Biofilms are surface-attached aggregates of microbes embedded in extracellular polymeric substances (EPS) produced by attached cells. Found ubiquitously in aqueous environments, biofilms are up to 1,000 times more resistant to antimicrobial agents than planktonic (free-swimming) bacteria of the same genotype. These multicellular structures are involved in 80% of bacterial infections in humans and play an important role in the spread of antimicrobial resistance due to biofilm-associated horizontal gene transfer. Initial attachment is the first step of biofilm formation and plays a critical role in biofilm development. This process can be significantly affected by the properties of the substratum material. For example, surfaces with certain roughness on micrometer, submicrometer, and nanometer length scale (comparable with or smaller than bacterial cells) have been found to affect bacterial adhesion. However, how bacteria respond to these topographic features is not well understood.
To better understand the effect of micron scale surface topography on the early-stage biofilm formation and the underlying mechanism, polydimethylsiloxane (PDMS) surfaces with 5 μm tall line patterns and systematically varied pattern width (5, 10, and 20 μm) and inter-pattern distance (3, 5, 10, and 20 μm) were prepared via soft lithography. By following the adhesion of Escherichia coli RP437/pRSH103 on these patterned surfaces, we obtained results revealing that surface topography can affect the orientation and cluster formation of bacterial cells during the early-stage biofilm formation. Interestingly, the attached E. coli cells were found to preferentially align perpendicularly to the line patterns when the pattern width got narrower (5 μm); and the mutants of flagella, flagellar stator, and fimbriae genes exhibited defects in such adjustment. In addition, cell attachment and cluster formation on the protruding line patterns were found to increase with pattern width. For example, compared to the smooth PDMS, surfaces with 5 μm wide patterns and 3 μm inter-pattern distance reduced biofilm surface coverage by 5-fold and cell cluster formation by 10-fold. Collectively, these findings provided new information about the effects of surface topography on bacterial adhesion and following formation of multicellular biofilms.