Assessment of in vitro biofilm formation and antifungal susceptibility of Candida albicans isolates from vulvovaginal candidiasis

Objectives Vulvovaginal candidiasis (VVC) is an inflammation of the genital mucosa, which mainly affects the vulva and vagina. Candida spp. are considered commensal fungus, however, when there is imbalance in the microbiota or the host immune system is compromised, these can become pathogenic. C. albicans is responsible for most cases of VVC and is able of expressing mechanisms which allow the colonization or infection in the host. These factors related yeasts, including the growth of strains resistant to antifungal agents and virulence attributes (such as biofilm formation) are important in the development of VVC. In this sense, the objective of this study was to evaluate the in vitro biofilm formation and susceptibility to antifungal of C. albicans isolates from patients with vulvovaginal candidiasis. Methods For the study were analyzed 30 clinical isolates of Candida albicans. The clinical isolates were separated in groups of 10 samples of the according to symptoms presented by the patients: asymptomatic (AS), vulvovaginal candidiasis (VVC) and recurrent vulvo- vaginal candidiasis (RVVC). For all isolates were analyzed biofilm formation and minimal inhibitory concentration (MIC) for fluconazole and nystatin. The MIC was performed according to M27-A3 protocol of the Clinical Laboratory Standards Institute. Biofilm forming ability was assessed through quantification of total biomass by crystal violet (CV) staining, performed on 96-well microplates containing a cellular suspension of 1 9 107 cells ml1 and incubated for 24 h at 37°C. Results Antifungal susceptibility testing is showed in table 1. The isolates were tested to the two antifungals. The MIC raging from 0.125 to 2 lg ml1 for fluconazole and 1 to 4 lg ml1 to nystatin. The figure 1 show the quantification of the total biomass. It was evident that all the C. albicans isolates were able to form biofilm, although differences occurred depending on the isolated and consequently the group. Importantly it was noted that, in general, VVC and RVVC groups had similar capacity biofilm formation. On the other hand, these groups had less total biomass (average Abs = 1,091 ` 0.88) compared with AS group (average Abs = 1,521 ` 1.32). Conclusion Although all the samples analyzed are sensitive to anti- fungals tested research of resistant strains is relevant, since recurrences are related to cases of VVC. Nystatin and fluconazole were effective in small concentrations for the isolates analysed. All samples were able to form biofilm and the average of the group of asymptomatic patients greater than the others. Thus, the capacity to form- ing biofilm is an important virulence factor in the persistence of microorganisms in infectious processes and represent an increase in resistance to antifungal and host defense

Genotypic variability and antifungal susceptibility of Candida spp. isolated from hospital surfaces and hands of healthcare professionals

Objectives Candida spp. are responsible for 9095% of hematogenous fungal infections. In Brazil and Latin America, C. albicans is the most common specie, followed by C. parapsilosis and C. tropicalis. Infections caused by Candida spp. may have their origin in exogenous sources, transmitted to patients via contaminated infusions, biomedical devices or even by the hands of the hospital staff members. Molecular biology techniques such as Randomly Amplified Polymorphic DNA (RAPD) can show that the strains found in anatomical sites or abi- otic surfaces have the same pattern genome.Moreover, in the last decades it has been observed increasing the number of yeasts isolated from hospital environment resistant to antifungals. Thus, the aim of this study was to determine the susceptibility to antifungals and intraspecies similarity among isolates of different hospital surfaces and hands of healthcare professionals. Methods The study was conducted with 25 isolates of Candida spp.: 5 strains of C. albicans and 5 strains of C. parapsilosis isolated from hospital surfaces. 5 strains of C. albicans, 5 strains of C. parapsilosis and 5 strains of C. tropicalis isolated from hands of healthcare professionals. Professionals and surfaces belonged to intensive care units. The minimal inhibitory concentration (MIC) was determined to voriconazole (VOR), fluconazole (FLZ), amphotericin B (AMB) and micafungin (MFG) according to M27-A3 of the Clinical and Labora- tory Standards Institute (CLSI). To determine the intra-species similarity, 3 primers were used: P4 (50 -AAGAGCCCGT-30 ), OPA-18 (50AGCTGACCGT30) and OPE-18 (50GGACTGCAGA 30). RAPD pro- files were analyzed using BioNumerics software version 4.6. The study was approved by the Ethics in research involving human subjects, CAAE 0448.0.093.000-11 protocol. Results In relation to susceptibility testing (Table 1), it is important to highlight that C. parapsilosis showed 80% of MFG resistance. C. albicans and C. tropicalis showed reduced susceptibility to VOR, and resistence of the AMB was observed for C. albicans (20%). All amplifi- cations revealed distinct polymorphic bands. Genetic distances between each of the isolates were calculated and cluster analysis was used to generate a dendrogram showing relationships between them. The analysis of all primers showed similarity greater than 80% between strains of hands and hospital surfaces for intraspecies. Conclusion Our work shows that, healthy people and hospital surfaces may be colonized by different species yeast. Furthermore, the strains studied had relative resistance to antifungal drugs most frequently used in clinical practice. Finally, there was a high similarity between samples from hands (hospital staff members) and surfaces, providing an infection risk to susceptible individuals. Healthy people working in hospitals can carry yeasts on their hands with the same potential virulence, and which therefore offer the same risk of infection. This information should be considered when preventive measures are established. Attention to the colonization of hands and surfaces should not be restricted to high-risk units such as NICUs, but should also include other sections of hospitals

