Rosário Oliveira

Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance

The incidence of infections caused by Candida species (candidosis) has increased considerably over the past three decades, mainly due to the rise of the AIDS epidemic, an increasingly aged population, higher numbers of immunocompromised patients and the more widespread use of indwelling medical devices. Candida albicans is the main cause of candidosis; however, non-C. albicans Candida (NCAC) species such as Candida glabrata, Candida tropicalis and Candida parapsilosis are now frequently identified as human pathogens. The apparent increased emergence of these species as human pathogens can be attributed to improved identification methods and also associated with the degree of diseases of the patients, the interventions that they were subjected and the drugs used. Candida pathogenicity is facilitated by a number of virulence factors, most importantly adherence to host surfaces including medical devices, biofilm formation and secretion of hydrolytic enzymes (e.g. proteases, phospholipases and haemolysins). Furthermore, despite extensive research to identify pathogenic factors in fungi, particularly in C. albicans, relatively little is known about NCAC species. This review provides information on the current state of knowledge on the biology, identification, epidemiology, pathogenicity and antifungal resistance of C. glabrata, C. parapsilosis and C. tropicalis.

Biofilms of non-Candida albicans Candida species: quantification, structure and matrix composition

Most cases of candidiasis have been attributed to C. albicans, but recently, non- Candida albicans Candida (NCAC) species have been identified as common pathogens. The ability of Candida species to form biofilms has important clinical repercussions due to their increased resistance to antifungal therapy and the ability of yeast cells within the biofilms to withstand host immune defenses. Given this clinical importance of the biofilm growth form, the aim of this study was to characterize biofilms produced by three NCAC species, namely C. parapsilosis, C. tropicalis and C. glabrata. The biofilm forming ability of clinical isolates of C. parapsilosis, C. tropicalis and C. glabrata recovered from different sources, was evaluated by crystal violet staining. The structure and morphological characteristics of the biofilms were also assessed by scanning electron microscopy and the biofilm matrix composition analyzed for protein and carbohydrate content. All NCAC species were able to form biofilms although these were less extensive for C. glabrata compared with C. parapsilosis and C. tropicalis. It was evident that C. parapsilosis biofilm production was highly strain dependent, a feature not evident with C. glabrata and C. tropicalis. Scanning electron microscopy revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Candida parapsilosis biofilm matrices had large amounts of carbohydrate with less protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrate and protein. Interestingly, C. glabrata biofilm matrix was high in both protein and carbohydrate content. The present work demonstrates that biofilm forming ability, structure and matrix composition are highly species dependent with additional strain variability occurring with C. parapsilosis.

Exopolymers in bacterial adhesion: interpretation in terms of DLVO and XDLVO theories

Exopolymers have an important role in bacterial adhesion and are associated with irreversible adhesion. Moreover, they can coat surfaces enhancing or avoiding bacterial colonisation. To study the role of exopolymers in the adhesion of bacteria to glass, three mutants of Sphingomonas paucimobilis (which are high (TR), medium (CV) and low (F72) exopolymer producers), were used. The adhesion tests were performed in phosphate saline buffers and in solutions of the exopolymer produced by each mutant. The DLVO theory was able to explain the results in phosphate saline buffers, although this theory could not explain the results obtained in the presence of the exopolymer. The XDLVO theory enabled the interpretation of the results in the presence of the exopolymer, where hydrophobic interactions played an important role. However, polymeric interactions that are not taken into account in these two theories are also expected to be determinant in the adhesion process.

Evaluation of Fenton and ozone-based advanced oxidation processes as mature landfill leachate pre-treatments.

Fenton treatment (Fe(2+)/H(2)O(2)) and different ozone-based Advanced Oxidation Processes (AOPs) (O(3), O(3)/OH(-) and O(3)/H(2)O(2)) were evaluated as pre-treatment of a mature landfill leachate, in order to improve the biodegradability of its recalcitrant organic matter for subsequent biological treatment. With a two-fold diluted leachate, at optimised experimental conditions (initial pH 3, H(2)O(2) to Fe(2+) molar ratio of 3, Fe(2+) dosage of 4 mmol L(-1), and reaction time of 40 min) Fenton treatment removed about 46% of chemical oxygen demand (COD) and increased the five-day biochemical oxygen demand (BOD(5)) to COD ratio (BOD(5)/COD) from 0.01 to 0.15. The highest removal efficiency and biodegradability was achieved by ozone at higher pH values, solely or combined with H(2)O(2). These results confirm the enhanced production of hydroxyl radical under such conditions. After the application for 60 min of ozone at 5.6 g O(3)h(-1), initial pH 7, and 400 mg L(-1) of hydrogen peroxide, COD removal efficiency was 72% and BOD(5)/COD increased from 0.01 to 0.24. An estimation of the operating costs of the AOPs processes investigated revealed that Fe(2+)/H(2)O(2) was the most economical system (8.2 € m(-3)g(-1) of COD removed) to treat the landfill leachate. This economic study, however, should be treated with caution since it does not consider the initial investment, prices at plant scale, maintenance and labour costs.

