(714g) Elucidating Material-Dependent Bacterial Surface Adhesion Using CRISPR Interference and Translation to a Clinical Uropathogen

Biofilms are multicellular communities that enable survival in harsh environments and can form on both abiotic and biotic surfaces. However, biofilm formation also poses a significant threat to human health by contributing to antibiotic resistance and biofilm-associated infections, often due to bacterial adhesion on surfaces of implantable medical devices. Here, we sought to investigate the interplay between how differential gene regulation can control bacterial adhesion to diverse biomaterial surfaces, using a model silicone, polydimethylsiloxane (PDMS), and polyethylene glycol dimethacrylate (PEG) hydrogel. For clinical relevance, we studied the adhesion of clinical uropathogenic Escherichia coli (using the UPEC CFT073 strain), which are a leading cause of catheter-associated urinary tract infections. By silencing the expression of genes associated with biofilm formation and adhesion, we aimed to determine how surface chemical properties, particularly hydrophobicity, interact with genotypic regulation to influence adhesion. We designed a custom CRISPR interference (CRISPRi) system to strongly silence gene repression in the UPEC CFT073 strain, utilizing gRNA multiplexing. From characterized libraries, we demonstrate 17 total genes that significantly control adhesion to these biomaterials. Of these, seven gene targets were selected based on previously classification of biofilm-associated genes for laboratory E. coli strains and eleven additional genes were identified from our prior genome-wide CRISPRi adhesion screen in E. coli MG1655 for these biomaterials, which are novel genes. Highlights from this work are the observed contrasting adhesion phenotypic effects that can result for adhesion to each biomaterial, emphasizing the material-dependent nature of bacterial adhesion, and the potential for multiplexing to enhance the magnitude of effect on adhesion. In this work, we developed a highly multiplexed CRISPR array with up to 12 gRNA in a single array, which is among the largest reported to our knowledge, and studied the effects of multiple gene silencing and identity of the selected gene set. Repression of the reported biofilm-associated genes resulted in reduced adhesion on both PEG and PDMS, with only one exception for one biomaterial. In contrast, silencing genes identified from genome-wide CRISPRi screens revealed distinct adhesion behaviors on the two surfaces, evidenced by a weak correlation between adhesion to PEG and PDMS for gene repression in CFT073. These findings suggest that specific gene functions influence bacterial adhesion in a surface-dependent manner. However, adhesion trends in CFT073 were generally consistent with those observed in MG1655 for silencing of each gene showing strong correlations between adhesion for each strain. Together, this study provides new insights into the genetic basis of bacterial adhesion, highlighting the importance of genotypic contributions in bacteria-material interactions and offering a framework for translating genomic tools to clinical bacteria.