Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.

Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex.

Isolates of the Trichophyton mentagrophytes complex vary phenotypically. Whether the closely related zoophilic and anthropophilic anamorphs currently associated with Arthroderma vanbreuseghemii have to be considered as members of the same biological species remains an open question. In order to better delineate species in the T. mentagrophytes complex, we performed a mating analysis of freshly collected isolates from humans and animals with A. benhamiae and A. vanbreuseghemii reference strains, in comparison to internal transcribed spacer (ITS) and 28S rDNA sequencing. Mating experiments as well as ITS and 28S sequencing unambiguously allowed the distinction of A. benhamiae and A. vanbreuseghemii. We have also shown that all the isolates from tinea pedis and tinea unguium identified as T. interdigitale based on ITS sequences mated with A. vanbreuseghemii tester strains, but had lost their ability to give fertile cleistothecia. Therefore, T. interdigitale has to be considered as a humanized species derived from the sexual relative A. vanbreuseghemii.

Effect of irrigation water on the incidence of Salmonella spp. on lettuces produced by urban agriculture and sold on the markets in Dakar, Senegal

The aim of our survey was to assess the effect of irrigation water of the microbiological quality on the production chain of lettuce in the Dakar area. Microbiological analysis showed that 35% of irrigation water was contaminated by Salmonella spp. between the two water-types used for irrigation (groundwater and wastewater), no significant difference (p>0.05) in their degree of contamination was found. The incidence of different types of irrigation water on the contamination rate of lettuces from the farm (Pikine and Patte d'Oie) was not different either (p>0.05). However, the contamination rate of lettuce from markets of Dalifort and Grand-Yoff that were supplied by the area of Patte d'Oie was greater than those of Sham and Zinc supplied by Pikine (p<0.05). Comparison of serotypes of Salmonella isolated from irrigation water and lettuce showed that irrigation water may affect the microbiological quality of lettuce. Manures, frequently used as organic amendment in cultivating lettuce are another potential source of contamination. These results showed that lettuce may constitute effective vectors for the transmission of pathogens to consumers. Extensive treatment of the used wastewater and/or composting of manure could considerably reduce these risks.

Les Chalcidiens (Hymenoptera, Chalcidoidea) associés au genre Ficus spp. (Moracee) du Sénégal : répartition, abondance et diversité

Cette étude est la première portant sur la biodiversité écologique des Chalcidiens associés au genre Ficus au Sénégal. Sur les 26 espèces de figuiers signalées au Sénégal, 11 ont été trouvées. Chaque espèce de Ficus possède une microfaune Chalcidienne caractéristique. La comparaison des niveaux d'infestation révèle une différence très significative entre espèces de Ficus (P<0.0001). Les figues de F.sycomorus du domaine Nord-Soudanien sont, en valeurs absolues, plus infestées que celles des domaines Sud-Soudanien et Soudanien Atlantique. Les tests statistiques révèlent une différence d'infestation non significative des figues de F.sycomorus entre les 3 domaines (P>0.05). Cependant, entre le domaine Nord-Soudanien et le domaine Soudanien Atlantique, cette différence est significative (P=0.0091).

Clinal variation in a brown lemur (Eulemur spp.) hybrid zone: Combining morphological, genetic and climatic data to examine stability.

Studies of hybrid zones can inform our understanding of reproductive isolation and speciation. Two species of brown lemur (Eulemur rufifrons and E. cinereiceps) form an apparently stable hybrid zone in the Andringitra region of south-eastern Madagascar. The aim of this study was to identify factors that contribute to this stability. We sampled animals at 11 sites along a 90-km transect through the hybrid zone and examined variation in 26 microsatellites, the D-loop region of mitochondrial DNA, six pelage and nine morphological traits; we also included samples collected in more distant allopatric sites. Clines in these traits were noncoincident, and there was no increase in either inbreeding coefficients or linkage disequilibrium at the centre of the zone. These results could suggest that the hybrid zone is maintained by weak selection against hybrids, conforming to either the tension zone or geographical selection-gradient model. However, a closer examination of clines in pelage and microsatellites indicates that these clines are not sigmoid or stepped in shape but instead plateau at their centre. Sites within the hybrid zone also occur in a distinct habitat, characterized by greater seasonality in precipitation and lower seasonality in temperature. Together, these findings suggest that the hybrid zone may follow the bounded superiority model, with exogenous selection favouring hybrids within the transitional zone. These findings are noteworthy, as examples supporting the bounded superiority model are rare and may indicate a process of ecologically driven speciation without geographical isolation.

