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With the antibiotic development pipeline running dry, many fear that we might soon run out of treatment options. High-density infections are particularly difficult to treat due to their adaptive multidrug-resistance and currently there are no therapies that adequately address this important issue. Here, a large-scale in vivo study was performed to enhance the activity of antibiotics to treat high-density infections caused by multidrug-resistant Gram-positive and Gram-negative bacteria. It was shown that synthetic peptides can be used in conjunction with the antibiotics ciprofloxacin, meropenem, erythromycin, gentamicin, and vancomycin to improve the treatment outcome of murine cutaneous abscesses caused by clinical hard-to-treat pathogens including all ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae) pathogens and Escherichia coli. Promisingly, combination treatment often showed synergistic effects that significantly reduced abscess sizes and/or improved clearance of bacterial isolates from the infection site, regardless of the antibiotic mode of action. In vitro data suggest that the mechanisms of peptide action in vivo include enhancement of antibiotic penetration and potential disruption of the stringent stress response., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The peptides described here have been filed for patent protection, assigned to REWH’s employer the University of British Columbia, and licenced to ABT Innovations Inc. in which the University of British Columbia and REWH own shares.

Biofilms represent a multicellular growth state of bacteria that are intrinsically resistant to conventional antibiotics. It was recently shown that a synthetic immunomodulatory cationic peptide, 1018 (VRLIVAVRIWRR-NH 2 ), exhibits broad-spectrum antibiofilm activity but the sequence determinants of antibiofilm peptides have not been systematically studied. In the present work, a peptide library consisting of 96 single amino acid substituted variants of 1018 was SPOT-synthesized on cellulose arrays and evaluated against methicillin resistant Staphylococcus aureus (MRSA) biofilms. This dataset was used to establish quantitative structure-activity relationship (QSAR) models relating the antibiofilm activity of these peptides to hundreds of molecular descriptors derived from their sequences. The developed 3D QSAR models then predicted the probability that a peptide would possess antibiofilm activity from a library of 100,000 virtual peptide sequences in silico. A subset of these variants were SPOT-synthesized and their activity assessed, revealing that the QSAR models resulted in ~85% prediction accuracy. Notably, peptide 3002 (ILVRWIRWRIQW-NH 2 ) was identified that exhibited an 8-fold increased antibiofilm potency in vitro compared to 1018 and proved effective in vivo, significantly reducing abscess size in a chronic MRSA mouse infection model. This study demonstrates that QSAR modeling can successfully be used to identify antibiofilm specific peptides with therapeutic potential.

As bacterial biofilms display extreme tolerance to conventional antibiotic treatments, it has become imperative to develop new antibacterial strategies with alternative mechanisms of action. Herein, we report the synthesis of a series of ciprofloxacin-nitroxide conjugates and their corresponding methoxyamine derivatives in high yield. This was achieved by linking various nitroxides or methoxyamines to the secondary amine of the piperazine ring of ciprofloxacin using amide bond coupling. Biological evaluation of the prepared compounds on preformed P. aeruginosa biofilms in flow cells revealed substantial dispersal with ciprofloxacin-nitroxide hybrid 25, and virtually complete killing and removal (94%) of established biofilms in the presence of ciprofloxacin-nitroxide hybrid 27. Compounds 25-28 were shown to be non-toxic in both human embryonic kidney 293 (HEK 293) cells and human muscle rhabdomyosarcoma (RD) cells at concentrations up to 40 μM. Significantly, these hybrids demonstrate the potential of antimicrobial-nitroxide agents to overcome the resistance of biofilms to antimicrobials via stimulation of biofilm dispersal or through direct cell killing., (Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.)

