Aída Verónica Rodríguez-Tovar

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×106 chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.

Ultrastructural changes on clinical isolates of Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum caused by Solanum chrysotrichum saponin SC-2.

Worldwide, dermatophytoses represent a high percentage of all superficial mycoses. The most frequently isolated dermatophyte is Trichophyton rubrum. Solanum chrysotrichum is a vegetal species widely used in Mexican traditional medicine to treat skin infections; its extract has been used to formulate an herbal medicinal product that is used successfully to treat Tinea pedis. Spirostanic saponin SC-2 from S. Chrysotrichum possesses high activity against dermatophytes. The present study reports the ultrastructural changes observed by means of transmission electron microscopy (TEM) in clinical isolates of T. rubrum, T. mentagrophytes, and Microsporum gypseum induced by saponin SC-2. Strains were grown in RPMI 1640 containing SC-2 (1600 microg/mL). Fungi were harvested at 6, 12, 24, and 48 h; controls without SC-2 were included. T. mentagrophytes was the most susceptible to the SC-2 saponin, followed by M. gypseum, while T. rubrum was the most resistant. The main alterations caused by the SC-2 saponin were as follows: i) loss of cytoplasmic membrane continuity; ii) organelle degradation; iii) to a lesser extent, irreversible damage to the fungal wall; and iv) cellular death.

Isolation and characterization of yeasts associated with plants growing in heavy-metal- and arsenic-contaminated soils.

Yeasts were quantified and isolated from the rhizospheres of 5 plant species grown at 2 sites of a Mexican region contaminated with arsenic, lead, and other heavy metals. Yeast abundance was about 10(2) CFU/g of soil and 31 isolates were obtained. On the basis of the phylogenetic analysis of 26S rRNA and internal transcribed spacer fragment, 6 species were identified within the following 5 genera: Cryptococcus (80.64%), Rhodotorula (6.45%), Exophiala (6.45%), Trichosporon (3.22%), and Cystobasidium (3.22%). Cryptococcus spp. was the predominant group. Pectinases (51.6%), proteases (51.6%), and xylanases (41.9%) were the enzymes most common, while poor production of siderophores (16.1%) and indole acetic acid (9.67%) was detected. Isolates of Rhodotorula mucilaginosa and Cystobasidium sloffiae could promote plant growth and seed germination in a bioassay using Brassica juncea. Resistance of isolates by arsenic and heavy metals was as follows: As(3+) ≥ 100 mmol/L, As(5+) ≥ 30 mmol/L, Zn(2+) ≥ 2 mmol/L, Pb(2+) ≥ 1.2 mmol/L, and Cu(2+) ≥ 0.5 mmol/L. Strains of Cryptococcus albidus were able to reduce arsenate (As(5+)) into arsenite (As(3+)), but no isolate was capable of oxidizing As(3+). This is the first study on the abundance and identification of rhizosphere yeasts in a heavy-metal- and arsenic-contaminated soil, and of the reduction of arsenate by the species C. albidus.

Exploratory Study on the Clinical and Mycological Effectiveness of a Herbal Medicinal Product from Solanum chrysotrichum in Patients with Candida Yeast-Associated Vaginal Infection

Mexican traditional medicine uses Solanum chrysotrichum to treat fungi-associated dermal and mucosal illness; its methanolic extract is active against dermatophytes and yeasts. Different spirostanic saponins (SC-2-SC-6) were identified as the active molecules; SC-2 was the most active in demonstrating a fungicidal effect against Candida albicans and non-albicans strains. The aim of the present study was to compare the clinical (elimination of signs and symptoms) and mycological effectiveness (negative mycological studies) of an S. chrysotrichum herbal medicinal product (Sc-hmp), standardized in 1.89 mg of SC-2, against ketoconazole (400 mg) in the topical treatment of cervical and/or vaginal infection by Candida. Both treatments (vaginal suppositories) were administered daily during 7 continuous nights. The study included 101 women (49 in the experimental group) with a confirmed clinical condition and positive mycological studies (direct examination and/or culture) of Candida infection. Basal conditions did not show differences between the groups; a moderate clinical picture was present in 62% of the cases, direct examination was positive in 69%, and the culture was positive with C. albicans predominating (65%). At the end of the administration period, both treatments demonstrated 100% tolerability, and clinical cure in 57.14% of S. chrysotrichum-treated cases and in 72.5% of ketoconazole-treated cases (p = 0.16), as well as 62.8% and 97.5% of mycological effectiveness, respectively (p = 0.0 001). We conclude that, at the doses used, Sc-hmp exhibits the same clinical effectiveness as ketoconazole, but with lower percentages of mycological eradication. Additional clinical studies with Sc-hmp are necessary, with increasing doses of SC-2, for improving the clinical and mycological effectiveness.

Candida glabrata survives and replicates in human osteoblasts

Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell.

Disseminated penicilliosis due to Penicillium chrysogenum in a pediatric patient with Henoch-Schönlein syndrome.

