A biofilm-targeting lipo-peptoid to treat Pseudomonas aeruginosa and Staphylococcus aureus co-infections

Abstract

Antibiotic-resistant bacterial infections are a significant clinical challenge, especially when involving multiple species. Antimicrobial peptides and their synthetic analogues, peptoids, which target bacterial cell membranes as well as intracellular components, offer potential solutions. We evaluated the biological activities of novel peptoids TM11-TM20, which include an additional charged NLys residue, against multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus, both in vitro and in vivo. Building on insights from previously reported compounds TM1-TM10, the lipo-peptoid TM18, which forms self-assembled ellipsoidal micelles, demonstrated potent antimicrobial, anti-biofilm, and anti-abscess activity. Transcriptome sequencing (RNA-seq) revealed that TM18 disrupted gene expression pathways linked to antibiotic resistance and tolerance, and biofilm formation in both pathogens. Under dual-species conditions, TM18 induced overlapping but attenuated transcriptional changes, suggesting a priming effect that enhances bacterial tolerance. In a murine skin infection model, TM18 significantly reduced dermonecrosis and bacterial burden in mono-species infections. When combined with the antibiotic meropenem, they synergistically nearly cleared co-infections. Our findings highlight that TM18 has potential as a novel therapeutic for combating antibiotic-resistant pathogens and associated biofilm-driven tolerance.

Keywords: Abscess model; Biofilms; Host-mimicking conditions; Peptides; Peptoids; Polymicrobial.

Fig. 1

Antibiofilm activities of peptoids against mono-species and dual-species biofilms. Peptoids (31.25 μg/mL) were used to treat mono-species biofilms of (A, B) P. aeruginosa (purple circles), and (C, D) S. aureus (red triangles), as well as (E, F) dual-species biofilms of P. aeruginosa-S. aureus. Biofilms were grown for 20–24 h in DFG in 96-well plates before peptoid treatment, followed by re-incubation for 24 h. (A, C, E) Biofilm mass was quantified by staining with 0.1 % crystal violet and (B, D, F) bacterial survivors were determined on selective agar plates. TM12 data (grey circle/triangle; n = 1) was excluded from statistical analysis due to limited sample availability. Significance levels are indicated as ∗p < 0.05 and ∗∗p < 0.01 based on the Kruskal−Wallis test with Dunn's correction. Dashed line represents the limit of detection. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Structural and SAXS analysis of TM18. A) Chemical structure of TM18. B) SAXS data for TM18 measured at 1 mM, 2 mM, and 4 mM plotted together with the best fit (red line) using an ellipsoidal core shell-micellar model described in [17]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

In vivo activity of peptoid TM18 treatment in a high-density mono-species P. aeruginosa or S. aureus infection model. Female Swiss Webster mice were subcutaneously injected with 2.5 × 10⁷ CFU of (A) P. aeruginosa LESB58, or (B) S. aureus USA300 LAC. One-hour post-infection, mice were treated intra-abscess with 125 μg (5 mg/kg) peptoid or PBS as a vehicle control. After three days, the mice were euthanized. Abscesses were measured, including the area of skin dermonecrosis, and collected for bacterial enumeration. Results are presented as median with whiskers indicating min and max values, or as geometric mean ± geometric SD. Statistical significance is denoted as ∗∗p < 0.01, according to Kruskal−Wallis test with Dunn's correction. Dashed line represents limit of detection.

Fig. 4

Peptoid TM18 synergizes with meropenem against P. aeruginosa and S. aureus co-infections in vivo. Female Swiss Webster mice were subcutaneously injected with 2.5 × 10⁷ CFU of P. aeruginosa LESB58 and S. aureus USA300 LAC. After 1 h, mice were treated intra-abscess with 5 mg/kg of peptoid TM18, meropenem, 5 mg/kg, a combination of peptoid TM18 and meropenem, or vehicle (PBS) control. After three days, mice were euthanized, abscesses were measured and then collected for bacterial enumeration, and (A) skin dermonecrosis area was measured; (B) P. aeruginosa survivors, and (C) S. aureus survivors. Results are displayed as geometric mean ± geometric SD. ∗p < 0.05, ∗∗p < 0.01, according to Kruskal−Wallis test with Dunn's correction. Sum of CFU log reduction obtained for each single treatment and a Mann-Whitney test performed to compare PAE of the individual treatments to the CFU counts obtained by the drug combination ⊕ p =<0.05 is synergistic. Dashed line indicates limit of detection.