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The recent trend in the early admittance to long-term care facilities (LTCFs) of severely injured patients transferred from general hospitals has given a new dynamic to the incidence of healthcare-associated infections, including biofilm-based infections related to the implant of urinary and intravascular catheters, and the onset of pressure ulcers. Catheter-associated urinary tract infections lead in most of the surveys on LTCFs, approximately 80% of urinary tract infections in these settings being due to the short- or long-term insertion of a urinary catheter. Furthermore, the implantation of intravascular catheters is often responsible for catheter-related bloodstream infections caused by the development of an intraluminal biofilm. Pressure ulcers, frequently occurring in bedridden patients admitted to LTCFs, are also susceptible to infection by biofilm-growing aerobic and anaerobic bacteria, the biofilm formation on the wound being the main reason for its delayed healing.

Photodynamic therapy (PDT) is the application of a photoactive dye followed by irradiation that leads to the death of microbial cells in the presence of oxygen. Its use for controlling biofilms has been documented in many areas, particularly oral care. However, the potential use of PDT in the treatment of chronic wound-associated microbial biofilms has sparked much interest in the field of wound care. The aim of this article is to provide an overview on the effectiveness of PDT on in vitro and in vivo biofilms, their potential application in both the prevention and management of wound biofilm infections and their prospective role in the enhancement of wound healing.

Scanning electron microscopy (SEM) provides useful information on the shape, size, and localization within the biofilm of single bacteria as well as on the steps of biofilm formation process, on bacterial interactions, and on production of extracellular polymeric substances.When biofilms are constituted by microbial species involved in health care-associated infections, information provided by SEM can be fruitfully used not only for basic researches but also for diagnostic purposes.The protocols currently used in our laboratory for biofilm investigation by SEM are reported here. Particularly, the procedures to fix, dehydrate, and metalize in vitro-developed biofilms or ex vivo clinical specimens colonized by biofilm-growing microorganisms are described as well as the advantages of the observation of these samples by field emission scanning electron microscopy.

Probiotics are defined as 'live microorganisms that when administered in adequate amounts confer health benefits on the host', underlining the need of microbial viability and the requirement of a suitable dose to obtain a health benefit. The dose and the administration regimen are critical issues for probiotics either ingested as foods claiming health benefits or used as drugs in clinics. In fact, regulatory authorities demand to guarantee consumers that a probiotic is effective in the recommended conditions of use and responds to its specific claims. Thus, a proper identification of probiotic strain(s), a definition of the amount of microorganisms surviving by the end of the product shelf-life, and a demonstration of their beneficial effects by appropriate human trials are required. The current knowledge on the effective dose of different probiotic strains used for several disorders is here reviewed.

The use of probiotic products, especially for humans, requires an unequivocal taxonomical definition of their microbial content, in order to assign the probiotic effects to well identified and characterized microbial strains. In the absence of this, the labeling of some marketed probiotics may be misleading, both in terms of microbiological contents and possible beneficial effects. Currently, the 'polyphasic taxonomy' based on the integration of phenotypic and genotypic data seems to be the most appropriate approach. In fact, even if phenotypic characters often overlap among genetically different species, the molecular methods alone are frequently not able to establish distinct boundaries among phylogenetically related species. Thus, a valid scheme for the identification of a probiotic strain should be currently based on its morphological, physiological, and biochemical features as well as on aspects of its genetic profile. It is important that the identity of specific probiotic strains appearing on the product label is the result of a carefully selected combination of suitable phenotypic and genotypic analytical methods. Only adoption of such a policy could give the right emphasis to the significance of strain-specificity and thus provide health authorities with accurate tools to better evaluate the health benefits claimed by each probiotic-based product. The most common phenotypic and genotypic methods are briefly reviewed here with the aim of highlighting the suitable techniques which can be used to differentiate among microorganisms of probiotic interest, particularly those claiming beneficial health effects for humans.

The characterization of a broad representative sample of ST131 Escherichia coli isolates from different origins and settings (1991 to 2010) revealed that this clonal group has likely diversified recently and that the expansion of particular variants has probably been favored by the capture of diverse, multidrug-resistant IncFII plasmids (pC15-1a, pEK499, pKF3-140-like). The low ability to adhere and to grow as biofilm that was detected in this study suggests unknown mechanisms for the persistence of this clonal group which need to be further explored.

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Biofilms play a pivotal role in healthcare-associated infections, especially those related to the implant of medical devices, such as intravascular catheters, urinary catheters and orthopaedic implants. This paper reviews the most successful approaches for the control and prevention of these infections as well as promising perspectives for the development of novel devices refractory to microbial adhesion, colonization and biofilm formation.

Objectives: The study was undertaken to investigate vancomycin-resistant (vanA) Enterococcus faecalis isolates carrying aggregation substance (AS) gene(s) for their ability to co-transfer vanA and AS genes., Methods: Six vanA clumping-positive E. faecalis isolates (five human and one food sample) carrying one or more AS genes (prgB, asa1, asa373) were analysed for co-transfer of vanA and AS genes to E. faecalis JH2-2 and Enterococcus faecium 64/3., Results: E. faecalis isolates harboured one or multiple plasmids carrying vanA, one or more AS gene(s) or both. vanA was transferred to JH2-2 (frequencies of 10(-3)-10(-6)) from all donors and to 64/3 (10(- 6)-10(- 8)) only from donors from humans. AS genes were detected in 51/60 (85%) of JH2-2 and in 20/50 (40%) of 64/3 vanA transconjugants (prgB, asa1, asa373 or prgB asa373), of which a total of 53.6% were clumping-positive. The plasmid content of JH2-2 transconjugants from the same donor was either identical to that of the donor or it was completely different, suggesting different mechanisms for co-transfer (location on the same pheromone plasmid, mobilization of vanA plasmids by the pheromone-inducible conjugation system, rearrangement of plasmid content during matings). After induction with pheromones, a marked increase in adhesion to Caco-2 cells was observed in four isolates and in some JH2-2 transconjugants, all clumping-positive., Conclusions: Findings suggest that co-transfer of vanA and AS genes may be a common feature of E. faecalis isolates. Since AS is recognized as a virulence factor, this feature might contribute to the emergence of strains with enhanced ability to cause infection and disease in humans.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.

