Antifungal activity of [K3]temporin-SHa against medically relevant yeasts and moulds.

Few antifungal agents are currently available for the treatment of fungal infections. Antimicrobial peptides (AMPs), which are natural molecules involved in the innate immune response of many organisms, represent a promising research method because of their broad killing activity. The aim of this study was to assess the activity of a frog AMP, [K3]temporin-SHa, against some species of yeasts and moulds, and to further explore its activity against Candida albicans. MIC determinations were performed according to EUCAST guidelines. Next, the activity of [K3]temporin-SHa against C. albicans was explored using time-killing curve experiments, membrane permeabilization assays, and electron microscopy. Finally, chequerboard assays were performed to evaluate the synergy between [K3]temporin-SHa and amphotericin B or fluconazole. [K3]temporin-SHa was found to be active in vitro against several yeasts with MIC between 5.5 and 45 µM. [K3]temporin-SHa displayed rapid fungicidal activity against C. albicans (inoculum was divided into two in less than an hour and no viable colonies were recovered after 5 h) with a mechanism that could be due to membrane permeabilization. [K3]temporin-SHa was synergistic with amphotericin B against C. albicans (FICI = 0.303). [K3]temporin-SHa could represent an additional tool to treat several Candida species and C. neoformans.

In vitro sensitivity of 30 anaerobic bacterial strains of the human intestinal core microbiota to antibiotics: Culture and LC-MS/MS approaches.

We have a vast knowledge on human intestinal microbiota but it can still be regarded incomplete. One of the objectives of scientists using so-called "omics" techniques is to be interested in the consequences that drugs can have on the composition of the intestinal microbiota and inversely. To date, few publications have reported the effects of drugs on the growth of bacteria composing this microbiota using a "culturomics" approach. We focused on antibiotics commonly prescribed for which the only published are the susceptibility of the pathogenic strains and not that of the commensal strains. The aim of our study was to determine the sensitivity of 30 strains considered to represent the intestinal core microbiota to 8 antibiotics and to study the possible modification of these molecules by bacteria. The 30 bacterial strains were cultured under anaerobic conditions in order to determine their sensitivity to the antibiotics. After 48 h of culture, the supernatants were also analyzed via UHPLC-MS/MS in order to determine if the antibiotics have been chemically modified. Under the current experimental conditions, cefpodoxime, metronidazole, erythromycin, sulfamethozaxole, trimethoprim and the trimethoprim/sulfamethozaxole combination have little impact on the core microbiota strain growth. On the contrary, moxifloxacin and amoxicillin inhibit the growth of numerous strains of our panel. Using UHPLC-MS/MS analyses, we have shown that some antibiotics can be modifed by the bacteria composing the intestinal core microbiome. The bacteria that make up the intestinal microbiota core are impacted by the antibiotics most commonly prescribed in clinics today and inversely.

Invasive Candida bovina Infection, France.

New Candida species such as Candida auris have emerged recently as important invasive fungal diseases. We report a case of C. bovina bloodstream infection in a 94-year-old patient in France. The species led to identification issues because it was misidentified by phenotypic and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods.

Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila.

Aeromonas hydrophila, considered as an emerging pathogen, is increasingly involved in opportunistic human infections. This bacterium, mainly present in aquatic environments, can therefore develop relationships with the free-living amoeba Vermamoeba vermiformis in hospital water networks. We showed in this study that the joint presence of V. vermiformis and A. hydrophila led to an increased bacterial growth in the first 48 h of contact and moreover to the protection of the bacteria in adverse conditions even after 28 days. These results highlight the fact that strategies should be implemented to control the development of FLA in hospital water systems.

Vermamoeba vermiformis: a Free-Living Amoeba of Interest.

