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.

No abstract

No abstract

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.

Biofilm formation on contact surfaces contributes to persistence of foodborne pathogens all along the food and feed chain. The specific physiological features of bacterial cells embedded in biofilms contribute to their high tolerance to environmental stresses, including the action of antimicrobial compounds. As membrane lipid adaptation is a vital facet of bacterial response when cells are submitted to harsh or unstable conditions, we focused here on membrane fatty acid composition of biofilm cells as compared to their free-growing counterparts. Pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium) were cultivated in planktonic or biofilm states and membrane fatty acid analyses were performed on whole cells in both conditions. The percentage of saturated fatty acids increases in biofilm cells in all cases, with a concomitant decrease of branched-chain fatty acids for Gram-positive bacteria, or with a decrease in the sum of other fatty acids for Gram-negative bacteria. We propose that increased membrane saturation in biofilm cells is an adaptive stress response that allows bacteria to limit exchanges, save energy, and survive. Reprogramming of membrane fluidity in biofilm cells might explain specific biofilm behavior including bacterial recalcitrance to biocide action.

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...

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of didecyldimethyl ammonium bromide (DDAB) was investigated. In this study, the strain was sub‐cultured in a medium with increasing DDAB concentrations. After adaptation, Ps. aeruginosa was able to grow until the DDAB concentration in the medium was about five times greater than the Minimal Inhibitory Concentration. Resistance of cells to the bactericidal activity of DDAB also increased gradually during adaptation. This resistance was dependent on the presence of the biocide, as it quickly decreased when the cells were transferred to medium without biocide. Adapted cells showed changes in membrane fatty acid composition. The modifications mainly affected lauric, β‐hydroxylauric and palmitic acids, and they underlined the implication of the membranes in the cell response to the presence of the biocide. Simple linear regression analysis showed that the membrane fatty acid composition of Ps. aeruginosa played an important part in the resistance mechanisms of cells to the bactericidal activity of DDAB.

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.