Evaluation of propolis and its subproduct as an inhibitor of growth and biofilm formation in vaginal yeast from pregnant women

Objectives The treatment of vulvovaginal candidiasis (VVC) is still unsatisfactory, especially in pregnant women, being promising to the utilization of alternative therapies. Propolis extract solution (PES) has demonstrated antifungal efficacy and low toxicity. In addition, the subproduct of propolis extract solution (SPES) is produced during the process of preparing PES and is usually discarded, but can still sub- mit substances responsible for biological effects, such as the polyphenols, responsible for the therapeutic activity of propolis. SPES have not been investigated or used as an antimicrobial agent. Thus, the objective of the present study was to investigate the effect of PES and SPES on Candida spp. isolated from the vaginal material of pregnant women. Methods Vaginal samples from 291 pregnant women were collected and cultivated for yeasts, which were identified by the classical method and performing susceptibility tests against PES, SPES and conventional antifungal agents. The anti-biofilm effect and cytotoxicity tests of the PES and SPES were evaluated. Results In 38.48% (112/291) of culture was positive for Candida species. There were patients with two different species, being a total of 115 yeasts (82.61% C. albicans; 6.08% C. glabrata; 5.22% C. tropi- calis; 5.22% C. parapsilosis and 0.87% C. krusei). PES and SPES were effective, even against isolates resistant to conventional antifungal (Table 1) and reduced about 25% C. tropicalis biofilm, besides presenting its low toxicity in the concentrations of fungicides. Conclusion Thus, in addition to the PES, SPES can also be a promising alternative treatment, especially in this population

Virulence potential of Candida albicans isolated from oral cavity of patients with chronic renal failure on hemodialysis

Objective In patients with chronic renal failure (PCRF), the frequency of colonization of the oral cavity by yeasts of genus Candida spp. is high compared with healthy individuals. These yeasts have virulence factors that may contribute to the persistence of colonization and the development of these infections. The aim of this study was evaluate aspects of virulence from Candida albicans isolated from oral cavity of PCRF on dialysis. Methods This study was initially conducted with 49 clinical samples of C. albicans. The virulence factors assayed were produce of biofilm, germ tube, determination of adherence in oral epithelial cells and evaluation of resistance to the antimicrobial action of neutrophils and mononuclear cells. Results All isolates were highly efficient in forming biofilms on poly- styrene microplates, where 94% of the samples formed 4 + biofilm. Used as a screening test, of which three isolates were selected with different degrees of ability to form biofilm to assess other indicators of virulence. Overall, the isolates exhibited different characteristics regarding the virulence factors analyzed. It was also observed that the hypophosphorous acid (HOCl), production, one of leading inflammatory mediators with fungicidal action, also varied especially when the neutrophils, and not mononuclear cells, were stimulated with different samples. (Figure 1). Conclusion Therefore, our results indicate that C. albicans, is not only the most common species in the oral cavity of CRFP on dialysis, but also it presents the main virulence attributes, which reinforces the importance of monitoring of these patients towards the prevention of fungal infections