Free-living amoebae are protists that are widely distributed in the environment including water, soil, and air. Although the amoebae of the genus Acanthamoeba are still the most studied, other species, such as Vermamoeba vermiformis (formerly Hartmannella vermiformis), are the subject of increased interest. Found in natural or man-made aquatic environments, V. vermiformis can support the multiplication of other microorganisms and is able to harbor and potentially protect pathogenic bacteria or viruses. This feature is to be noted because of the presence of this thermotolerant amoeba in hospital water networks. As a consequence, this protist could be implicated in health concerns and be indirectly responsible for healthcare-related infections. This review highlights, among others, the consequences of V. vermiformis relationships with other microorganisms and shows that this free-living amoeba species is therefore of interest for public health.

Vermamoeba vermiformis-Aspergillus fumigatus relationships and comparison with other phagocytic cells.

Free living amoebae (FLA) are protists ubiquitously present in the environment. Aspergillus fumigatus is a mould responsible for severe deep-seated infections, and that can be recovered in the same habitats as the FLA. By conducting coculture experiments and fungal incubation with amoebal supernatants, we report herein that Vermamoeba vermiformis, a FLA present in hospital water systems, promotes filamentation and growth of A. fumigatus. This finding is of particular importance to institutions whose water systems might harbor FLA and could potentially be used by immunocompromised patients. Also, the relationships between V. vermiformis and A. fumigatus were compared to those between this fungus and two other phagocytic cells: Acanthamoeba castellanii, another FLA, and macrophage-like THP-1 cells. After 4 h of coincubation, the percentages of the three phagocytic cell types with adhered conidia were similar, even though the types of receptors between FLA and macrophagic cell seemed different. However, the percentage of THP-1 with internalized conidia was considerably lower (40 %) in comparison with the two other cell types (100 %). Thus, this study revealed that interactions between A. fumigatus and these three phagocytic cell types show similarities, even though it is premature to extrapolate these results to interpret relationships between A. fumigatus and macrophages.

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits.

Free-living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.

Cellular, biochemical, and molecular changes during encystment of free-living amoebae.

Free-living amoebae are protozoa found in soil and water. Among them, some are pathogenic and many have been described as potential reservoirs of pathogenic bacteria. Their cell cycle is divided into at least two forms, the trophozoite and the cyst, and the differentiation process is named encystment. As cysts are more resistant to disinfection treatments than trophozoites, many studies focused on encystment, but until recently, little was known about cellular, biochemical, and molecular modifications operating during this process. Important signals and signaling pathways at play during encystment, as well as cell responses at the molecular level, have been described. This review summarizes our knowledge and focuses on new findings.

Involvement of minerals in adherence of Legionella pneumophila to surfaces.

Legionella pneumophila is the causative agent of 90 % of Legionnaires' disease cases. This bacterium lives naturally in fresh water and can colonize biofilms, which play an important role in the protection of Legionella against environmental stress factors. Relationship between the presence of minerals in water and Legionella adherence to surfaces is not well-known. In this study, we studied influence of minerals on bacterial adherence. For the first time, to our knowledge, this report shows that calcium and magnesium in a less extent, enhances the adherence of Legionella to surfaces compared to the bacteria behavior in distilled water. Treatment with proteinase K of live cells showed that surface proteins do not seem to play a crucial role in bacteria adherence to surfaces. Our results represent a first step in understanding effect of ions on Legionella adherence to surfaces. Such field of research could be helpful to better understand biofilm colonization by this bacterium to improve Legionella risk management in water networks.

Fatal-mixed cutaneous zygomycosis-aspergillosis: a case report.

Mucorales and Aspergillus are molds responsible for infections in immunocompromised patients. In this report, we describe a case of a rare extensively mixed cutaneous infection caused by Lichtheimia ramosa, Aspergillus fumigatus and Aspergillus terreus in a neutropenic patient suffering from an acute leukemia. The fatal outcome of this patient can be attributed to its hematologic malignancy, the extensive nature of the lesions and the resistance of the strains to antifungals.

In vitro reciprocal interactions between yeasts from human cutaneous mycobiota and parabens used in cosmetics.