Background: Catheter-related sepsis is a clinical syndrome characterized by the presence of a catheter-associated infection along with a systemic inflammatory response. The continual increase in the use of central venous catheters (CVCs) has been associated with a substantial risk of infectious complications that prolong the hospital stay and increase costs., Methods: The literature on CVCs was reviewed to determine the incidence of catheterrelated sepsis, its diagnosis, and the role of biofilms in pathogenesis., Results: The European Sepsis Group recently reported that 28% of CVC infections in intensive care unit patients were associated with sepsis, 24% with severe sepsis, and 30% with septic shock. Clinical diagnosis remains difficult. After CVC insertion, the intravascular portion of the device is covered rapidly by a thrombin layer, rich in host-derived proteins, that forms a conditioning film and promotes surface adherence of microbial colonizers. These microorganisms then enter their sessile mode of growth, secreting an exopolysaccharide slime within which organism density is regulated by quorum-sensing molecules. Microorganisms are dispersed in clumps that can become septic emboli. Antiadhesive, antiseptic, and antibiotic coatings of catheters have demonstrated only modest clinical efficacy., Conclusion: Our group is involved in the design and in vitro assessment of new polymeric matrices for controlled release of antimicrobial molecules, and in comparative clinical studies of conventional versus antibiotic-coated or -impregnated catheters.

Bile-resistant bacteria, particularly gram-positive Enterococcus faecalis and Enterococcus faecium, play an important role in biliary stent occlusion, because their sessile mode of growth protects them against host defenses and antimicrobial agents. Twelve E. faecalis and seven E. faecium strains isolated from occluded biliary stents have been investigated for slime production, presence of aggregation substance genes, and ability to adhere to Caco-2 cells. Ten isolates were strong producers of slime, and seven isolates produced clumps when exposed to pheromones of E. faecalis JH2-2 and/or OG1RF. The small E. faecium clumps differed from the large clumps of E. faecalis and were similar to those of E. faecium LS10(pBRG1) carrying a pheromone response plasmid. After induction with pheromones, the adhesion to Caco-2 cells of clumping-positive strains was found to increase from two- to fourfold. Amplicons of the expected size were detected in three clumping-positive and three clumping-negative E. faecalis isolates by using primers (agg) internal to a highly conserved region of the E. faecalis pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to prgB of the E. faecalis plasmid pCF10. The agg/prgB-positive E. faecalis strains were also positive in Southern hybridization experiments with a prgB-specific probe. No PCR products were obtained with the same primers from four clumping-positive isolates (one E. faecalis and three E. faecium strains), which were also Southern hybridization negative. Our results demonstrate that slime production and pheromone response are both present in isolated enterococci, suggesting that clinical strains with these features might have a selective advantage in colonizing biliary stents.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 0.5 mT can induce variations in the expression of cell adhesion molecules (CAMs) in two human osteosarcoma cell lines (MG-63 and Saos-2) was investigated. In particular, the expression of two important integrins, VLA-2, the receptor for collagen, and VLA-5, the receptor for fibronectin, as well as CD44, were examined in both cell lines after these had been exposed for 7 and 14 days to a 50 Hz, 0.5 mT field. Cell surface morphology (scanning electron microscopy), cell growth characteristics (growth curves and cell cycle phase distribution), and cell death (necrosis and apoptosis) were also examined. The results demonstrate that no variations in surface morphology and cell death occurred between control and exposed cells in both MG-63 and Saos-2 cells, while significant changes were noted in cell growth and fibronectin and CD44 expression in MG-63 cells. The results are discussed in view of the important role that CAMs play in controlling various cancer cell functions, particularly proliferation and metastasis., (Copyright 2003 Wiley-Liss, Inc.)

Uropathogenic Escherichia coli strains frequently produce a Rho-activating protein toxin named cytotoxic necrotizing factor type 1 (CNF1). We herein report that CNF1 promotes transcription and release of tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), and IL-8 proinflammatory cytokines and increases the production of reactive oxygen species (ROS) in uroepithelial T24 cells. The antioxidant N-acetyl-L-cysteine counteracts these phenomena, a fact which suggests a role for ROS-mediated signaling in CNF1-induced proinflammatory cytokine production.

To investigate the mode of action of Clostridium perfringens epsilon-toxin, MDCK cells were treated with purified toxin and incubated at 37 degrees C for up to 24h. Exposure to epsilon-toxin caused a time-dependent decrease in cell-cell and cell-substrate interactions. After 30min of treatment retraction of the cell body and the emission of filopodia were detectable in a number of cells. Longer exposure resulted in cell rounding and cell blebbing which reached a maximum after 5h of toxin treatment. A parallel modification in the cytoskeleton was also detected. Actin marginalization and the entanglement of microtubules and intermediate filaments were observed by fluorescence microscopy after 30min of toxin exposure. Functional alterations of the plasma membrane of MDCK cells were assessed by flow cytometry. After 10 or 30min of intoxication an increase in cell volume was detected, indicating an alteration in plasma membrane permeability. These findings provide evidence for cytoskeletal changes and plasma membrane functional alterations in the in vitro cell response to C. perfringens epsilon-toxin.