A case of disseminated infection caused by Penicillium chrysogenum in a 10-year-old boy with a history of Henoch-Schönlein purpura and proliferative glomerulonephritis, treated with immunosuppressors, is reported herein. The patient had a clinical picture of 2 weeks of fever that did not respond to treatment with broad-spectrum antibiotics and amphotericin B. Computed tomography imaging showed diffuse cotton-like infiltrates in the lungs, hepatomegaly, mesenteric lymphadenopathy, and multiple well-defined round hypodense lesions in the spleen. His treatment was changed to caspofungin, followed by voriconazole. One month later, a splenic biopsy revealed hyaline septate hyphae of >1μm in diameter. Fungal growth was negative. However, molecular analysis showed 99% identity with P. chrysogenum. A therapeutic splenectomy was performed, and treatment was changed to amphotericin B lipid complex and caspofungin. The patient completed 2 months of treatment with resolution of the infection. P. chrysogenum is a rare causative agent of invasive fungal infections in immunocompromised patients, and its diagnosis is necessary to initiate the appropriate antifungal treatment.

Morphological and molecular characterization, enzyme production and pathogenesis of Fusarium temperatum on corn in Mexico

Abstract A Fusarium sp. from a corn field in Chapingo, Estado de Mexico, Mexico was identified and characterized by morphological and molecular approaches. Colony growth on Czapeks medium and potato dextrose agar and pigment formation was assessed. Microscopically, the isolate displayed typical hyaline septate mycelium with long-beaked macroconidia with one to six septa, with a curved apical cell and a foot-like basal cell. Molecular characterization was performed by PCR of the ITS1-5.8S rDNA-ITS2 region and a portion of the elongation factor 1 alpha (EF-1α) followed by sequencing. The isolate was identified as Fusarium temperatum, an important corn pathogen in Europe, Asia and South America. A phylogenetic analysis employing the EF-1α sequence showed the isolate grouped with several species of the Gibberella fujikuroi species complex. Additionally, the isolate produced pectinase, xylanase, cellulase and lipase in culture and at 15 days post-inoculation, was able to infect the roots of corn. To our knowledge, this is the first confirmed identification and characterization of a phytopathogenic F. temperatum isolate from Mexico.

Effect of the inoculation of Axonopus affinis plantlets with immobilized rhizobacteria exposed to cadmium

The interactions between plants with rhizosphere and root associated microorganisms have been considered because they are potentially useful in phytoremediation and are used as microbial inoculates (biofertilizers) that give bioprotection to plants against biotic and abiotic stresses. One of the methods safe and effective for introducing bioinoculants is the encapsulation of cells in biodegradable gel matrices like alginate. The aim of this study is to evaluate the effect of three rhizobacteria (

The phosphorelay signal transduction system in Candida glabrata: an in silico analysis

Signaling systems allow microorganisms to sense and respond to different stimuli through the modification of gene expression. The phosphorelay signal transduction system in eukaryotes involves three proteins: a sensor protein, an intermediate protein and a response regulator, and requires the transfer of a phosphate group between two histidine-aspartic residues. The SLN1-YPD1-SSK1 system enables yeast to adapt to hyperosmotic stress through the activation of the HOG1-MAPK pathway. The genetic sequences available from Saccharomyces cerevisiae were used to identify orthologous sequences in Candida glabrata, and putative genes were identified and characterized by in silico assays. An interactome analysis was carried out with the complete genome of C. glabrata and the putative proteins of the phosphorelay signal transduction system. Next, we modeled the complex formed between the sensor protein CgSln1p and the intermediate CgYpd1p. Finally, phosphate transfer was examined by a molecular dynamic assay. Our in silico analysis showed that the putative proteins of the C. glabrata phosphorelay signal transduction system present the functional domains of histidine kinase, a downstream response regulator protein, and an intermediate histidine phosphotransfer protein. All the sequences are phylogenetically more related to S. cerevisiae than to C. albicans. The interactome suggests that the C. glabrata phosphorelay signal transduction system interacts with different proteins that regulate cell wall biosynthesis and responds to oxidative and osmotic stress the same way as similar systems in S. cerevisiae and C. albicans. Molecular dynamics simulations showed complex formation between the response regulator domain of histidine kinase CgSln1 and intermediate protein CgYpd1 in the presence of a phosphate group and interactions between the aspartic residue and the histidine residue. Overall, our research showed that C. glabrata harbors a functional SLN1-YPD1-SSK1 phosphorelay system.

Antagonistic Interaction of Staphylococcus aureus Toward Candida glabrata During in vitro Biofilm Formation Is Caused by an Apoptotic Mechanism

Background: Infections caused by Candida species and Staphylococcus aureus are associated with biofilm formation. C. albicans–S. aureus interactions are synergistic due to the significant increase in mixed biofilms and improved resistance to vancomycin of S. aureus. C. glabrata and S. aureus both are nosocomial pathogens that cause opportunistic infections in similar host niches. However, there is scarce information concerning the interaction between these last microorganisms. Results: The relationship between C. glabrata and S. aureus was evaluated by estimating the viability of both microorganisms in co-culture of planktonic cells and in single and mixed biofilms. An antagonistic behavior of S. aureus and their cell-free bacterial supernatant (CFBS) toward C. glabrata, both in planktonic form and in biofilms, was demonstrated. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) images showed yeast cells surrounded by bacteria, alterations in intracytoplasmic membranes, and non-viable blastoconidia with intact cell walls. Concomitantly, S. aureus cells remained viable and unaltered. The antagonistic activity of S. aureus toward C. glabrata was not due to cell-to-cell contact but the presence of CFBS, which causes a significant decrement in yeast viability and the formation of numerous lipid droplets (LDs), reactive oxygen species (ROS) accumulation, as well as nuclear alterations, and DNA fragmentation indicating the induction of an apoptotic mechanism. Conclusion: Our results demonstrate that the S. aureus CFBS causes cell death in C. glabrata by an apoptotic mechanism.