Parabens are substances with antifungal and antibacterial properties, suspected to be endocrine disruptors and widely used as preservatives in cosmetics. In this case, exposure to these compounds is mainly dermal and interactions may occur with skin components including cutaneous mycobiota. In this work, we have explored the in vitro reciprocal interactions between three parabens (methylparaben, ethylparaben, and propylparaben) and yeasts from the human cutaneous mycobiota (Candida parapsilosis, Cryptococcus uniguttulatus, and Rhodotorula mucilaginosa) by studying the effect of these parabens on fungal growth and the fungal ability to metabolize the tested compounds. Our results showed that, at the tested concentrations, the growth of three strains of C. parapsilosis was not influenced by the presence of parabens. Whereas, using the same parabens concentrations, growth of C. uniguttulatus and R. mucilaginosa was completely inhibited by ethylparaben since the first day of contact, whereas these same fungi were not sensitive to the two other parabens, even after seven days of incubation. The presence of a lamellar wall in these basidiomycete fungi as well as the physico-chemical properties of ethylparaben could explain this selective inhibition. Additionally, C. parapsilosis and R. mucilaginosa degraded 90% to 100% of propylparaben after seven days of incubation but had no effect on the other tested parabens. Thus, their enzymes seem to only degrade long chain parabens. In the same conditions, C. uniguttulatus did not degrade any paraben. This inability may be due to the absence of fungal enzymes able to degrade parabens or to the possible inaccessibility of intracellular enzymes due to the polysaccharide capsule. Our work has shown that parabens can act differently from one fungus to another within the cutaneous mycobiota. These preliminary results have evidenced that in vitro parabens, contained in cosmetic products, could be involved in the occurrence of a state of dysbiosis. The tested yeasts from the cutaneous mycobiota can also be involved in the degradation of parabens and thereby reduce, according to the produced metabolites and their activities, the risk of endocrine disruption they can induce.

[Could antifungal lock be useful in the management of candidiasis linked with catheters?]. / Candidoses associées aux cathéters : quelle place pour les verrous antifongiques?

Fungal biofilms associated with inserted medical devices such as catheters, represent a major risk factor for candidemia. In addition, these biofilm yeasts show a decreased susceptibility to antifungal agents. Recently, a new therapeutic approach has emerged, the "lock therapy", based on the use of high concentrations of antimicrobials, instilled into the lumen of the catheter and left in place for 8 to 12 h. In vitro or in vivo studies have evaluated the interest of antifungal locks using amphotericin B, an azole or echinocandins. The promising results will permit us to discuss the relevance of this technique.

Development of an in vitro Model of Human Gut Microbiota for Screening the Reciprocal Interactions With Antibiotics, Drugs, and Xenobiotics.

Altering the gut microbiota can negatively affect human health. Efforts may be sustained to predict the intended or unintended effects of molecules not naturally produced or expected to be present within the organism on the gut microbiota. Here, culture-dependent and DNA-based approaches were combined to UHPLC-MS/MS analyses in order to investigate the reciprocal interactions between a constructed Human Gut Microbiota Model (HGMM) and molecules including antibiotics, drugs, and xenobiotics. Our HGMM was composed of strains from the five phyla commonly described in human gut microbiota and belonging to Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria. Relevantly, the bacterial diversity was conserved in our constructed human gut model through subcultures. Uneven richness distribution was revealed and the sensitivity of the HGMM was mainly affected by antibiotic exposure rather than by drugs or xenobiotics. Interestingly, the constructed model and the individual cultured strains respond with the same sensitivity to the different molecules. UHPLC-MS/MS analyses revealed the disappearance of some native molecules in the supernatants of the HGMM as well as in those of the individual strains. These results suggest that biotransformation of molecules occurred in the presence of our gut microbiota model and the coupled approaches performed on the individual cultures may emphasize new bacterial strains active in these metabolic processes. From this study, the new HGMM appears as a simple, fast, stable, and inexpensive model for screening the reciprocal interactions between the intestinal microbiota and molecules of interest.

Cellular response of the amoeba Acanthamoeba castellanii to chlorine, chlorine dioxide, and monochloramine treatments.

Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii.

Acanthamoeba castellanii: proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation.

Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis.