Microbiome-derived antimicrobial peptides offer therapeutic solutions for the treatment of *Pseudomonas aeruginosa* infections

Microbiomes, particularly the rumen microbiome, represent a largely untapped resource for novel bioactive compounds due to their complexity and diversity. The rumen, a highly competitive environment, harbors microbes that produce antimicrobials, many of which are antimicrobial peptides (AMPs). AMPs are short peptide sequences with broad-spectrum activity against pathogenic bacteria; they can also modulate innate defenses and the inflammatory process. The rise of multidrug-resistant (MDR) bacteria, including the ESKAPE pathogens (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Enterobacter* species), necessitates the development of novel antimicrobials. *P. aeruginosa*, a major cause of nosocomial and wound infections, often forms biofilms that render many antibiotic treatments ineffective. This study investigated the in vitro and in vivo efficacy of four rumen microbiome-derived AMPs—Lynronne 1, Lynronne 2, Lynronne 3, and P15s—against *P. aeruginosa* strains, employing biochemical and ‘omics technologies. Previous work characterized Lynronne 1-3 as effective against MRSA and other pathogens, while P15s was less characterized. This study aimed to provide the pre-clinical data necessary to assess the viability of these AMPs as alternative therapeutics.

The literature extensively documents the challenges posed by multidrug-resistant (MDR) bacteria, particularly the ESKAPE pathogens. *Pseudomonas aeruginosa*, a key member of this group, forms biofilms that significantly hinder treatment. Antimicrobial peptides (AMPs) are emerging as a potential solution, due to their broad-spectrum activity and mechanisms of action that differ from traditional antibiotics. Previous research has explored AMPs derived from various sources, demonstrating their efficacy against diverse bacterial pathogens. However, the rumen microbiome remains a relatively understudied source of novel AMPs. Several studies have highlighted the rumen's potential as a reservoir of bioactive compounds, including AMPs with activity against clinically relevant bacteria like MRSA and *P. aeruginosa*. This study builds upon these previous findings by focusing specifically on the *in vitro* and *in vivo* activity of four specific rumen-derived AMPs against multiple *P. aeruginosa* strains.

The study employed several methods to assess the antimicrobial and therapeutic properties of the four rumen-derived AMPs. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using a modified broth microdilution method. Time-kill kinetics assays assessed the bactericidal activity of the AMPs against *P. aeruginosa* strains PAO1 and LES431. A 96-well plate method was used to evaluate the AMPs' ability to prevent and disrupt biofilm formation. Serial-passage assays determined the potential for AMP resistance development. Cytotoxicity assays evaluated the AMPs' toxicity against human lung cells (IMR90 and BEAS-2B). The therapeutic index (TI) was calculated by comparing the IC50 values with MIC values. Transmission electron microscopy (TEM) visualized the effects of AMPs on bacterial cell morphology. Peptide-lipid interactions were measured using a Langmuir balance. Transcriptomic analysis (RNA-seq) investigated the AMPs' effects on gene expression in PAO1 and LES431. Metabolomic analysis using flow injection electrospray high-resolution mass spectrometry (FIE-HRMS) analyzed the metabolic changes following AMP treatment. Finally, *in vivo* efficacy was evaluated using a *Galleria mellonella* infection model.

All four AMPs (Lynronne 1, 2, 3, and P15s) showed antimicrobial activity against seven clinical *P. aeruginosa* strains, with MICs ranging from 4 to 512 µg/mL. Lynronne 1 demonstrated the lowest MIC values across all strains. Time-kill kinetics revealed that Lynronne 1 and 2 exhibited rapid bactericidal activity against PAO1 and LES431, achieving significant reductions in CFU/mL within the first hour to two hours. P15s showed bactericidal effects against LES431 but not PAO1. All three AMPs significantly inhibited *P. aeruginosa* biofilm formation, but not established biofilms. No resistant mutants were observed in serial passage assays. Cytotoxicity assays revealed that P15s was the least cytotoxic to human lung cells (BEAS-2B and IMR90), followed by Lynronne 2, and then Lynronne 1. The therapeutic index (TI) was highest for P15s and Lynronne 2 and lowest for Lynronne 1. TEM showed that Lynronne 1 caused significant disruption of PAO1 cell morphology, while P15s had less impact. Peptide-lipid interaction studies revealed varying affinities of the AMPs to bacterial and eukaryotic lipids. Transcriptomic and metabolomic analyses showed that the AMPs affected numerous genes and metabolic pathways, primarily those related to bacterial membrane function, energy metabolism and stress response (e.g., arginine metabolism, respiratory chain components). In the *Galleria mellonella* infection model, Lynronne 1 and 2 displayed significant efficacy, leading to 100% survival rates at specific concentrations. P15s also exhibited some efficacy at higher concentrations.

This study demonstrates the potent antimicrobial activity of rumen microbiome-derived AMPs against *P. aeruginosa*. The differences in activity and mechanism of action between the α-helical Lynronne peptides and the β-sheet P15s peptide are noteworthy. The rapid bactericidal activity of Lynronne 1 and 2, coupled with their biofilm inhibition properties and lack of resistance development, make them promising candidates for therapeutic development. P15s, despite slower activity, also shows potential but needs further investigation. The observed changes in gene expression and metabolic profiles suggest multiple cellular targets, potentially explaining the lack of resistance development. The *in vivo* efficacy in the *G. mellonella* model provides further support for the potential of these AMPs as novel therapeutics. The cytotoxic effects of Lynronne 1 highlight the importance of further modification to improve the therapeutic index.

This study reveals the significant therapeutic potential of rumen microbiome-derived AMPs, particularly Lynronne 1 and 2, against *P. aeruginosa* infections. Their rapid bactericidal activity, biofilm inhibition properties, and lack of resistance development are highly promising. Further research should focus on modifying Lynronne 1 to reduce its cytotoxicity and optimizing the dosage and delivery methods for all three AMPs. Exploration of combination therapies with existing antibiotics could also enhance their efficacy against established biofilms. The findings strongly support further development of these AMPs as alternative therapeutics for *P. aeruginosa* infections.

The *in vivo* studies were conducted using a *Galleria mellonella* model, which may not perfectly reflect human physiology. The limited number of *P. aeruginosa* strains studied could restrict the generalizability of the findings. Further studies are needed to evaluate the long-term efficacy and safety of these AMPs in mammalian models before clinical trials can be considered. The mechanisms of action deduced from the 'omics data are based on correlative observations and require further validation.