Biofilms are three-dimensional bacterial communities that adhere to medical devices, exhibiting resistance to antibiotics and immune cells. These infections are challenging to treat, often requiring surgical removal of the device. Enteric bacteria like *Salmonella enterica* and *Escherichia coli* frequently form biofilms, causing bloodstream infections and other complications. Biofilms contribute to antibiotic tolerance and resistance by creating a physical barrier, hindering immune response, and promoting the formation of persister cells. Curli amyloid fibers are a major component of Enterobacteriaceae biofilms, including those of *E. coli* and *Salmonella*. Curli, composed of CsgA and CsgB subunits, facilitates bacterial adhesion and biofilm formation. The amyloid nature of curli, with its cross-beta sheet structure, presents conformational epitopes that can be targeted by antibodies. The human monoclonal antibody 3H3, known for its pan-amyloid binding activity (binding to various amyloid structures), was investigated for its potential to disrupt curli-mediated biofilms. This study aims to determine if 3H3 can inhibit curli polymerization, alter biofilm architecture, and enhance bacterial susceptibility to antibiotics and immune clearance.

Extensive research highlights the challenges posed by bacterial biofilms in healthcare-associated infections. The recalcitrance of biofilms to conventional antibiotic treatment is well-documented, stemming from factors including reduced antibiotic penetration, immune evasion, and the presence of persister cells. The role of curli amyloid fibers in biofilm formation by Enterobacteriaceae has been established, showcasing their contribution to bacterial adhesion, biofilm structure, and protection from host defenses. Previous studies have shown the potential of targeting amyloid structures with antibodies, but the application to prokaryotic amyloids like curli remained largely unexplored. The antibody 3H3, previously shown to bind and inhibit the polymerization of various eukaryotic amyloid proteins, offered a promising candidate for this investigation.

The study employed a multi-faceted approach to evaluate the efficacy of mAb 3H3 against *S. Typhimurium* biofilms. In vitro experiments involved assessing the impact of 3H3 on biofilm formation and architecture using confocal microscopy and crystal violet assays. Biofilm thickness, curli content (Congo Red staining), and bacterial density were quantified. The effects of 3H3 on established biofilms were also investigated. To assess the impact of 3H3 on biofilm permeability, the movement of fluorescent beads within the biofilm matrix was tracked using time-lapse confocal microscopy. The synergistic effect of 3H3 with different classes of antibiotics (ampicillin, ciprofloxacin, and streptomycin) on biofilm eradication was evaluated. In vivo studies utilized a murine catheter implantation model to assess the impact of 3H3 alone or in combination with ampicillin on the clearance of *S. Typhimurium* biofilms from pre-colonized catheters. Macrophage assays were conducted to investigate the role of 3H3 in enhancing the phagocytic uptake of biofilm bacteria and curli fibers by immune cells. The study also included in vitro experiments to investigate the direct effects of 3H3 on curli fibrillization using synthetic CsgA peptides and the Thioflavin T (ThT) assay. Additionally, other human monoclonal antibodies with amyloid binding specificity (4A6, 4G1, and 2C10) were investigated alongside 3H3 to understand the correlation between amyloid binding affinity and biofilm disruption activity. Statistical analysis using Student's t-tests was performed to determine statistical significance.

The study demonstrated that mAb 3H3 significantly inhibits *S. Typhimurium* biofilm formation in vitro. Treatment with 3H3 resulted in thinner, less dense biofilms with reduced curli content. 3H3 also disrupted the architecture of pre-established biofilms, loosening the bacterial association and increasing the release of bacteria into the supernatant. Time-lapse microscopy revealed increased penetration of fluorescent beads into 3H3-treated biofilms, indicating altered permeability. Importantly, 3H3 showed a synergistic effect with ampicillin, ciprofloxacin, and streptomycin in eradicating biofilms in vitro. In vivo studies demonstrated that 3H3 treatment significantly reduced biofilm formation on vascular catheters in mice. Combination therapy with 3H3 and ampicillin resulted in near complete clearance of the catheter-associated biofilms. Macrophage assays showed that 3H3 enhanced the phagocytic uptake of both biofilm bacteria and curli fibers by macrophages. In vitro experiments confirmed 3H3's ability to inhibit curli fibrillization. Among the other monoclonal antibodies tested, 4G1 also showed biofilm disruption activity, correlating with its pan-amyloid binding ability.

The findings of this study strongly support the concept of targeting curli amyloid fibers as a strategy for combating bacterial biofilms. The potent activity of mAb 3H3 against *S. Typhimurium* biofilms, both in vitro and in vivo, highlights the therapeutic potential of pan-amyloid antibodies. The mechanism likely involves disrupting curli polymerization and altering the biofilm matrix structure, making bacteria more susceptible to antibiotics and immune clearance. The synergistic effect observed with antibiotics suggests a combination therapy approach could be highly effective in treating biofilm-associated infections. The enhanced macrophage uptake of biofilm components in the presence of 3H3 further underscores the importance of the immune response in biofilm eradication. The study suggests that targeting pan-amyloid epitopes shared by both prokaryotic and eukaryotic amyloids could offer a broad-spectrum approach to combatting various biofilm infections.

This study demonstrates the effectiveness of the human monoclonal antibody 3H3 in disrupting *Salmonella Typhimurium* biofilms. The antibody's pan-amyloid binding activity, coupled with its synergistic effects with antibiotics and its enhancement of immune clearance, positions it as a promising therapeutic agent for biofilm-related infections. Future research should focus on optimizing antibody formulations and investigating its efficacy against a broader range of bacterial species and biofilm types.

The study focused primarily on *S. Typhimurium*. While the results suggest broad applicability due to the pan-amyloid nature of the antibody, further investigations are needed to confirm its efficacy against other bacterial species and biofilm types. The in vivo model used catheter-associated biofilms, which might not fully represent all biofilm infections. Long-term effects of 3H3 treatment and potential off-target effects require further evaluation.