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...

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Aims:  Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. Methods and Results:  Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. Conclusion:  A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. Significance and Impact of the Study:  The DMMB–resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.

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Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H2O2) and stabilized H2O2 combination products. Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate‐based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H2O2 combined with silver possessed a higher bactericidal and fungicidal activity compared to native H2O2 with and without glycerol. Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H2O2 combined with silver was clearly a more potent disinfectant compared to H2O2 in killing bacteria and fungi. Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost‐effective alternative to the more laborious PCCT reference method. H2O2 with silver may replace native H2O2 to increase overall disinfection efficiency.

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