Escherichia coli repellence via laser-induced periodic surface structures (LIPSS) on titanium: Critical role of biofilm-associated gene expression

• Laser-induced periodic nanotextures on Ti repel Escherichia coli. • In situ monitoring of genetic response via fluorescent reporter protein. • Low spatial frequency LIPSS stimulate biofilm related genes. • High spatial frequency LIPSS and polished surfaces have no biofilm stimulating effect. The inconsistency in the observed bacteria-repellent effects of laser-induced surface textures remains a subject of scientific debates. With E. coli TG1 as test strain, we performed biofilm formation assays on ultrashort pulse laser textured titanium. Samples covered with low spatial frequency LIPSS (LSFL) of periods around 750 nm (LSFL) or with high spatial frequency LIPSS (HSFL) featuring smaller periods around 100 nm (HSFL) were processed on polished samples. While LSFL significantly reduce the number of adhering bacteria compared to the polished reference, HSFL do not. Simultaneously, we concentrate our study on bacterial strategies employed for biofilm formation, that is the transcription of the biofilm-associated genes csgB , involved in primary adhesion to a surface, and wza , participating in biofilm maturation. Promoter regions of these genes were cloned into a promotor probe plasmid with the green fluorescent protein GFP+ as reporter and transferred into the test strain. As indicated by the fluorescence intensity of the reporter protein, among the bacteria colonizing LSFL-covered areas, a markedly higher proportion of cells transcribing csgB or wza were detected, compared to the bacterial populations colonizing the polished or HSFL-covered areas. To our knowledge, this is the first study indicating that the antifouling effect of LSFL topographies can be counteracted when bacteria activate biofilm-associated genes.