Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

High colonization by Candida parapsilosis sensu stricto on hands and surfaces in an adult intensive care unit.

BACKGROUND: Yeasts of the Candida parapsilosis complex have frequently been reported as agents of fungal infection in Brazil and worldwide, most of the cases are related to hospital-acquired infection. C. parapsilosis is the third most common cause of candidemia, and the hands of hospital workers as well as hospital surfaces have been suggested as possible sources. OBJECTIVES: In this study we verified the frequency of C. parapsilosis on the hands of workers and on surfaces in the adult intensive care unit (AICU) of a tertiary hospital in Paraná-Brazil. METHODS: Surface samples were collected with swabs moistened with saline, and a plastic bag with distilled water was used to collect samples from hands. The yeasts were identified by morphology, MALDI-TOF mass spectrometry and PCR-RFLP of the secondary alcohol dehydrogenase-encoding gene (SADH) after digestion with the restriction enzyme BanI. RESULTS AND CONCLUSIONS: A total of 223 yeast were found, of which 101 (45.29%) were identified as C. parapsilosis sensu stricto. Of these, 46.66% (n=35) were found on surfaces and 44.59% (n=66) on the hands of the employees. The analysis of C. parapsilosis strains by microsatellite loci (CP1, CP4, CP6 and B5) showed 80 different genotypes. Their antifungal susceptibility profile, evaluated by the microdilution broth method, revealed that C. parapsilosis was sensitive to amphotericin B, fluconazole and voriconazole, but not to micafungin. The results revealed the heterogeneity of the yeast population, suggesting that there is no common source of contamination in the AICU of this hospital.

Candidemia by Candida parapsilosis in a neonatal intensive care unit: human and environmental reservoirs, virulence factors, and antifungal susceptibility.

The Candida parapsilosis complex has emerged as one of the main causes of candidemia worldwide. This study aims to evaluate possible C. parapsilosis sensu stricto reservoirs in a NICU, the expression of virulence factors, and antifungal susceptibility, and to analyze their genetic and phenotypic similarity. The study included 17 isolates of C. parapsilosis: seven environmental, one from a newborn's mother, and nine samples from six newborns. We used molecular and phenotypic tests to characterize the isolates and to trace possible routes of infection. The genetic similarity was determined by random amplified polymorphic DNA. The hemolytic and DNAse activity was determined using sheep's blood and DNAse agar, biofilm production by XTT method, and the susceptibility to antifungals through microdilution methodology. Two environmental strains isolated in the same month had high similarity. The 17 isolates expressed at least one of the three virulence factors studied, and one environmental isolate was resistant to fluconazole. This study shows that environmental contamination can be an important reservoir of potentially pathogenic microorganisms, since isolates of C. parapsilosis sensu stricto collected from the hospital environment were able to express virulence factors. Therefore, we emphasized the importance of determining the transmission routes in NICU in order to detect pathogen sources and reservoirs, as well as to establish prevention measures, such as adequate disinfection of the environment.

In vitro characterization of virulence factors among species of the Candida parapsilosis complex.

INTRODUCTION: Candida parapsilosis complex species differ from each other with regard to their prevalence and virulence. METHODS: The hydrolytic enzyme activity, biofilm production, and adhesion to epithelial cells were analyzed in 87 C. parapsilosis complex strains. RESULTS: Among the studied isolates, 97.7%, 63.2%, and 82.8% exhibited very strong proteinase, esterase, and hemolysin activity, respectively. All the C. parapsilosis complex isolates produced biofilms and presented an average adherence of 96.0 yeasts/100 epithelial cells. CONCLUSIONS: Our results show that Candida parapsilosis complex isolates showed different levels of enzyme activity, biofilm production, and adhesion to epithelial cells.

In vitro characterization of virulence factors among species of the Candida parapsilosis complex

Abstract INTRODUCTION: Candida parapsilosis complex species differ from each other with regard to their prevalence and virulence. METHODS: The hydrolytic enzyme activity, biofilm production, and adhesion to epithelial cells were analyzed in 87 C. parapsilosis complex strains. RESULTS: Among the studied isolates, 97.7%, 63.2%, and 82.8% exhibited very strong proteinase, esterase, and hemolysin activity, respectively. All the C. parapsilosis complex isolates produced biofilms and presented an average adherence of 96.0 yeasts/100 epithelial cells. CONCLUSIONS: Our results show that Candida parapsilosis complex isolates showed different levels of enzyme activity, biofilm production, and adhesion to epithelial cells.

