Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches.

BACKGROUND: Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES: The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS: Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS: Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS: The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Spathaspora boniae sp. nov., a D-xylose-fermenting species in the Candida albicans/Lodderomyces clade.

Two yeast isolates producing asci-containing elongate ascospores with curved ends typical of the genus Spathaspora were isolated from rotting wood samples collected in an Atlantic rainforest ecosystem in Brazil. Phylogenetic analysis of the LSU rRNA gene D1/D2 domain sequences demonstrated that the strains represent a new species and placed it next to Candida blackwellae, in a clade that also contains Candida albicans and Candida dubliniensis. Other sequences of the ribosomal gene cluster supported same placementin the same clade, and a phylogenomic analysis placed this new species in an early emerging position relative to the larger C. albicans/Lodderomyces clade. One interpretation is that the genus Spathaspora is, in fact, paraphyletic. In conformity with this view, we propose the novel species Spathaspora boniae sp. nov. to accommodate the isolates. The type strain of Spathaspora boniae sp. nov. is UFMG-CM-Y306T (=CBS 13262T). The MycoBank number is MB 821297. A detailed analysis of xylose metabolism was conducted for the new species.

Saturnispora bothae sp. nov., isolated from rotting wood.

Two strains representing a novel species of the genus Saturnispora were isolated from rotting wood samples collected in an Atlantic Rainforest site in Brazil. Analyses of the sequences of the D1/D2 domains of the rRNA gene showed that this novel species belongs to a subclade in the Saturnispora clade formed by Saturnispora sanitii, Saturnispora sekii, Saturnispora silvae and Saturnisporasuwanaritii. The novel species differed in D1/D2 sequences by 60 or more nucleotide substitutions from these species. The strains produced asci with one to four hemispherical ascospores. A novel species named Saturnispora bothae sp. nov. is proposed to accommodate these isolates. The type strain is UFMG-CM-Y292T (=CBS 13484T). The MycoBank number is MB 817127.

Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis.

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

Identification of lactic acid bacteria associated with traditional cachaça fermentations.

During the production of traditional cachaça (alembic´s cachaça), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaça production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaça fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Antifungal Activity Directed Toward the Cell Wall by 2-Cyclohexylidenhydrazo- 4-Phenyl-Thiazole Against Candida albicans.

BACKGROUND: The increasing incidence of invasive forms of candidiasis and resistance to antifungal therapy leads us to seek new and more effective antifungal compounds. OBJECTIVE: To investigate the antifungal activity and toxicity as well as to evaluate the potential targets of 2- cyclohexylidenhydrazo-4-phenyl-thiazole (CPT) in Candida albicans. METHODS: The antifungal activity of CPT against the survival of C. albicans was investigated in Caenorhabditis elegans. Additionally, we determined the effect of CPT on the inhibition of C. albicans adhesion capacity to buccal epithelial cells (BECs), the toxicity of CPT in mammalian cells, and the potential targets of CPT in C. albicans. RESULTS: CPT exhibited a minimum inhibitory concentration (MIC) value of 0.4-1.9 µg/mL. Furthermore, CPT at high concentrations (>60 x MIC) showed no or low toxicity in HepG2 cells and <1% haemolysis in human erythrocytes. In addition, CPT decreased the adhesion capacity of yeasts to the BECs and prolonged the survival of C. elegans infected with C. albicans. Analysis of CPT-treated cells showed that their cell wall was thinner than that of untreated cells, especially the glucan layer. We found that there was a significantly lower quantity of 1,3-ß-D-glucan present in CPT-treated cells than that in untreated cells. Assays performed on several mutant strains showed that the MIC value of CPT was high for its antifungal activity on yeasts with defective 1,3-ß-glucan synthase. CONCLUSION: In conclusion, CPT appears to target the cell wall of C. albicans, exhibits low toxicity in mammalian cells, and prolongs the survival of C. elegans infected with C. albicans.

Effects of two 6-quinolinyl chalcones on the integrity of plasma membrane of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, yet few therapeutic options exist. Our aim was to search for new compounds with high efficacy, low toxicity, shorter treatment time and affordable cost. We studied two synthetic 6-quinolinyl chalcones, 3b and 3e, to determine their effects on VERO cells, antifungal activity, survival curve, interaction with other drugs and phenotypic effects against several isolates of Paracoccidioides spp. In this study, we verified that the compounds were not toxic, exhibited superior in vitro activity compared with that shown by trimethoprim-sulfamethoxazole, and after 5 days of treatment, decreased the fungal cell viability by approximately 70%. Additionally, no interactions were observed between the tested compounds and other drugs. We also found that these compounds induced morphological changes, such as shriveling of cells, fragmentation of the plasma membrane and cytoplasmic disorganization in vitro. The changes observed by microscopy assays corroborate the observation made with propidium iodide, where the number of cells stained with the compounds was higher than that observed after amphotericin B treatment. We observed an increase in the efflux of K+ and a loss of intracellular contents in cells treated with 3b and 3e, confirming their effects on fungal membranes. However, damage to the membrane was not associated with a decrease in membrane ergosterol levels. The experimental evidences showed no direct indications of cellular wall damage caused by these compounds. Thus, these results confirm the antifungal potential of 3b and 3e against Paracoccidioides spp. with possible action on the membrane.