As bacterial biofilms are often refractory to conventional antimicrobials, the need for alternative and/or novel strategies for the treatment of biofilm related infections has become of paramount importance. Herein, we report the synthesis of novel hybrid molecules comprised of two different hindered nitroxides linked to the piperazinyl secondary amine of ciprofloxacin via a tertiary amine linker achieved utilising reductive amination. The corresponding methoxyamine derivatives were prepared alongside their radical-containing counterparts as controls. Subsequent biological evaluation of the hybrid compounds on preformed P. aeruginosa flow cell biofilms divulged significant dispersal and eradication abilities for ciprofloxacin-nitroxide hybrid compound 10 (up to 95% eradication of mature biofilms at 40 μM). Importantly, these hybrids represent the first dual-action antimicrobial-nitroxide agents, which harness the dispersal properties of the nitroxide moiety to circumvent the well-known resistance of biofilms to treatment with antimicrobial agents.

The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1β production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1β suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Host defense (antimicrobial) peptides, produced by all complex organisms, typically contain an abundance of positively charged and hydrophobic amino acid residues. A small synthetic peptide termed innate defense regulator (IDR-)1018 was derived by substantial modification of the bovine neutrophil host defense peptide bactenecin. Here, we review its intriguing properties that include anti-infective, anti-inflammatory, wound healing, and anti-biofilm activities. It was initially developed as an immune modulator with an ability to selectively enhance chemokine production and polarize cellular differentiation while suppressing/balancing the pro-inflammatory response. In this regard, it has demonstrated in vivo activity in murine models including enhancement of wound healing and an ability to protect against Staphylococcus aureus, multidrug resistant Mycobacterium tuberculosis, herpes virus, and inflammatory disorders, including cerebral malaria and neuronal damage in a pre-term birth model. More recently, IDR-1018 was shown, in a broad-spectrum fashion, to selectively target bacterial biofilms, which are adaptively resistant to many antibiotics and represent the most common growth state of bacteria in human infections. Furthermore, IDR-1018 demonstrated synergy with conventional antibiotics to both prevent biofilm formation and treat pre-existing biofilms. These data are consistent with a strong potential as an adjunctive therapy against antibiotic-resistant infections., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Biofilm-related infections account for at least 65% of all human infections, but there are no available antimicrobials that specifically target biofilms. Their elimination by available treatments is inefficient since biofilm cells are between 10- and 1,000-fold more resistant to conventional antibiotics than planktonic cells. Here we describe the synergistic interactions, with different classes of antibiotics, of a recently characterized antibiofilm peptide, 1018, to potently prevent and eradicate bacterial biofilms formed by multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Combinations of peptide 1018 and the antibiotic ceftazidime, ciprofloxacin, imipenem, or tobramycin were synergistic in 50% of assessments and decreased by 2- to 64-fold the concentration of antibiotic required to treat biofilms formed by Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus. Furthermore, in flow cell biofilm studies, combinations of low, subinhibitory levels of the peptide (0.8 μg/ml) and ciprofloxacin (40 ng/ml) decreased dispersal and triggered cell death in mature P. aeruginosa biofilms. In addition, short-term treatments with the peptide in combination with ciprofloxacin prevented biofilm formation and reduced P. aeruginosa PA14 preexisting biofilms. PCR studies indicated that the peptide suppressed the expression of various antibiotic targets in biofilm cells. Thus, treatment with the peptide represents a novel strategy to potentiate antibiotic activity against biofilms formed by multidrug-resistant pathogens., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Summary In many infections, bacteria form surface-associated communities known as biofilms that are substantially more resistant to antibiotics than their planktonic counterparts. Based on the design features of active antibiofilm peptides, we made a series of related 12-amino acid L-, D- and retro-inverso derivatives. Specific D-enantiomeric peptides were the most potent at inhibiting biofilm development and eradicating preformed biofilms of seven species of wild-type and multiply antibiotic-resistant Gram-negative pathogens. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. As shown previously for 1018, these D-amino acid peptides targeted the intracellular stringent response signal (p)ppGpp. The most potent peptides DJK-5 and DJK-6 protected invertebrates from lethal Pseudomonas aeruginosa infections and were considerably more active than a previously described L-amino acid peptide 1018. Thus, the protease-resistant peptides produced here were more effective both in vitro and in vivo. [ABSTRACT FROM AUTHOR]