Resistance of Candida spp. to antifungal drugs in the ICU: where are we now?

Current increases in antifungal drug resistance in Candida spp. and clinical treatment failures are of concern, as invasive candidiasis is a significant cause of mortality in intensive care units (ICUs). This trend reflects the large and expanding use of newer broad-spectrum antifungal agents, such as triazoles and echinocandins. In this review, we firstly present an overview of the mechanisms of action of the drugs and of resistance in pathogenic yeasts, subsequently focusing on recent changes in the epidemiology of antifungal resistance in ICU. Then, we emphasize the clinical impacts of these current trends. The emergence of clinical treatment failures due to resistant isolates is described. We also consider the clinical usefulness of recent advances in the interpretation of antifungal susceptibility testing and in molecular detection of the mutations underlying acquired resistance. We pay particular attention to practical issues relating to ICU patient management, taking into account the growing threat of antifungal drug resistance.

Isothermal microcalorimetry for antifungal susceptibility testing of Mucorales, Fusarium spp., and Scedosporium spp.

We evaluated isothermal microcalorimetry for real-time susceptibility testing of non-Aspergillus molds. MIC and minimal effective concentration (MEC) values of Mucorales (n = 4), Fusarium spp. (n = 4), and Scedosporium spp. (n = 4) were determined by microbroth dilution according to the Clinical Laboratory Standard Institute M38-A2 guidelines. Heat production of molds was measured at 37 °C in Sabouraud dextrose broth inoculated with 2.5 × 10(4) spores/mL in the presence of amphotericin B, voriconazole, posaconazole, caspofungin, and anidulafungin. As determined by microcalorimetry, amphotericin B was the most active agent against Mucorales (MHIC 0.06-0.125 μg/mL) and Fusarium spp. (MHIC 1-4 μg/mL), whereas voriconazole was the most active agent against Scedosporium spp. (MHIC 0.25 to 8 μg/mL). The percentage of agreement (within one 2-fold dilution) between the MHIC and MIC (or MEC) was 67%, 92%, 75%, and 83% for amphotericin B, voriconazole, posaconazole, and caspofungin, respectively. Microcalorimetry provides additional information on timing of antifungal activity, enabling further investigation of drug-mold and drug-drug interaction, and optimization of antifungal treatment.

Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease.

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

De Novo Production of Metabolites by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca.

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato.

Summary Background Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. Objectives The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. Methods One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction-amplified internal transcribed spacer (ITS) region of the rDNA. Results Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. Conclusions Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.

Declining diversity of egg-associated bacteria during development of naturally spawned whitefish embryos (Coregonus spp.)

Fish eggs are associated with microbes, whose roles range from mutualism to parasitism. Recent laboratory experiments have shown that the taxonomic composition of associated microbial communities on the egg influences embryonic development. Host genetics also plays an important role in determining the consequences for embryonic growth and survival in this interaction. Moreover, it has been found that the importance of host genetics increases during embryogenesis. These findings suggest that during embryogenesis, the host increasingly influences the composition of its associated microbial community. However, little is known about the composition of microbial communities associated with naturally spawned eggs in the wild. We sampled fertilized whitefish eggs (Coregonus spp.) of different developmental stages from six sub-Alpine lakes and used a universal primer pair and 454 pyrosequencing in order to describe the taxonomic composition of egg-associated bacterial communities. We found bacterial communities on early embryos to be very diverse and to resemble the bacterial composition of the surrounding water environment. The bacterial communities on late embryos were significantly less diverse than on early embryos and displayed a clear shift in taxonomic composition that corresponded poorly with the bacterial composition of the surrounding water environment. The main bacterial components on whitefish eggs in this study were Proteobacteria, Actinobacteria, and Firmicutes, while the five most common families were Leuconostocaceae, Streptococcaceae, Comamonadaceae, Oxalobacteraceae and Moraxellaceae. Their putative relationships with the host are discussed. We conclude that natural symbiotic bacterial communities become more specialized during embryogenesis because of specific interactions with their embryo host.