Comparative Antibody-Mediated Phagocytosis of Staphylococcus epidermidis Cells Grown in a Biofilm or in the Planktonic State

ABSTRACT Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to elaboration of biofilms on medical surfaces that protect the organisms from immune system clearance. Even though leukocytes can penetrate biofilms, they fail to phagocytose and kill bacteria. The properties that make biofilm bacteria resistant to the immune system are not well characterized. In order to better understand the mechanisms of resistance of bacteria in biofilms to the immune system, we evaluated antibody penetration throughout the biofilm and antibody-mediated phagocytic killing of planktonic versus biofilm cells of S. epidermidis by using a rabbit antibody to poly-N-acetylglucosamine (PNAG). These antibodies are opsonic and protect against infection with planktonic cells of PNAG-positive Staphylococcus aureus and S. epidermidis. Antibody to PNAG readily penetrated the biofilm and bound to the same areas in the biofilm as did wheat germ agglutinin, a lectin known to bind to components of staphylococcal biofilms. However, biofilm cells were more resistant to opsonic killing than their planktonic counterparts in spite of producing more PNAG per cell than planktonic cells. Biofilm extracts inhibited opsonic killing mediated by antibody to PNAG, suggesting that the PNAG antigen within the biofilm matrix prevents antibody binding close to the bacterial cell surface, which is needed for efficient opsonic killing. Increased resistance of biofilm cells to opsonic killing mediated by an otherwise protective antibody was due not to a biofilm-specific phenotype but rather to high levels of antigen within the biofilm that prevented bacterial opsonization by the antibody.

Understanding Adhesion: A Means for Preventing Fouling

Adhesion of particulate materials is an important step in the formation of fouling. Because the size of such materials is generally less than 1 μm, the phenomenon can be described in terms of colloid chemistry. Accordingly, the net force of interaction between foulants and the surface has been described in terms of DLVO theory (van der Waals attraction and electrostatic double-layer repulsion). However, those forces are sometimes not sufficient to describe the formation of fouling. Recent works have made it possible to calculate the effect of hydrophobic interactions and steric forces, which can also be taken into account. In aqueous media, the various types of interactions can be strongly affected by the pH, the ionic strength, the type of ions, and the presence of polymeric molecules. The objective of this work is to give a general overview of the basic physicochemical factors playing a role in fouling and to outline some practical aspects related to the theoretical reasoning to help prevent or at least mitigate fouling.

Guidelines to cell engineering for monoclonal antibody production

Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Thus, this review describes the progress made for the improvement of productivity in mammalian expression systems for the high production of mAbs. Advances in the development of mAb-producing cell lines are being made, particularly regarding expression vector design and methods used for transfection, with the intent to create a reproducible methodology. Selection of the most suitable clones is also a critical step that can be improved, by including variables other than the expression level, which is still the common practice. Furthermore, strategies of cell engineering, although still mostly based on trial-and-error experimentation and not in standard protocols, hold great interest to improve cell growth and productivity, as well as product quality in the future. Improvements of the initial steps of the production process would not only result in cells with higher expression ability, but would also speed-up the process development.

Adhesion to and viability of Listeria monocytogenes on food contact surfaces

Listeria monocytogenes is an important pathogen responsible for major outbreaks associated with food products. Adhesion to surfaces leads to significant modifications in cell physiology. The aim of this work was to determine the adhesion ability of 10 isolates of L. monocytogenes to eight materials commonly used in kitchens and to evaluate the viability of the adhered cells. The materials assayed were stainless steel 304, marble, granite, glass, polypropylene from a bowl and from a cutting board, and two kinds of silestone. All L. monocytogenes strains attached to all surfaces, although to different extents. L. monocytogenes adhered most tightly to granite and marble, followed by stainless steel 304, glass, silestones, and finally polypropylene surfaces. Surfaces at the threshold between hydrophobicity and hydrophilicity, with high electron acceptor capability and a regular pattern of roughness, were more prone to attachment. Polypropylene surfaces displayed the highest percentage of viable bacteria (nearly 100%), whereas marble and granite had a lower percentage of cultivable cells, 69.5 and 78.7%, respectively. The lowest percentage of culturable bacteria was found on white silestone (18.5%). These results indicate that there are differences in adhered cell viability on different materials. Cell viability assays are important to better understand the cross-contamination process because only adhered bacteria that remain viable are responsible for postprocess contamination.

Isolation and partial characterization of a biosurfactant produced by Streptococcus thermophilus A

Isolation and characterization of the surface active components from the crude biosurfactant produced by Streptococcus thermophilus A was studied. A fraction rich in glycolipids was obtained by the fractionation of crude biosurfactant using hydrophobic interaction chromatography. Molecular (by Fourier transform infrared spectroscopy) and elemental compositions (by X-ray photoelectron spectroscopy) were determined. Critical micelle concentration achieved was 20 g/l, allowing for a surface tension value of 36 mJ/m(2). Moreover, this glycolipid rich fraction was found to be an anti-adhesive and antimicrobial agent against several bacterial and yeast strains isolated from explanted voice prostheses. Further purification steps should be carefully analyzed as each purification step will increase the costs and decreases the amounts of biosurfactants recovered.

Influence of Biosurfactants from Probiotic Bacteria on Formation of Biofilms on Voice Prostheses

ABSTRACT Biofilms were grown on preconditioned voice prostheses with biosurfactants obtained from probiotic bacteria Lactococcus lactis 53 and Streptococcus thermophilus A in an artificial throat model. Both biosurfactants greatly reduced microbial numbers on prostheses and also induced a decrease in the airflow resistance that occurs on voice prostheses after biofilm formation. This study presents a promising strategy for prolonging the lifespan of voice prostheses.