However, there are also some cases in which resistance to AmB can occur in cells with normal ergosterol content (15, 16). Aspergillus terreus, which is a mold that presents reduced susceptibility to AmB, contains similar ergosterol content to Aspergillus fumigatus, which is fully susceptible to this antifungal. In this case, increase in catalase and reduction of ROS production have been associated with increased resistance to AmB (17). Moreover, mechanisms that protect against the production of ROS also confer reduced susceptibility to AmB (6, 9). These results indicate that AmB exerts multiple responses in the cells and that resistance to this antifungal can be acquired by different mechanisms.The correlation between the cell wall and resistance to AmB has been poorly described. A relationship between resistance to AmB and changes in the cell wall has been observed in different fungi (18)(19)(20), although it is not known to what extent changes in the cell wall contribute to amphotericin B res...

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

TE or PG are widely expressed among Candida spp., although they do not seem to influence clinical outcome.

No abstract

(2015) Cryptococcusneoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleriamellonella, Virulence, 6:1,[66][67][68][69][70][71][72][73][74]