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 are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

Vulvovaginal candidiasis (VVC) is an infection caused by Candida species that affects millions of women every year. Although Candida albicans is the main cause of VVC, the identification of non-Candida albicans Candida (NCAC) species, especially Candida glabrata, as the cause of this infection, appears to be increasing. The development of VVC is usually attributed to the disturbance of the balance between Candida vaginal colonization and host environment by physiological or nonphysiological changes. Several host-related and behavioral risk factors have been proposed as predisposing factors for VVC. Host-related factors include pregnancy, hormone replacement, uncontrolled diabetes, immunosuppression, antibiotics, glucocorticoids use and genetic predispositions. Behavioral risk factors include use of oral contraceptives, intrauterine device, spermicides and condoms and some habits of hygiene, clothing and sexual practices. Despite a growing list of recognized risk factors, much remains to be elucidated as the role of host versus microorganisms, in inducing VVC and its recurrence. Thus, this review provides information about the current state of knowledge on the risk factors that predispose to VVC, also including a revision of the epidemiology and microbiology of VVC, as well as of Candida virulence factors associated with vaginal pathogenicity.

SUMMARYSoon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans).

Most cases of candidosis have been attributed to Candida albicans, but recently non-C. albicans Candida species have been identified as frequent human pathogens. Candida pathogenicity has been attributed to several factors, including adhesion to medical devices and/or host cells, biofilm formation, and secretion of hydrolytic enzymes (proteases, phospholipases and haemolysins). Although 'new'Candida species are emerging, there is still a lack of information about their pathogenicity. This review discusses recent advances in our knowledge of Candida glabrata, Candida parapsilosis and Candida tropicalis virulence factors, specifically those of adhesion and biofilm formation, which are key components in Candida pathogenicity.

Staphylococcus epidermidis is now well established as a major nosocomial pathogen associated with infections of indwelling medical devices. The major virulence factor of these organisms is their ability to adhere to devices and form biofilms. However, it has not been established that adherence and biofilm formation are closely linked phenotypes for clinical isolates. In this study, the initial adhesion to different materials (acrylic and glass) of 9 clinical isolates of S. epidermidis, along with biofilmpositive and biofilm-negative control strains, was assayed using physico-chemical interactions to analyze the basis for bacterial adherence to the substratum. X-ray photo electron spectroscopy (XPS) analysis of the cell surface elemental composition was also performed in an attempt to find a relationship between chemical composition and adhesion capabilities. Biofilm formation on the two surfaces was evaluated by dry weight measurements. Human erythrocytes were used to evaluate the ability of S. epidermidis strains to cause hemagglutination, an indicator of the production of a poly-N -acetyl glucosamine cell surface polysaccharide also involved in biofilm formation. The clinical isolates exhibited different cell wall physico-chemical properties, resulting in differing abilities to adhere to surfaces. Adhesion to hydrophobic substrata for all strains occurred to a greater extent than that to hydrophilic surfaces. Bacterial cell hydrophobicity seemed to have little or no influence on adhesion. X-ray photoelectron spectroscopy analysis showed a high ratio of oxygen/carbon for all strains, which is a common characteristic of S. epidermidis species. No relevant relationship was found between XPS data and adhesion values. All strains forming biofilms were able to agglutinate erythrocytes. However, no direct relationship was found between the amount of biofilm formed and the initial adhesion extent. These results indicate that high levels of initial adherence do not necessarily lead to thick biofilm formation. These two aspects of the pathogenesis of medical device related-infection may need to be evaluated independently to ascertain the contribution of each to the virulence of S. epidermidis causing device-related infections.