Since the 1960's much research has focused on biofilms, i.e. microbial-derived populations irreversibly attached to a surface and embedded in a self-produced polymeric matrix. In this matrix, microbial cells are protected from detrimental external factors such as heat, UV radiation and the host immune system. The most relevant biofilm-related property is the unusual high resistance to antimicrobial therapy, although the origin of this extreme resistance is still the subject of debate. Besides an overview of the main characteristics of biofilms, this review discusses the different resistance mechanisms that lead to increased biofilm-related morbidity and mortality. Adherent communities are involved in at least 65% of all human bacterial infections, particularly in cystic fibrosis and several nosocomial device- related infections. Even in healthy immunocompetent individuals, biofilm infections are rarely resolved and usually persist until the colonized surface is removed from the body. Fundamental research aiming to develop new anti-biofilm strategies will largely depend on the availability of appropriate in vitro models for production and quantification of biofilms. This review describes the most frequently used in vitro biofilm models with respect to the different pitfalls that can emerge from in vitro biofilm research. Despite extensive efforts, no antimicrobial drug has yet been found that completely eradicates adherent microbial populations. The advantages and disadvantages of the currently available therapies are described with a particular focus on antibiotics and biocides. The options and benefits of future antibiofilm therapies are discussed.

Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides.Microbial communities irreversibly attached to a surface and encapsulated in a self-produced polymeric matrix are known as biofilms. A particular characteristic is their extreme resistance to antimicrobial treatment (6). This resistance is mediated by several mechanisms that can act together: (i) poor penetration or inactivation of antimicrobials in the matrix, (ii) an altered bacterial metabolic state, (iii) the formation of persister cells, and (iv) resistance induced by the antimicrobial itself following the use of sublethal concentrations and the upregulation of efflux pumps (2, 7). Hence, biofilms are hard to eradicate and are claimed to be responsible for up to 60% of all infections in humans (1, 5). Staphylococcus aureus and Pseudomonas aeruginosa are notorious biofilm producers, the first being nosocomial and responsible mainly for medical device-associated infections (13, 28) and the latter being an opportunistic pathogen causing life-threatening infections mainly in cystic fibrosis patients (13,19).Looking at the high biofilm-related morbidity and mortality, the antibiofilm properties of antimicrobials have been studied extensively, with a main focus on the activity of antibiotics (1,17,20,26). However, a recent study indicated that the prospect of using solely antibiotics to achieve complete biofilm destruction is limited, since the biofilm matrix persists (25). As their mechanisms of action are not limited to the bacterial metabolism, biocides should also be considered as valuable...

Longitudinal quantification of free radicals during IRI reveals the occurrence of two major radical bursts. The radical peak in peripheral blood after ischaemia may be related to systemic hypoxia. After 1 h of reperfusion, the lung tissue shows a significant increase of superoxide, NO and their reaction products, which are probably involved in IRI pathogenesis.

The ability of Porphyromonas gingivalis to cause adult periodontitis is determined by its arsenal of virulence factors. Here, we investigated the importance of biofilm formation and bacterial dipeptidyl peptidase IV (DPPIV) for the pathogenicity of clinical P. gingivalis isolates. In our study, the isolates with biofilm-forming capacity also showed high DPPIV activity in vitro. Moreover, DPPIV activity increased in P. gingivalis biofilms compared to planktonic cells. In a murine subcutaneous abscess model, the biofilm-forming isolates with high DPPIV activity proved to be pathogenic, while the nonbiofilm formers with low DPPIV activity did not induce abscesses. The biofilm-forming ATCC 33277 strain with low DPPIV activity was not pathogenic in mice either. Our results suggest that biofilm formation and DPPIV activity contribute to the pathogenic potential of P. gingivalis. Furthermore, we show that biofilm formation may enhance P. gingivalis virulence through an increased DPPIV activity. Because of their importance for bacterial colonization and growth, biofilm formation and DPPIV activity could present interesting therapeutic targets to tackle periodontitis.

Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O(2)(.-) as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)(2)] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O(2)(.-) and NO produced by different types of activated macrophages.

The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.

Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO ® -3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r 2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 10 4 to 10 8 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.

The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.