Only a few, relatively cumbersome animal models enable long-term Gram-negative bacterial infections that mimic human situations, where untreated infections can last for weeks. Here, we describe a simple murine cutaneous abscess model that enables chronic or progressive infections, depending on the subcutaneously injected bacterial strain. In this model, Pseudomonas aeruginosa cystic fibrosis epidemic isolate LESB58 caused localized high-density skin and soft tissue infections and necrotic skin lesions for up to 10 days but did not disseminate in either CD-1 or C57BL/6 mice. The model was adapted for use with four major Gram-negative nosocomial pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli. This model enabled noninvasive imaging and tracking of lux-tagged bacteria, the influx of activated neutrophils, and production of reactive oxygen-nitrogen species at the infection site. Screening antimicrobials against high-density infections showed that local but not intravenous administration of gentamicin, ciprofloxacin, and meropenem significantly but incompletely reduced bacterial counts and superficial tissue dermonecrosis. Bacterial RNA isolated from the abscess tissue revealed that Pseudomonas genes involved in iron uptake, toxin production, surface lipopolysaccharide regulation, adherence, and lipase production were highly upregulated whereas phenazine production and expression of global activator gacA were downregulated. The model was validated for studying virulence using mutants of more-virulent P. aeruginosa strain PA14. Thus, mutants defective in flagella or motility, type III secretion, or siderophore biosynthesis were noninvasive and suppressed dermal necrosis in mice, while a strain with a mutation in the bfiS gene encoding a sensor kinase showed enhanced invasiveness and mortality in mice compared to controls infected with wild-type P. aeruginosa PA14.

Antimicrobial peptides have been the focus of considerable research; however, issues associated with toxicity and aggregation have the potential to limit clinical applications. Here, a derivative of a truncated version of aurein 2.2 (aurein 2.2Δ3), namely peptide 73, was investigated, along with its D-amino acid counterpart (D-73) and a retro-inverso version (RI-73). A version that incorporated a cysteine residue to the C-terminus (73c) was also generated, as this form is required to covalently attach antimicrobial peptides to polymers (e.g., polyethylene glycol (PEG) or hyperbranched polyglycerol (HPG)). The antimicrobial activity of the 73-derived peptides was enhanced 2-to 8-fold, and all the derivatives eradicated preformed Staphylococcus aureus biofilms. Formulation of the peptides with compatible polyethylene glycol (PEG)-modified phospholipid micelles alleviated toxicity toward human cells and reduced aggregation. When evaluated in vivo, the unformulated D-enantiomers aggregated when injected under the skin of mice, but micelle encapsulated peptides were well absorbed. Pegylated micelle formulated peptides were investigated for their potential as therapeutic agents for treating highdensity infections in a murine cutaneous abscess model. Formulated peptide 73 reduced abscess size by 36% and bacterial loads by 2.2-fold compared to the parent peptide aurein 2.2Δ3. Micelle encapsulated peptides 73c and D-73 exhibited superior activity, further reducing abscess sizes by 85% and 63% and lowering bacterial loads by 510-and 9-fold compared to peptide 73.

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Synthetic peptides derived from naturally occurring host defence peptides (HDPs) have garnered significant attention as novel pharmaceuticals, particularly as alternatives to antibiotics and for immunomodulatory applications. One of the barriers to advancing synthetic peptides for therapeutic applications is their tendency to aggregate under specific ionic conditions similar to those they would encounter in vivo. Formulating peptides with biocompatible excipients that prevent solvent‐induced peptide aggregation represents a possible solution to this aggregation issue; however, this strategy has not been systematically explored. In the present work, we describe the screening of various polymeric substances (including hyaluronic acid, carboxymethyl cellulose and hydroxypropyl methyl cellulose) as formulation candidates for HDPs and identified derivatized hyperbranched polyglycerols (dHPGs) as a biocompatible excipient that limits peptide aggregation. Notably, the immunomodulatory activity of a synthetic innate defence regulator peptide, IDR‐1018, formulated with dHPG was retained when evaluated against both peripheral blood mononuclear cells and human bronchiolar epithelial cells in vitro. Further characterization of dHPG polymers revealed that decreasing the negative charge density on the polymer surface potentiated the cytotoxic effects of IDR‐1018, emphasizing the need to optimize this parameter for future peptide delivery formulations. Importantly, the dHPG formulated IDR‐1018 inhibited peptide aggregation in vitro in the presence of mucin as well as in vivo when injected subcutaneously into CD1 mice. This study highlights the potential use of dHPGs as formulation candidates for synthetic HDPs and identifies important considerations regarding their physico‐chemical properties and relevance to immunomodulatory applications.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.