Influence of Laminarin in colonisation process of Candida albicans

Objective Candida albicans is responsible for the majority of cases of vulvovaginal candidiasis (VVC), one of the most important candidal virulence factors is the ability to adhere to host surfaces. Chemotherapies that seek to improve the host immune response are an alterna- tive to control fungal infections. b-glucans are polymeric carbohydrates that have been reported to modulate human inflammatory responses in vitro and in vivo. The aim of this study was to determine the influence of Laminarin (LAM) a b-glucan on C. albicans virulence, namely colonisation of HeLa cells. Methods To assess the role of LAM in the cell colonization process, HeLa cells were previously treated or not with 3 mg mL1 of LAM (b-glucan extracted from Laminarina digitata) for 30 min at 37 °C, 5% CO2. Three clinical isolates (5V, 7V and 9V) obtained from female vaginal secretions and one reference strain (ATCC 90028) were used in the study. These strains were separated according to symptoms presented by the patients. Colonization assays were assessed for 2 h incubation at 37 °C, 5% CO2, with 2x105 HeLa cells mL1 treated or not with LAM and 1x107 yeast mL1 of different clinical isolates of C. albicans. After colonization assays, adherent C. albicans cells were harvested by detaching the cervical cells mono- layer to evaluation of viable cells (colony forming units). Results In this study, LAM significantly decreased the interaction of VVC clinical isolates with Hela cells (Figure 1). For ASS and VVC iso- lates, there was a similar reduction in the number of viable cells during colonization process, approximately one log (P < 0.05). Moreover, RVVC isolate showed a reduction more expressive, approximately two log (P < 0.05). Conclusion The pathogenesis of VVC involves the initial adherence of the yeast to the vaginal mucosa, followed by asymptomatic colonisation, ultimately leading to infection (symptomatic vaginitis). This study was able to show that LAM a b-glucan can negatively modulate the process of interaction between HeLa cells and Candida albicans. These results show that this carbohydrate might be a promising agent for preventing the first contact between yeast and vaginal epithelium, and consequently the development of VVC

Propolis potential activity against Candida tropicalis adhered cells and its biofilms

Objectives: Invasive fungal infections, such as candidiasis, represent a public health problem of major importance, and Candida tropicalis has been highlighted among the main agents of candidiasis. One of the major contributions to C. tropicalis virulence is its versatility in adapting to a variety of different habitats and the formation of surface attached microbial communities known as biofilms. Moreover, from the clinical perspective, the most important feature of Candida biofilms is its role in increasing tolerance to conventional antifungal therapy. This scenario encourages the search for alternative therapies. Natural matrixes, such as propolis, compromise a multitude of bioactive properties, in particular phenolic extracts have evidenced significant antimicrobial properties against a multiple of opportunist invaders, including Candida species. Thus, the main objective of the present work was to evaluate the potential antifungal effect of propolis against Candida tropicalis biofilms. Methods: This study was conducted with four strains of C. tropicalis and one reference strain, from the American Type Culture Collection (ATCC 40042). Biofilm formation were carried out on 96-well microplates containing a cellular suspension of 1x105 cells/mL and incubated for 24 h at 37°C. Pre-formed C. tropicalis biofilms were treated with propolis (ranging from 0.47 to 1.42 mg/ml), during 24 h at 37°C and its effect assessed through quantification of the number of colony forming unit (CFU). Results: It was evident that all C. tropicalis strains tested were able to form biofilm and that propolis was able to reduce around 40% and 50% of the pre-formed biofilm. Moreover, in general the propolis effect was similar among all the C. tropicalis clinical isolates strains Conclusions: These data are promising, since they open important perspectives regarding new antifungal agents, much more effective and safer than the currently available to treat and to prevent C. tropicalis infections

Examination of potential virulence factors of Candida tropicalis clinical isolates from hospitalized patients

Candida tropicalis has been reported to be one of the Candida species which is most likely to cause bloodstream and urinary tract infections in hospitalized patients. Accordingly, the aim of this study was to characterize the virulence of C. tropicalis by assessing antifungal susceptibility and comparing the expression of several virulence factors. This study was conducted with seven isolates of C. tropicalis from urine and blood cultures and from central venous catheter. C. tropicalis ATCC 750 was used as reference strain. Yeasts adhered (2 h) to epithelial cells and silicone and 24 h biofilm biomass were determined by crystal violet staining. Pseudohyphae formation ability was determined after growth in fetal bovine serum. Enzymes production (hemolysins, proteases, phospholipases) was assessed by halo formation on agar plates. Susceptibility to antifungal agents was determined by E-test. Regarding adhesion, it can be highlighted that C. tropicalis strains adhered significantly more to epithelium than to silicone. Furthermore, all C. tropicalis strains were able to form biofilms and to express total hemolytic activity. However, protease was only produced by two isolates from urine and by the isolates from catheter and blood. Moreover, only one C. tropicalis (from catheter) was phospholipase positive. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. Four strains were susceptible-dose dependent to itraconazole and one clinical isolate was found to be resistant

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion