Clinicians prescribe hundreds of millions of β-lactam antibiotics to treat the majority of patients presenting with bacterial infections. Patient outcomes are positive unless resistant bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), are present. P. aeruginosa has both intrinsic and acquired antibiotic resistance, making clinical management of infection a real challenge, particularly when these bacteria are sequestered in biofilms. These problems would be alleviated if, upon the initial presentation of bacterial infection symptoms, clinicians were able to administer an antibiotic that kills both susceptible and otherwise resistant bacteria and eradicates biofilms. As the most common class of antibiotics, β-lactams could be used in a new drug if the leading causes of βlactam antibiotic resistance, permeation barriers from lipopolysaccharide, efflux pumps, and βlactamase enzymes, were also defeated. Against P. aeruginosa and their biofilms, the potency of βlactam antibiotics is restored with 600 Da branched polyethylenimine (600 Da BPEI). Checkerboard assays using microtiter plates demonstrate the potentiation of piperacillin, cefepime, Meropenem, and erythromycin antibiotics. Growth curves demonstrate that only a combination of 600 Da BPEI and piperacillin produces growth inhibition antibiotic resistant P. aeruginosa.Scanning electron microscopy (SEM) was used to confirm that the combination treatment leads to abnormal P. aeruginosa morphology. Data collected with isothermal titration calorimetry and fluorescence spectroscopy demonstrate a mechanism of action in which potentiation at low concentrations of 600 Da BPEI reduces diffusion barriers from lipopolysaccharides without disrupting the outer membrane itself. Coupled with the ability to overcome a reduction in antibiotic activity created by biofilm exopolymers, targeting anionic sites on lipopolysaccharides Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.9b00486. Antibiotic susceptibility, MIC and FICI values, checkerboard assays, incubation of PA BAA-47 cells at high cell density with resazurin, uptake of H33342, calcium ions occupying LPS binding sites, an illustration of using excess metal ions to occupy the anionic site of LPS and prevent the binding of 600 Da BPEI, ITC data, illustration of the dye 1-N-phenylnaphthylamine, HPLC chromatogram, mass spectrum of 600 Da BPEI, FTIR spectrum (PDF)

Advances in biotechnology to treat and cure human disease have markedly improved human health and the development of modern societies. However, substantial challenges remain to overcome innate biological factors that thwart the activity and efficacy of pharmaceutical therapeutics. Until recently, the importance of extracellular DNA (eDNA) in biofilms was overlooked. New data reveal its extensive role in biofilm formation, adhesion, and structural integrity. Different approaches to target eDNA as anti-biofilm therapies have been proposed, but eDNA and the corresponding biofilm barriers are still difficult to disrupt. Therefore, more creative approaches to eradicate biofilms are needed. The production of eDNA often originates with the genetic material of bacterial cells through cell lysis.However, genomic DNA and eDNA are not necessarily structurally or compositionally identical. Variations are noteworthy because they dictate important interactions within the biofilm. Interactions between eDNA and biofilm components may as well be exploited as alternative anti-biofilm strategies. In this review, we discuss recent developments in eDNA research, emphasizing potential ways to disrupt biofilms. This review also highlights proteins, exopolysaccharides, and other molecules interacting with eDNA that can serve as anti-biofilm therapeutic targets.Overall, the array of diverse interactions with eDNA is important in biofilm structure, architecture, and stability. K E Y W O R D Santi-biofilm therapies, biofilms, eDNA, eDNA therapy, eDNA-interactions | BACKGROUNDA global health crisis is growing due to antibiotic resistance. Antibiotics were first prescribed to treat severe infections after Alexander Fleming discovered penicillin in the 1940's. Over time, various classes of antibiotics that target different bacterial machineries were introduced to the market. However, in recent years, the rate of discovery of new antibiotic classes has stagnated whereas the rate of antibiotic consumption has continued to increase (Silver, 2011). As a result, antibiotic resistance can emerge as bacteria respond to therapeutic treatments. The Centers for Disease Control and Prevention reports that at least 2.8 million people get infected and at least 35,000 die because of antibiotic resistant bacteria in the United States annually (CDC, 2019). In Europe, an estimated number of 25,000 deaths are associated to antibiotic resistant bacteria ("Annual report of the European Medicines Agency," 2010). Greater than 30,000 deaths per year are reported in countries like Thailand as well as increasing variants of antibiotic resistant bacteria emerging in South America, the Middle East, and Asia (Howell, 2013). In addition to these statistics, neonatal sepsis attributed to antibiotic resistance are increasing in Tanzania and Mozambique, and approximately 58,000 neonatal sepsis deaths related to antibiotic resistance are reported in India (Hellen et al., 2015

Bacterial biofilms, often impenetrable to antibiotic medications, are a leading cause of poor wound healing. The prognosis is worse for wounds with biofilms of antimicrobial-resistant (AMR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant S. epidermidis (MRSE), and multi-drug resistant Pseudomonas aeruginosa (MDR-PA). Resistance hinders initial treatment of standard-of-care antibiotics. The persistence of MRSA, MRSE, and/or MDR-PA often allows acute infections to become chronic wound infections. The water-soluble hydrophilic properties of low-molecular-weight (600 Da) branched polyethylenimine (600 Da BPEI) enable easy drug delivery to directly attack AMR and biofilms in the wound environment as a topical agent for wound treatment. To mitigate toxicity issues, we have modified 600 Da BPEI with polyethylene glycol (PEG) in a straightforward one-step reaction. The PEG–BPEI molecules disable β-lactam resistance in MRSA, MRSE, and MDR-PA while also having the ability to dissolve established biofilms. PEG-BPEI accomplishes these tasks independently, resulting in a multifunction potentiation agent. We envision wound treatment with antibiotics given topically, orally, or intravenously in which external application of PEG–BPEIs disables biofilms and resistance mechanisms. In the absence of a robust pipeline of new drugs, existing drugs and regimens must be re-evaluated as combination(s) with potentiators. The PEGylation of 600 Da BPEI provides new opportunities to meet this goal with a single compound whose multifunction properties are retained while lowering acute toxicity.

Infections from antibiotic‐resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial‐resistant (AMR) pathogens, 600‐Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β‐lactam antibiotics against Gram‐positive bacteria. BPEI binds cell‐wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram‐positive pathogens but not Gram‐negative bacteria. 600‐Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non‐β‐lactam co‐drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL‐8 by human epithelial keratinocytes. This enables BPEI to function as a broad‐spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a serious threat worldwide. MRSA is the predominant species isolated from medical-device-related biofilm infections and chronic wounds. Its ability to form biofilms grants it resistance to almost all antibiotics on the market. Answering the call for alternative treatments, our lab has been investigating the efficacy of 600 Da branched polyethylenimine (BPEI) as a β-lactam potentiator against bacterial biofilms. Our previous study showed promise against methicillin-resistant Staphylococcus epidermidis biofilms. This study extends our previous findings to eradicate a more virulent pathogen: MRSA biofilms. Microtiter minimum biofilm eradication concentration models, crystal violet assays, and electron microscopy images show synergistic effects between BPEI and ampicillin as a two-step mechanism: step one is the removal of the extracellular polymeric substances (EPS) to expose individual bacteria targets, and step two involves electrostatic interaction of BPEI with anionic teichoic acid in the cell wall to potentiate the antibiotic.