Rafał Kolenda

Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH , ffp , pilV , spnT, and yggT, affected motility, adhesion, or antibiotic stress. Δ flgH exhibited 80% decrease and Δ yggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. Δ flgH lost motility and was regenerated when complemented, whereas Δ ffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δ ffp and Δ spnT were more resistant and protected the bacteria, with Δ spnT showing the best clump formation in terms of ampicillin stress protection. Δ yggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH , ffp , pilV , spnT , and yggT, and respective complementations in trans cemented their roles in multiple capacities.