Breaking membrane barriers to neutralize E. coli and K. pneumoniae virulence with PEGylated branched polyethylenimine.

Bacterial infections caused by Gram-negative pathogens, such as those in the family Enterobacteriaceae, are among the most difficult to treat because effective therapeutic options are either very limited or non-existent. This raises serious concern regarding the emergence and spread of multi-drug resistant (MDR) pathogens in the community setting; and thus, creates the need for discovery efforts and/or early-stage development of novel therapies for infections. Our work is directed towards branched polyethylenimine (BPEI) modified with polyethylene glycol (PEG) as a strategy for targeting virulence from Gram-negative bacterial pathogens. Here, we neutralize lipopolysaccharide (LPS) as a barrier to the influx of antibiotics. Data demonstrate that the ß-lactam antibiotic oxacillin, generally regarded as ineffective against Gram-negative bacteria, can be potentiated by 600 Da BPEI to kill some Escherichia coli and some Klebsiella pneumoniae. Modification of 600 Da BPEI with polyethylene glycol (PEG) could increase drug safety and improves potentiation activity. The ability to use the Gram-positive agent, oxacillin, against Gram-negative pathogens could expand the capability to deliver effective treatments that simplify, reduce, or eliminate some complicated treatment regimens.

Recent Advances in the Development and Characterization of Electrochemical and Electrical Biosensors for Small Molecule Neurotransmitters.

Neurotransmitters act as chemical messengers, determining human physiological and psychological function, and abnormal levels of neurotransmitters are related to conditions such as Parkinson's and Alzheimer's disease. Biologically and clinically relevant concentrations of neurotransmitters are usually very low (nM), so electrochemical and electronic sensors for neurotransmitter detection play an important role in achieving sensitive and selective detection. Additionally, these sensors have the distinct advantage to potentially be wireless, miniaturized, and multichannel, providing remarkable opportunities for implantable, long-term sensing capabilities unachievable by spectroscopic or chromatographic detection methods. In this article, we will focus on advances in the development and characterization of electrochemical and electronic sensors for neurotransmitters during the last five years, identifying how the field is progressing as well as critical knowledge gaps for sensor researchers.

Eradicating Biofilms of Carbapenem-Resistant Enterobacteriaceae by Simultaneously Dispersing the Biomass and Killing Planktonic Bacteria with PEGylated Branched Polyethyleneimine.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging pathogens that cause variety of severe infections. CRE evade antibiotic treatments because these bacteria produce enzymes that degrade a wide range of antibiotics including carbapenems and ß-lactams. The formation of biofilms aggravates CRE infections, especially in a wound environment. These difficulties lead to persistent infection and non-healing wounds. This creates the need for new compounds to overcome CRE antimicrobial resistance and disrupt biofilms. Recent studies in our lab show that 600 Da branched polyethyleneimine (BPEI) and its derivative PEG350-BPEI can overcome antimicrobial resistance and eradicate biofilms in methicillin-resistant S. aureus, methicillin-resistant S. epidermidis, P. aeruginosa, and E. coli. In this study, the ability of 600 Da BPEI and PEG350-BPEI to eradicate carbapenem-resistant Enterobacteriaceae bacteria and their biofilms is demonstrated. We show that both BPEI and PEG350-BPEI have anti-biofilm efficacy against CRE strains expressing Klebsiella pneumoniae carbapenemases (KPCs) and metallo-ß-lactamases (MBLs), such as New Delhi MBL (NDM-1). Furthermore, our results illustrate that BPEI affects planktonic CRE bacteria by increasing bacterial length and width from the inability to proceed with normal cell division processes. These data demonstrate the multi-functional properties of 600 Da BPEI and PEG350-BPEI to reduce biofilm formation and mitigate virulence in carbapenem-resistant Enterobacteriaceae.

Dimerization of 600 Da branched polyethylenimine improves ß-lactam antibiotic potentiation against antibiotic-resistant Staphylococcus epidermidis and Pseudomonas aeruginosa.

Antibiotic resistance is a growing concern in the medical field. Drug-susceptible infections are often treated with ß-lactam antibiotics, which bind to enzymes known as penicillin-binding proteins (PBPs). When the PBPs are disabled, the integrity of the cell wall is compromised, leading to cell lysis. Resistance renders ß-lactam antibiotics ineffective, and clinicians turn to be more effective, but often more toxic, antibiotics. An alternative approach is combining antibiotics with compounds that disable resistance mechanisms. Previously, we have shown that low-molecular-weight 600 Da branched polyethylenimine restores ß-lactam susceptibility to Gram-positive and Gram-negative pathogens with antibiotic resistance. In this study, this approach is extended to the homodimers of 600 Da BPEI that have improved potentiation properties compared to monomers of 600 Da BPEI and 1200 Da BPEI. The homodimers are synthesized by linking two 600 Da BPEI molecules with methylenebisacrylamide (MBAA). The resulting product was characterized with FTIR spectroscopy, 1 H NMR spectroscopy, checkerboard microbroth dilution assays, and cell toxicity assays. These data show that the 600 Da BPEI homodimer is more effective than 1200 Da BPEI toward the potentiation of oxacillin against methicillin-resistant Staphylococcus epidermidis and the potentiation of piperacillin against Pseudomonas aeruginosa.

