In vitro method to study antifungal perfusion in Candida Biofilms

Antimycotic perfusion through Candida biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different Candida species (i.e., Candida albicans, Candida parapsilosis, and Candida krusei) that were developed on microporous filters. Scanning electron microscopy revealed that C. albicans formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with C. parapsilosis and C. krusei, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by C. albicans biofilms in comparison to C. parapsilosis and C. krusei. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in Candida is dependent both on the antimycotic and the Candida species in question, and in clinical terms, these phenomena could contribute to the failure of Candida biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.published_or_final_versio

The role of saliva and serum in Candida albicans biofilm formation on denture acrylic surfaces

The long term effect of either a salivary or a serum pellicle on Candida albicans biofilm formation on denture acrylic surfaces was investigated both by quantifying the ATP (adenosine triphosphate) content of the resultant biofilms and by scanning electron microscopy. When the biofilm formation on saliva-coated acrylic strips was examined, the yeasts initially colonised this surface at a slower rate than the controls although with increasing incubation time, at 72 h, the ATP content was almost ten-fold higher than the protein-free control strips. Ultrastructural studies revealed this to be due to cell aggregation and hyphal emergence, phenomena not observed in the controls. As compared with the control strips, biofilm activity of the serum-coated strips was almost 100-fold greater within 48 h incubation, and scanning electron microscopy revealed multilayer blastospore-blastospore co-adhesion, germ tube, hyphal and pseudohyphal emergence and blastospore-hyphal coadherence. Further immunocytochemical observation revealed that concanavalin-A binding material and fibronectin were involved in biofilm formation on both saliva and serum coated specimens and, in addition, mannan-binding protein and protein-A binding material also contributed to the biofilm formation on serum coated specimens.link_to_OA_fulltex

Identification of differential pharyngeal cytokine profiles during HIV infection

Significantly higher pharyngeal shedding of Epstein-Barr virus (EBV) is observed during HIV infection. Increased EBV shedding in pharynx is not affected even during highly active antiretroviral theyrapy (HAART). EBV positive monocyte populations have been shown to carry EBV to pharyngeal mucosa. Human cytokine profiles are often altered to facilitate herpes virus infection. Thus pharyngeal cytokine profiles may influence EBV reactivation and shedding during HIV infection. Our objective was to compare 37 pharyngeal cytokine profiles of HIV-seropositive patients who were or were not receiving HAART therapy

Fluconazole resistance in Candida albicans is induced by Pseudomonas aeruginosa quorum sensing

Microorganisms employ quorum sensing (QS) mechanisms to communicate with each other within microbial ecosystems. Emerging evidence suggests that intraspecies and interspecies QS plays an important role in antimicrobial resistance in microbial communities. However, the relationship between interkingdom QS and antimicrobial resistance is largely unknown. Here, we demonstrate that interkingdom QS interactions between a bacterium, Pseudomonas aeruginosa and a yeast, Candida albicans, induce the resistance of the latter to a widely used antifungal fluconazole. Phenotypic, transcriptomic, and proteomic analyses reveal that P. aeruginosa’s main QS molecule, N-(3-Oxododecanoyl)-L-homoserine lactone, induces candidal resistance to fluconazole by reversing the antifungal’s effect on the ergosterol biosynthesis pathway. Accessory resistance mechanisms including upregulation of C. albicans drug-efflux, regulation of oxidative stress response, and maintenance of cell membrane integrity, further confirm this phenomenon. These findings demonstrate that P. aeruginosa QS molecules may confer protection to neighboring yeasts against azoles, in turn strengthening their co-existence in hostile polymicrobial infection sites

Candida species exhibit differential in vitro hemolytic activities

A total of 80 Candida isolates representing 14 species were examined for their respective responses to an in vitro hemolytic test. A modification of a previously described plate assay system where the yeasts are incubated on glucose (3%)-enriched sheep blood agar in a carbon dioxide (5%)-rich environment for 48 h was used to evaluate the hemolytic activity. A group of eight Candida species which included Candida albicans (15 isolates), C. dubliniensis (2), C. kefyr (2), C. krusei (4), C. zeylanoides (1), C. glabrata (34), C. tropicalis (5), and C. lusitaniae (2) demonstrated both alpha and beta hemolysis at 48 h postinoculation. Only alpha hemolysis was detectable in four Candida species, viz., C. famata (3), C. guilliermondii (4), C. rugosa (1), and C. utilis (1), while C. parapsilosis (5) and C. pelliculosa (1) failed to demonstrate any hemolytic activity after incubation for 48 h or longer. This is the first study to demonstrate the variable expression profiles of hemolysins by different Candida species.published_or_final_versio

Kandyan home gardens: Faunal repositories in Sri Lanka.

