The flagellar mutant exhibited lower biofilm levels compared to the wild type, as reported previously. The level of biofilm formation for the flagellar mutant stayed the same in the presence of PIP and RES, while biofilm levels for the wild type strain increased significantly. This could VE-821 suggest that PIP and RES affect UPEC biofilm formation mainly through interaction with bacterial flagellar protein FliC, or the associated gene. This theory is reinforced by the fact that both alkaloids resulted in a decrease in bacterial motility and a decreased expression of flagellar genes. There is no report in the literature describing how or why alkaloids might result in a decreased expression of flagella-associated genes. It has been suggested that under stress conditions, shutting down the flagellar gene expression is one of the strategies cells adopt to avoid waste of energy for making new flagella. Moreover, it has been reported that inactivation of genes responsible for flagella production also triggers expression of adhesion-associated genes, which is in accordance with our results. Formation of biofilms on urinary catheters significantly hampers the treatment of infection. Biofilms can decrease the access of antimicrobial agents to cells by a number of mechanisms, the most important of which is modulating the penetration of the drugs or degrading drug molecules. Our results indicated that adding antibiotics does not fully disperse biofilms; it can even result in an increase in the ratio of cells that adopted a sessile lifestyle. This effect has been reported before for antibiotics and other stressors where stress caused the bacterial cells to adopt a biofilm lifestyle as a defensive mechanism. Interestingly, adding PIP and RES along with the antibiotics resulted in a decrease in the levels of biofilm, suggesting that the presence of alkaloids decreased the ability of the cells in the biofilm matrix to escape the inhibitory action of antibiotics. Antibiotic penetration studies indicated that PIP significantly increased the penetration of antibiotics through the E. coli biofilm which could partially explain the enhanced antibiotic action in the presence of this alkaloid. The alkaloids PIP and RES used in the present investigation are known bioavailability enhancers. Many studies have reported the physiological effects of black pepper, its extracts, or its major active component, PIP. Furthermore, oral administration of P. longum containing PIP to poultry has been reported to enhance the therapeutic efficacy of the antibiotic oxytetracycline. Our results support the reports in the literature and further present a possible mechanism for the therapeutic effects of PIP, namely enhancing the diffusion of antibiotics in bacterial biofilms. Mutations that interfere with splicing are a frequent cause of human disease. The majority of these mutations affect premRNA splice sites or regulatory elements in cis.