Dual-Function Potentiation by PEG-BPEI Restores Activity of Carbapenems and Penicillins against Carbapenem-Resistant Enterobacteriaceae.

The rise of life-threatening carbapenem-resistant Enterobacteriaceae (CRE) infections has become a critical medical threat. Some of the most dangerous CRE bacteria can produce enzymes that degrade a wide range of antibiotics, including carbapenems and ß-lactams. Infections by CRE have a high mortality rate, and survivors can have severe morbidity from treatment with toxic last-resort antibiotics. CRE have mobile genetic elements that transfer resistance genes to other species. These bacteria also circulate throughout the healthcare system. The mobility and spread of CRE need to be curtailed, but these goals are impeded by having few agents that target a limited range of pathogenic CRE species. Against CRE possessing the metallo-ß-lactamase NDM-1, Klebsiella pneumoniae ATCC BAA-2146 and Escherichia coli ATCC BAA-2452, the potentiation of meropenem and imipenem is possible with low-molecular weight branched polyethylenimine (600 Da BPEI) and its poly(ethylene glycol) (PEG)ylated derivative (PEG-BPEI) that has a low in vivo toxicity. The mechanism of action is elucidated with fluorescence assays of drug influx and isothermal calorimetry data showing the chelation of essential Zn2+ ions. These results suggested that 600 Da BPEI and PEG-BPEI may also improve the uptake of antibiotics and ß-lactamase inhibitors. Indeed, the CRE E. coli strain is rendered susceptible to the combination of piperacillin and tazobactam. These results expand the possible utility of 600 Da BPEI potentiators, where previously we have demonstrated the ability to improve antibiotic efficacy against antibiotic resistant clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.

PEGylation of Polyethylenimine Lowers Acute Toxicity while Retaining Anti-Biofilm and ß-Lactam Potentiation Properties against Antibiotic-Resistant Pathogens.

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 ß-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.

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa.

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 ß-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-ß-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.

Low-Molecular-Weight Branched Polyethylenimine Potentiates Ampicillin against MRSA Biofilms.

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 ß-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.

Overcoming Multidrug Resistance and Biofilms of Pseudomonas aeruginosa with a Single Dual-Function Potentiator of ß-Lactams.

Clinicians prescribe hundreds of millions of ß-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, ß-lactams could be used in a new drug if the leading causes of ß-lactam antibiotic resistance, permeation barriers from lipopolysaccharide, efflux pumps, and ß-lactamase enzymes, were also defeated. Against P. aeruginosa and their biofilms, the potency of ß-lactam 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 against 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 and biofilm exopolysaccharides with the same compound provides new opportunities to counter the rise of multidrug-resistant infections.

Antibiofilm Synergy of ß-Lactams and Branched Polyethylenimine against Methicillin-Resistant Staphylococcus epidermidis.

Microbial biofilms are ubiquitous in nature, and they pose a serious threat to public health. Staphylococcus epidermidis is the most common clinical isolate from healthcare- and medical device-related biofilm infections. No antibiotic currently on the market can eradicate pathogenic biofilms, which contain complex defense mechanisms composed of slimelike extracellular polymeric substances. Understanding the need to develop alternative approaches, we examine 600 Da branched polyethylenimine (BPEI) against methicillin-resistant Staphylococcus epidermidis (MRSE) biofilms. Here, a microtiter biofilm model is used to test the synergistic effects between the two components of our combination treatment: BPEI and ß-lactam antibiotics. Electron microscopy was used to confirm the growth of MRSE biofilms from the model. Minimum biofilm eradication concentration assays, crystal violet assays, and biofilm kill curves suggest that BPEI exhibits antibiofilm activity and can potentiate ß-lactams to eradicate MRSE biofilms.

Cationic Branched Polyethylenimine (BPEI) Disables Antibiotic Resistance in Methicillin-Resistant Staphylococcus epidermidis (MRSE).

Staphylococcus epidermidis is one of the most prevalent prokaryotic species on human skin and mucosal membranes that constitute the commensal flora. S. epidermidis has become one of the most common causes of primary bacteremia. Infections are difficult to diagnose because the pathogen has natural niches on human skin and the ability to adhere to inanimate surfaces via biofilms. Alarmingly, S. epidermidis has acquired resistance to many antibiotics, which presents a danger to human health. Known as methicillin-resistant S. epidermidis (MRSE), most clinical isolates of MRSE in North America exhibit ß-lactam resistance primarily due to the presence of mecA, a gene that bestows ß-lactam antibiotic resistance in a manner similar to methicillin-resistant Staphylococcus aureus (MRSA). MecA encodes for expression of penicillin-binding protein 2a (PBP2a), which is absent in ß-lactam susceptible strains of S. epidermidis. We can disable this resistance factor in MRSE with 600-Da branched polyethylenimine (BPEI). Cationic BPEI targets anionic wall teichoic acid (WTA), an essential cofactor for proper functioning of PBP2a. We found that BPEI synergizes the activity of ß-lactam antibiotics against MRSE. Growth curves suggest that the combination of BPEI and oxacillin is bactericidal. Electron micrographs indicate abnormalities in the cellular septa and cell walls of treated samples. Therefore, first-line clinical treatments can be effective against MRSE when used in combination with BPEI.