The treatment of bacterial diseases is facing twin threats, with increasing bacterial antibiotic resistance and relatively few novel compounds or strategies under development or entering the clinic. Bacteria frequently grow on surfaces as biofilm communities encased in a polymeric matrix. The biofilm mode of growth is associated with 65 to 80% of all clinical infections. It causes broad adaptive changes; biofilm bacteria are especially (10- to 1,000-fold) resistant to conventional antibiotics and to date no antimicrobials have been developed specifically to treat biofilms. Small synthetic peptides with broad-spectrum antibiofilm activity represent a novel approach to treat biofilm-related infections. Recent developments have provided evidence that these peptides can inhibit even developed biofilms, kill multiple bacterial species in biofilms (including the ESKAPE [ Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species] pathogens), show strong synergy with several antibiotics, and act by targeting a universal stress response in bacteria. Thus, these peptides represent a promising alternative treatment to conventional antibiotics and work effectively in animal models of biofilm-associated infections.

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. There has been enormous publicity about the inexorable rise of resistance and the dearth of new therapies. However less attention has been placed on adaptively multidrug-resistant high density bacterial infections for which antibiotics are highly used but no effective therapies currently exist. Here we have provided new hope for this previously intractable class of infections as typified by abscess infections that are responsible for 3.2 million annual emergency room visits in the US alone. We show how to enhance the activity of antibiotics to treat multidrug-resistant Gram-positive and Gram-negative bacteria, using peptides that target the bacterial stress response, persister-based resistance and the outer membrane permeability barrier. In particular we have employed a new bacterial subcutaneous abscess mouse model to demonstrate that: (a) 7 of the society’s most recalcitrant pathogens formed cutaneous abscesses and even when antibiotics were directly delivered into abscess tissues, they showed poor efficacy; (b) By combining antibiotics with the local administration of anti-biofilm peptides that target cellular (stringent) stress responses, we could pharmacologically treat the infection and reduce the severity of cutaneous abscesses; (c) This synergy was due to increased outer membrane permeability as well as the disruption of the conserved stringent stress response that controls virulence and antibiotic resistance, particularly due to so-called persisters. These peptides have therefore the potential to broaden our limited antibiotic arsenal for a group of extremely difficult to treat infections.

The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii . Here, we investigated the mechanism of action of these molecules in A. baumannii . We identified two novel TonB-dependent receptors, termed Ab -PiuA and Ab -PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii , forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.