Lactic acid bacteria and yeasts associated with spontaneous fermentations during the production of sour cassava starch in Brazil.

Sour cassava starch is a traditional fermented food used in the preparation of fried foods and baked goods such as traditional cheese breads in Brazil. Thirty samples of sour cassava starch were collected from two factories in the state of Minas Gerais. The samples were examined for the presence of lactic acid bacteria, yeasts, mesophilic microorganisms, Bacillus cereus and faecal coliforms. Lactic acid bacteria and yeasts isolates were identified by biochemical tests, and the identities were confirmed by molecular methods. Lactobacillus plantarum and Lactobacillus fermentum were the prevalent lactic acid bacteria in product from both factories, at numbers between 6.0 and 9.0 log cfu g(-)(1). Lactobacillus perolans and Lactobacillus brevis were minor fractions of the population. Galactomyces geothricum and Issatchenkia sp. were the prevalent yeasts at numbers of 5.0 log cfu g(-)(1). A species similar to Candida ethanolica was frequently isolated from one factory. Mesophilic bacteria and amylolytic microorganisms were recovered in high numbers at all stages of the fermentation. B. cereus was found at low numbers in product at both factories. The spontaneous fermentations associated with the production of sour cassava starch involve a few species of lactic acid bacteria at high numbers and a variety of yeasts at relatively low numbers.

Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches

BACKGROUND Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6 percent of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8 percent of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9 percent of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75 percent of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9 percent of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaça (alembicïs cachaça), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaça production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaça fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Enteroxigenic Staphylococcus spp. and other microbial contaminants during production of Canastra cheese, Brazil

Canastra cheese is produced from raw cow's milk, and it is made at the farmhouse level using artisanal procedures and natural starters. The aim of this work was to determine the main hygienic-sanitary indicators and enterotoxigenic staphylococcal strains present during the manufacturing of traditional cheese of Serra da Canastra region, Minas Gerais state, Brazil. Samples from 10 farms were studied, and they included: water employed in the process, raw milk, natural starters, cheese curd before salting and cheese after five days of ripening. All water samples exhibited faecal coliform contamination above the maximum acceptable value recommended by Brazilian standards. Pseudomonas aeruginosa and sulfite-reducing clostridia were also isolated from the water samples. In five samples of raw milk faecal coliform were above the limits allowed by the Brazilian legislation. The counts of Staphylococcus spp. in milk were between <2.0 to 4.9 log.cfu.ml-1. The counts of microbiological indicators were higher in natural starters and curd. High levels of faecal and total coliform, as well as molds, were found in the cheese samples. In all cheeses analyzed Staphylococcus spp. were found in levels above 5.0 log.cfu.g-1. The enterotoxins (SE) most frequently produced by Staphylococcus spp. strains were SEB and SEC. A high number of coagulase negative Staphylococcus strains were also enterotoxin producers. None of the samples contained Salmonella spp. or Listeria spp. These results point out a need for improvements in the production process of the artisanal cheese produced at Serra da Canastra in Brazil.

O queijo Canastra é produzido a partir de leite cru, e é fabricado em fazendas utilizando procedimentos artesanais e culturas naturais de soro como iniciadoras. O objetivo deste trabalho foi determinar os principais indicadores higiênico-sanitário e as linhagens enterotoxigenicas de Staphylococcus presentes durante a fabricação do queijo tradicional da região da Serra da Canastra, Minas Gerais, Brasil. Amostras provenientes de 10 fazendas foram estudadas, e estas incluíram: a água utilizada no processo, o leite cru, o soro iniciador, a coalhada antes da salga, e o queijo após cinco dias de cura. Todas as amostras de água apresentaram contaminação por coliformes fecais acima do valor máximo recomendado pelos padrões brasileiros. Pseudomonas aeruginosa e clostrídios sufito-redutores também foram isolados das amostras de água. Em cinco amostras de leite cru os coliformes fecais apresentaram-se acima dos limites permitidos pela legislação brasileira. As contagens de Staphylococcus spp. no leite variaram de <2.0 a 4.9 log.ufc.ml-1. As contagens dos indicadores microbiológicos foram maiores no soro iniciador do que na massa coagulada. Níveis altos de contaminação por coliformes totais e fecais, como também bolores, foram observados nas amostras de queijo. Em todos os queijos estudados Staphylococcus spp. foram encontrados em níveis acima de 5.0 log.ufc.g-1. As enterotoxinas mais freqüentes produzidas pelas linhagens de Staphylococcus spp. foram B e C. Um número elevado de linhagens de Staphylococcus coagulase-negativa foram também produtores de enterotoxinas. Em nenhuma das amostras foi isolada Salmonella spp. ou Listeria spp. Estes resultados mostram a necessidade de melhorias no processo de produção do queijo artesanal produzido na Serra da Canastra, Brasil.