Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.
Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.
The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC 50 (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 mg ml "1 . For the three species tested, exposure to baicalein at the MIC 50 concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC 50 (fractional inhibitory concentration index50.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC 50 produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections. INTRODUCTIONCandidiasis is one of the most common fungal infections in humans caused by Candida species, most notably by Candida albicans. Currently, other species of this genus have been found to cause an increasing number of cases of candidiasis (Mokaddas et al., 2007;Pereira et al., 2010;Das et al., 2011).The therapy of invasive candidiasis remains a difficult medical problem. Despite the availability of extendedspectrum triazoles, the incidence of invasive infections and resistance to antifungal therapy continue to increase (Pfaller & Diekema, 2010). Widespread use of antifungal agents could be an explanation for the emergence of the more resistant non-albicans species of Candida, such as Candida glabrata (reviewed by Kothavade et al., 2010; Silva et al., 2012).The azole fluconazole is effective against most Candida species and is used currently as a first-line drug, although different degrees of susceptibility among species have been described. For instance, C. glabrata is inherently less susceptible to fluconazole. In addition, the emergence of fluconazole resistance has been reported in species typically susceptible to this agent, such as C. albicans, Candida tropicalis and Candida parapsilosis (Hajjeh et al., 2004;Yang et al., 2004;Lyon et al., 2010;Oxman et al., 2010; Arendrup et al., 2011), including that observed in Brazilian tertiarycare hospitals (Bruder-Nascimento et al., 2010;Favalessa et al., 2010;Pereira et al., 2010;Furlaneto et al., 2011).These factors present an urgent need to search for novel compounds with anticandidal activity. To this end, efforts have been made in the search for novel antifungal agents from various natural sources. In this regard, baicalein (5,6,7-trihydroxyflavone; Fig. 1) is a flavonoid originally isolated from the roots of the Chine...
In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.
The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a tertiary hospital in South Brazil and the in vitro antifungal susceptibility of isolates. Blood strains from 108 patients were identified by PCR-based method. Some 30.5 % of candidemia were caused by Candida tropicalis, 28.7 % were due to Candida albicans, 24.1 % with Candida parapsilosis sensu stricto, 8.3 % with Candida glabrata sensu lato, 1.8 % involved Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit settings (66 %). In vitro susceptibility to antifungal drugs was determined by a microdilution method; and new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for fluconazole, 98 % for voriconazole and 99 % for amphotericin B. Fluconazole resistance was mostly among C. parapsilosis sensu stricto isolates (26.9 %). Most of the findings reported here agreed with epidemiological features common to other tertiary hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis associated with candidemia. Besides, high rate of fluconazole resistance among C. parapsilosis stricto sensu isolates was obtained when applying the new species-specific clinical breakpoints.
The aim of this research was to study the incidence of antibiotic resistance in 56 Enterococcus strains isolated from dairy products. The identification of enterococci was detected by polymerase chain reaction (PCR) using specific primers to E. faecalis, E. faecium, E. gallinarum and E. casseliflavus, and antibiotic resistance was tested by the disk diffusion method. The most prevalent species was E. faecium with a rate of 58.33%, followed by 27.77% E. faecalis, 11.11% E. casseliflavus and 2.7% E. gallinarum. Distribution of resistance was found in different species. All isolates were susceptible to chloramphenicol, ampicillin, imipenem and amoxicillin/clavulanic acid. In addition, isolates resistant to tetracyclin, nalidixic acid, amikacin, erythromycin, vancomycin and cephalothin were detected. A total of 66.6% of E. faecium and 58.3% of E. faecalis strain were resistant to multiple drugs. The van(A) gene was detected in 100% of vancomycin resistant enterococci. Considering the results of our study, dairy enterococci can be considered a potential source for dissemination of antibiotic resistances.
Most cases of fungal bloodstream infections (BIs) are attributed to Candida albicans; however, non-Candida albicans Candida species have recently been identified as common pathogens. Although hemolytic factor is known to be putative virulence factor contributing to pathogenicity in Candida species, its production is poorly evaluated. The present study was undertaken to analyze the production of hemolytic factor by C. albicans (10), C. tropicalis (13), and C. parapsilosis (8) isolates associated with BIs. Data of hemolysis zones on plate assay revealed that the majority of C. albicans isolates produced mild hemolytic activity whereas the majority of C. tropicalis produced strong activity. None of the tested C. parapsilosis isolates exhibited hemolysis on plate assay. We also evaluated the hemolytic activity in the cell-free broth. There were no significant differences (P > 0.05) in the secreted hemolytic activity among intra-species isolates. Different levels of secreted hemolytic factor were observed for Candida species, where C. tropicalis exhibited the highest production of hemolytic factor (P < 0.05) followed by C. albicans and C. parapsilosis. Inhibition of hemolysis (up to 89.12 %) from culture supernatant, following incubation with the lectin Concanavalin A (Con A), was observed for all three Candida species. This finding suggests that the secreted hemolytic factor of C. tropicalis and C. parapsilosis may be a mannoprotein, similar to that described for C. albicans.
Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.
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