Antibiotic resistance is one of the greatest crises in human medicine. Increased incidents of antibiotic resistance are linked to clinical overuse and overreliance on antibiotics. Among the ESKAPE pathogens, Acinetobacter baumannii, especially carbapenem-resistant isolates, has emerged as a significant threat in the context of blood, urinary tract, lung, and wound infections. Therefore, new approaches that limit the emergence of antibiotic resistant A. baumannii are urgently needed. Recently, we have shown that random peptide mixtures (RPMs) are an attractive alternative class of drugs to antibiotics with strong safety and pharmacokinetic profiles. RPMs are antimicrobial peptide mixtures produced by incorporating two amino acids at each coupling step, rendering them extremely diverse but still defined in their overall composition, chain length, and stereochemistry. The extreme diversity of RPMs may prevent bacteria from evolving resistance rapidly. Here, we demonstrated that RPMs rapidly and efficiently kill different strains of A. baumannii, inhibit biofilm formation, and disrupt mature biofilms. Importantly, RPMs attenuated bacterial burden in mouse models of acute pneumonia and soft tissue infection and significantly reduced mouse mortality during sepsis. Collectively, our results demonstrate RPMs have the potential to be used as powerful therapeutics against antibiotic-resistant A. baumannii.

Antibiotic resistance is a daunting challenge in modern medicine, and novel approaches that minimize the emergence of resistant pathogens are desperately needed. Antimicrobial peptides are newer therapeutics that attempt to do this; however, they fall short because of low to moderate antimicrobial activity, low protease stability, susceptibility to resistance development, and high cost of production. The recently developed random peptide mixtures (RPMs) are promising alternatives. RPMs are synthesized by incorporating a defined proportion of two amino acids at each coupling step rather than just one, making them highly variable but still defined in their overall composition, chain length, and stereochemistry. Because RPMs have extreme diversity, it is unlikely that bacteria would be capable of rapidly evolving resistance. However, their efficacy against pathogens in animal models of human infectious diseases remained uncharacterized. Here, we demonstrated that RPMs have strong safety and pharmacokinetic profiles. RPMs rapidly killed both Pseudomonas aeruginosa and Staphylococcus aureus efficiently and disrupted preformed biofilms by both pathogens. Importantly, RPMs were efficacious against both pathogens in mouse models of bacteremia and acute pneumonia. Our results demonstrate that RPMs are potent broad-spectrum therapeutics against antibiotic-resistant pathogens.

The immune system plays a critical role in protecting the host against pathogens, including bacteria, viruses, and parasites. However, pathogens have evolved mechanisms to evade the immune system, for example by altering their surface proteins or by producing enzymes that can interfere with the immune response. These evasion strategies enable pathogens to escape detection and destruction by the immune system, which allows them to establish serious infections. Thus, there is a critical need for new strategies for developing antimicrobial agents. Here, we describe a novel strategy for targeting pathogens, by labeling them with a general peptide functioning as a bacterial binder, conjugated to a protein tag recognizable by the complement system, thereby activating the immune system against the target pathogen. To that end, we screened several pathogenic bacteria to find complement-resistant bacterial strain. A selected peptide binder was crosslinked with the C3b complement protein using glutaraldehyde. We show by an ELISA assay that the resulting complex binds the C5 complement protein with high affinity. We posited that by binding C5, this complex will be capable of initiating the alternative complement downstream proteolytic cascade, thereby inducing the formation of the membrane attack complex. Using this methodology, we were able to eradicate 90% of complement-resistant E. coli bacterial cells. By showing enhancement of complement sensitivity in complement-resistant pathogens, this work demonstrates the basis for new therapeutic approach capable of targeting pathogenic bacteria and activating the immune system against them.