Frequency, virulence factors and antifungal susceptibility of Candida parapsilosis species complex isolated from patients with candidemia in the central region of Argentina.

PURPOSE: Our objectives were to report species distribution and survival of patients with candidemia in Argentina's central region and to establish the prevalence of C.parapsilosis sensu lato species, their virulence factors and their antifungal susceptibility profiles. METHODS: Yeasts isolated from bloodstream infections in Córdoba (Argentina) (n=35) were molecularly identified. The production of lipase and acid aspartic protease (Sap), the adhesion capacity, and the isolates' ability to form biofilm were evaluated. The in vitro activity of 7 antifungal drugs was evaluated (CLSIdocument M27-4thed). RESULTS: C. albicans was the most prevalent species (48.57%) followed by C. parapsilosis sensu lato (28.57%). The 30-day survival rate for C. albicans candidemia was slightly lower than non-albicans blood infections (50.00% vs. 57.90%). C. parapsilosis sensu stricto and C. orthopsilosis account for 60% and 40% of the cryptic species. Sap production and biofilm formation capacity were higher in C. parapsilosis sensu strico than in C.orthopsilosis. All the strains were susceptible to caspofungin (CAS), anidulafungin (AFG), amphotericin B (AMB), posaconazole (POS) and voriconazole (VRC). Azoles were the most potent agent against C. parapsilosis sensu lato followed by echinocandins and AMB. There were no differences between MICs for fluconazole, VRC, POS and AMB. Contrarily, C. parapsilosis sensu stricto strains showed lower MIC than C. orthopsilopsis isolates for itraconazole and higher MIC values for echinocandins (P<0.01). CONCLUSIONS: We report a high frequency of isolation of C.orthopsilosis in candidemia patients of central region. Data on the prevalence, virulence capability and antifungal susceptibility of C. parapsilosis complex provide new epidemiological information about these cryptic species in Argentina.

Candida parapsilosis isolates from burn wounds can penetrate an acellular dermal matrix.

We isolated and identified yeasts from burn wounds and evaluated the ability of Candida parapsilosis isolates from burn wounds to penetrate an acellular dermal matrix (ADM). A prospective study was conducted with patients from the burn treatment center of North Paraná University Hospital in Londrina, Brazil from February 2015 to January 2016. Yeast cultures were obtained from the tissue of burn wounds that had been debrided and cleansed with 2% chlorhexidine. After identification and confirmation of the purity of the culture, the yeasts were placed on ADM fragments and incubated for three or seven days. During the study period, 273 patients were treated, and 36 of these patients fulfilled the inclusion criteria and provided samples for culture. Yeasts were isolated in 19.44% (n = 7) of the cultures, and the following species were identified: C. parapsilosis (57.1%), C. albicans (28.6%), and C. glabrata (14.3%). C. parapsilosis, the most frequent species, was chosen for the ADM tests. We demonstrated active penetration of the ADM by the yeast isolates from burn wounds. C. parapsilosis grew on ADM and penetrated the matrix, indicating that this yeast, which is common in skin and cutaneous wounds, has the potential to colonize and pass through ADM, a medical device that is frequently used to dress and regenerate burn wounds.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification.

In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Population Structure of Candida parapsilosis: No Genetic Difference Between French and Uruguayan Isolates Using Microsatellite Length Polymorphism.

Candida parapsilosis is a human commensal yeast, frequently involved in infection worldwide and especially in neonates. It is the second species responsible for bloodstream infections in Uruguay and the third species in France. We were interested in knowing whether the population structure of isolates responsible for candidemia in France and in Uruguay was different. Genotyping methods based on microsatellite length polymorphism (MLP) have been described and are especially used for investigation of local outbreaks. We therefore determined the genotypes of 159 C. parapsilosis isolates recovered from 122 patients (84 French patients from 43 hospitals and 38 Uruguayan patients from 10 hospitals) using three microsatellites markers previously described. Our results confirmed that C. parapsilosis population has a high genetic diversity, clonal inheritance and that majority of patients were infected by a single isolate. But we described recurrent infections due to related or unrelated genotypes resulting from isolates harboring loss or gain of heterozygosity. We also described three cases of coinfections due to unrelated genotypes. We did not uncover geographic specificity but observed two linked genotypes that seem to be associated with voriconazole resistance. Finally, among eight isolates involved in grouped cases, the genotypes were similar in six cases supporting the hypothesis of inter-patient transmission. These results confirmed the usefulness of performing MLP genotyping analysis for grouped cases of C. parapsilosis isolates in order to reinforce preventive hygiene measures.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.