Kandyan home gardens are one of the major designs of sustainable land use systems that have evolved over hundreds of years incorporating high biodiversity and providing a diverse and stable supply of socio-economic products and service benefits to householders in Sri Lanka. A study was carried out in three home gardens in the Kandy district from May to December 2009 with the objectives of (a) identifying spatial distribution of terrestrial vertebrates in different vegetation strata, (b) their diurnal and seasonal variations, (c) plant-animal interactions, (d) nesting habits and (e) diet composition. A total of 93 terrestrial vertebrate species including seven amphibians, 23 mammals, 43 birds, and 20 reptiles were recorded. These comprised 14 endemic species, 71 native, four introduced mammals and two migrant birds. Terrestrial vertebrate assemblages were similar in Home garden 1 and 3. According to the fully-nested ANOVA, statistically significant differences were found in the composition of terrestrial vertebrates and the home gardens (P=0.005<0.05), and their vegetation strata (P=0.005<0.05). However, no statistically significant differences were observed between richness of terrestrial vertebrates and the monsoonal periods (P=0.966>0.05), as well as the time of the day (P=0.775>0.05). A positive relationship was obtained between canopy cover and number of bird nests in all home gardens (rs = +1, α = 0.05).Commensalism was the most frequently recorded relationship in all three home gardens. Overall, the diversity of animals and their interactions were different in the three Kandyan Home Gardens studied. Preventing the degradation of home gardens can be considered as a means of conserving biodiversity. There is scope to develop the Kandyan Home Garden system as an important strategy to conserve biodiversity outside the natural and protected area systems

Impact of routine laboratory culture media on in-vitro biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis

Objectives: This study was aimed to determine the efficacy of four routine laboratory culture media onbiofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis.Methods: A sterile flat bottom 96 well plate was inoculated using 0.5 McFarland equivalent standardcell suspension of P. aeruginosa, S. aureus and E. feacalis and the growth rate of planktonic cells wasquantified by measuring the optical density (OD492) at two hour intervals. Influence of culture mediumon adhesion of bacteria as an initial step of biofilm formation in the presence of four culture media(Nutrient broth (NB), Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth and RPMI 1640) wasquantified using MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5- diphenyltetrazolium bromide) assay after90 minutes adhesion. Biofilms of P. aeruginosa, S. aureus, E. feacalis and their 1:1 mixed biofilmswere developed and the growth was quantified using MTT metabolic activity at 24 hour time intervals.Scanning electron microscopy (SEM) was performed to assess the ultrastructure.Results: On comparing the relative growth of the bacteria in different culture media, the maximumgrowth of all three planktonic cultures was achieved using BHI broth. All mono species and mixedspecies cultures exhibited their maximum adhesion in the presence of RPMI 1640. All biofilm exhibitedthe maximum growth in BHI broth. SEM imaging had shown the enhanced growth of ultrastructure ofthe biofilm with the presence of BHI broth.Conclusions: The maximum planktonic and biofilm growth was achieved with BHI broth. However,bacterial adhesion was enhanced in the presence of RPMI 1640

High dietary diversity is associated with obesity in Sri Lankan adults: An evaluation of three dietary scores.

Background: Dietary diversity is recognized as a key element of a high quality diet. However, diets that offer a greater variety of energy-dense foods could increase food intake and body weight. The aim of this study was to explore association of diet diversity with obesity in Sri Lankan adults. Methods: Six hundred adults aged > 18 years were randomly selected by using multi-stage stratified sample. Dietary intake assessment was undertaken by a 24 hour dietary recall. Three dietary scores, Dietary Diversity Score (DDS), Dietary Diversity Score with Portions (DDSP) and Food Variety Score (FVS) were calculated. Body mass index (BMI) ≥ 25 kg.m−2 is defined as obese and Asian waist circumference cut-offs were used diagnosed abdominal obesity. Results: Mean of DDS for men and women were 6.23 and 6.50 (p=0.06), while DDSP was 3.26 and 3.17 respectively (p=0.24). FVS values were significantly different between men and women 9.55 and 10.24 (p=0.002). Dietary diversity among Sri Lankan adults was significantly associated with gender, residency, ethnicity, education level but not with diabetes status. As dietary scores increased, the percentage consumption was increased in most of food groups except starches. Obese and abdominal obese adults had the highest DDS compared to non-obese groups (p<0.05). With increased dietary diversity the level of BMI, waist circumference and energy consumption was significantly increased in this population. Conclusion: Our data suggests that dietary diversity is positively associated with several socio-demographic characteristics and obesity among Sri Lankan adults. Although high dietary diversity is widely recommended, public health messages should emphasize to improve dietary diversity in selective food items