Patterned Biofilm Formation Reveals the Maximum Distance for Interaction Between Bacterial Clusters

Bacterial adhesion to a surface and subsequent formation of microcolonies play important roles in biofilm formation, which is a major cause of nosocomial infections and persistent biofouling. Despite the significance, these processes are still poorly understood due to the lack of well-defined surfaces and the heterogeneity in biofilm structure and gene expression. We recently reported that bacterial adhesion and biofilm formation can be confined in specific patterns by bio-inert surface chemistry afforded by micro-contact printed self-assembled monolayers (SAMs) presenting functional groups (Applied and Environmental Microbiology, 2007, 73: 4300-4307; Chemical Communications, 2009: 1207-1209). In this study, we applied such patterned surfaces to identify the maximum distance that allows communication and physical interactions between bacterial colonies.

The non-inhibitory alkanethiol [HS(CH₂)₁₄CH₃] was used to create square patterns with various dimensions including 2x2, 5x5, 10x10, 15x15, 20x20, 30x30, 40x40, and 50x50 μm². The background was filled with the inhibitory alkanethiol [HS(CH₂)₁₁(OCH₂CH₂)₃OH] and the distance between patterns (D) was varied to be 2, 5, 10, 15, 20, 30, 40, and 50 μm, respectively. Escherichia coli RP437 was labeled with the plasmid pRSH103 to constitutively express DsRed-express. Its biofilms formed on patterned surfaces were analyzed with confocal laser scanning microscopy. The biofilm images were then analyzed with the COMSTAT software to quantify surface coverage. Clear biofilm patterns were observed only on surfaces with patterns separated by 10 μm or more of inhibitory SAM, which suggests that biofilms expended from non-resistant patterns to resistant background when D<10 μm. Consistently, a significant decrease in surface coverage was observed when the patterns were separated by 10 μm or more. These data indicate that 10 mm is the maximum distance that allows communication and physical interaction between cell clusters under our experimental condition. This platform opens access to the study of fundamental questions such as how the physiology and gene expression of microbes respond to a restrained and well-defined microenvironment. It is useful for studying microbe-surface and microbe-host interactions and for developing antifouling and anticorrosion strategies.