Biofilms and the rapid evolution of multidrug resistance complicate the treatment of bacterial infections. Antibiofilm agents such as metallic-inorganic nanoparticles or peptides act by exerting antibacterial effects and, hence, do not combat biofilms of antibiotics-resistant strains. In this Letter, we show that the block copolymer DA95B5, dextran- block-poly((3-acrylamidopropyl) trimethylammonium chloride (AMPTMA)- co-butyl methacrylate (BMA)), effectively removes preformed biofilms of various clinically relevant multidrug-resistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE V583), and Enteroccocus faecalis (OG1RF). DA95B5 self-assembles into core-shell nanoparticles with a nonfouling dextran shell and a cationic core. These nanoparticles diffuse into biofilms and attach to bacteria but do not kill them; instead, they promote the gradual dispersal of biofilm bacteria, probably because the solubility of the bacteria-nanoparticle complex is enhanced by the nanoparticle dextran shell. DA95B5, when applied as a solution to a hydrogel pad dressing, shows excellent in vivo MRSA biofilm removal efficacy of 3.6 log reduction in a murine excisional wound model, which is significantly superior to that for vancomycin. Furthermore, DA95B5 has very low in vitro hemolysis and negligible in vivo acute toxicity. This new strategy for biofilm removal (nanoscale bacterial debridement) is orthogonal to conventional rapidly developing resistance traits in bacteria so that it is as effective toward resistant strains as it is toward sensitive strains and may have widespread applications.

The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.

Carbapenem‐resistant Gram‐negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer‐membrane or are excluded by efflux mechanisms. Here, we report a cationic block β‐peptide (PAS8‐b‐PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8‐b‐PDM12 does not only compromise the integrity of the bacterial outer‐membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8‐b‐PDM12 sensitizes carbapenem‐ and colistin‐resistant GNB to multiple antibiotics in vitro and in vivo. The β‐peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α‐peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

It is shown that Gram-type differentiation, an essential tool in microbiology, can now be easily accomplished by using the chain-elongated conjugated oligoelectrolyte COE-S6. Specifically, COE-S6 can be used as a fluorescent membrane probe that distinguishes between Gram-negative and Gram-positive bacteria in a single step. Confocal microscopy of labeled microorganisms and differential scanning calorimetry with phospholipidand lipopolysaccharide-containing model membranes suggest that lipopolysaccharides impede COE-S6 membrane intercalation in Gramnegative bacteria. Both Gram-types within a mixture are discretely labeled by counterstaining COE-S6 with the nonspecific and lipophilic membrane probe FM 4-64. This single-step procedure allows for the in situ visualization of individual Gram-types in complex polymicrobial biofilms. Given that COE-S6 fluorescence intensity increases considerably post intercalation, it can be used for the detection of Gram-positive bacteria by the naked eye. COE-S6 does not inhibit bacterial growth and is simple to use, making it a promising membrane-specific fluorescent probe.

Carbapenem-resistant Gram-negative bacteria (GNB) are heading the list of pathogens for whicha ntibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer-membrane or are excluded by efflux mechanisms.Here,wereport acationic block b-peptide (PAS8-b-PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8-b-PDM12 does not only compromise the integrity of the bacterial outermembrane,italso deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As ar esult, PAS8-b-PDM12 sensitizes carbapenem-and colistin-resistant GNB to multiple antibiotics in vitro and in vivo.The b-peptide allows the perfect alternation of cationic versus hydrophobic side chains,r epresenting as ignificant improvement over previous antimicrobial a-peptides sensitizing agents.T ogether, our results indicate that it is technically possible for as ingle adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group,i ncluding those resistant to last resort antibiotics. Singapore 637551 (Singapore) Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under https://doi.

Two membrane-intercalating conjugated oligoelectrolytes (COEs), namely COE-D8 and COE-S6, were combined to achieve enhanced antimicrobial efficacy. COE-D8 has a shorter molecular length than COE-S6 and is typical of effective antimicrobial COE molecules, presumably due to its prominent membrane disrupting function. In contrast, COE-D6 exhibits lower efficacy against bacteria and lower toxicity toward mammalian cells. Surprisingly, after supplementing 8 μM COE-S6, the minimum inhibitory concentration (MIC) of COE-D8 against methicillin-resistant Staphylococcus aureus (MRSA) was improved 8-fold, from 0.5 μM to 0.063 μM (0.050 μg mL−1). No increased toxicity toward mammalian cells was observed by the combination of COEs, as indicated by cytotoxicity measurements using the 3T3 cell line. Indeed, there is an extended ratio between the half maximal inhibitory concentration based on 3T3 cells to MIC against MRSA from 12 to greater than 256. Biophysical experiments using liposome models suggest that COE-S6 promotes the interactions between COE-D8 and lipid bilayers, which is in agreement with damages of cellular permeability and morphology, as observed by confocal microscopy and scanning electron microscopy. The application of a combined mixture of COEs further demonstrates their promising potential as a new class of antimicrobial agents with high efficacy and selectivity.

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