A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extrapolymeric substances. It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a multifactorial process involving the spatial organization of the biofilm. This review will discuss the mechanisms identified as playing a role in biofilm resistance to disinfectants, as well as novel anti-biofilm strategies that have recently been explored.

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SummaryLeuO, the regulator of leucine biosynthesis operon of Escherichia coli, is involved in the regulation of as yet unspecified genes affecting the stress response and pathogenesis expression. To get insights into the regulatory role(s) of LeuO, Genomic SELEX screening has been performed to identify the whole set of its regulation targets. A total of 140 LeuO-binding sites were identified on the E. coli genome, of which as many as 133 (95%) were found to contain the binding sites of H-NS, the universal silencer of stressresponse genes, supporting the concept that LeuO plays an antagonistic role with anti-silencing activity. Western blot analysis indicated that H-NS predominates in growing phase; however, after prolonged culture for 1 week, H-NS decreased instead LeuO increased, supporting the anti-silencing role of LeuO. In concert with this model, a set of stress-response genes including cryptic chaperone/usher-type fimbriae operons are under the control of antagonistic interplay between LeuO and H-NS. Confocal laser scanning microscopic observation in flow-chambers showed that the mutants lacking leuO and some fimbriae genes are defective in biofilm formation or form altered biofilm architecture. Taken together we propose that LeuO is a major player in antagonistic interplay against the universal silencer H-NS.

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The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter NDmedical) recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the NDmedical strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm). Evidences strongly suggest that the enhanced resistance of the NDmedical strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the NDmedical strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

The biocidal activity of peracetic acid (PAA) and benzalkonium chloride (BAC) on Pseudomonas aeruginosa biofilms was investigated by using a recently developed confocal laser scanning microscopy (CLSM) method that enables the direct and real-time visualization of cell inactivation within the structure. This technique is based on monitoring the loss of fluorescence that corresponds to the leakage of a fluorophore out of cells due to membrane permeabilization by the biocides. Although this approach has previously been used with success with various Gram-positive species, it is not directly applicable to the visualization of Gram-negative strains such as P. aeruginosa, particularly because of limitations regarding fluorescence staining. After adapting the staining procedure to P. aeruginosa, the action of PAA and BAC on the biofilm formed by strain ATCC 15442 was investigated. The results revealed specific inactivation patterns as a function of the mode of action of the biocides. While PAA treatment triggered a uniform loss of fluorescence in the structure, the action of BAC was first localized at the periphery of cell clusters and then gradually spread throughout the biofilm. Visualization of the action of BAC in biofilms formed by three clinical isolates then confirmed the presence of a delay in penetration, showing that diffusion-reaction limitations could provide a major explanation for the resistance of P. aeruginosa biofilms to this biocide. Biochemical analysis suggested a key role for extracellular matrix characteristics in these processes.The control of microbial surface contamination is a major concern in terms of public health. Pseudomonas aeruginosa is a Gram-negative bacterium that is well known to be involved in a large number of human infections (14, 30). Numerous outbreaks have been linked directly to its presence on medical equipment (11,15,16,25). The persistence of this bacterium in the environment can be attributed to its ability to form biofilms that increase its resistance to disinfection treatments. Numerous studies have indeed reported the high resistance of P. aeruginosa biofilms (compared to their planktonic counterparts) to numerous biocides, including chlorine, quaternary ammonium compounds, and aldehydes (5,10,13,26). Although the precise mechanisms underlying this resistance remain unclear, it appears to be a multifactorial process that is primarily related to the physiological and structural characteristics of the biofilm. It is now generally accepted that biofilms constitute heterogeneous structures that group subpopulations with distinct physiological states and resistance phenotypes (28).Data on biocide reactivity within these heterogeneous structures could provide a clearer understanding of the mechanisms involved in biofilm resistance and ultimately facilitate the development of new and more efficient treatments. Recently, a noninvasive technique based on confocal laser scanning microscopy (CLSM) was developed and used to investigate spatial and temporal patterns of antimicrobial...

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SP␤ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SP␤ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. Bacillus subtilis is a nonpathogenic Gram-positive bacterium that can be found in its natural habitats as free cells or associated with surfaces in biofilms. In the soil, B. subtilis strains have been shown to form surface-associated communities on plant tissues that protect them from infection by pathogens (1-3). Because of its ability to resist stress through biofilm or spore formation, the bacterium has been isolated from extreme environments, such as sand deserts, clouds, and the digestive tracts of animals (4 -6). As well as its ubiquitous presence in diverse habitats, B. subtilis has been used extensively in biotechnological applications, such as the production of natto, a traditional Japanese food made of fermented soybeans (7), and the production of industrial enzymes and pharmaceutical proteins (8, 9). As a generally recognized as safe (GRAS) organism, B. subtilis is also used as a biocontrol agent in agriculture and livestock buildings (10 -14) and as a probiotic agent to improve human and animal health by preventing gastrointestinal infections (15,16).In fundamental research, B. subtilis has emerged as the model organism for deciphering the complex genetic regulation involved in the biofilm mode of life of Gram-positive bacteria. The domesticated strain B. subtilis 168 has been widely used to dissect metabolic and cellular processes. However, this strain is not able to form robust pellicles and complex colonies like those of its parental strain, NCIB3610, a descendant of the original Marburg strain that was deposited in 1951 (17)....

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.