Acquisition of Resistance to PEGylated branched polyethylenimine Increases Pseudomonas aeruginosa Susceptibility to Aminoglycosides.

PEGylated branched polyethylenimine (PEG-BPEI) has antibacterial and antibiofilm properties. Exposure to PEG-BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG-BPEI) in the wild-type PAO1 strain is 16 µg/ml while, after 15 serial passages, the MIC increased to 1024 µg/mL. An additional 15 rounds of passage in the absence of BPEI or PEG-BPEI did not change the 1024 µg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16-32 fold reduction of MIC values in PEG350-BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self-mediated uptake mechanism, perhaps due to changes in the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O-antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest that PEG-BPEI resistance can be countered with antibiotics to prevent the emergence of PEG-BPEI resistant bacterial populations.

Low-molecular weight branched polyethylenimine reduces cytokine secretion from human immune system monocytes stimulated with bacterial and fungal PAMPs.

Adaptive immunity recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs.

Neutralizing Staphylococcus aureus PAMPs that Trigger Cytokine Release from THP-1 Monocytes.

Innate immunity has considerable specificity and can discriminate between individual species of microbes. In this regard, pathogens are "seen" as dangerous to the host and elicit an inflammatory response capable of destroying the microbes. This immune discrimination is achieved by toll-like receptors on host cells recognizing pathogens, such as Staphylococcus aureus, and microbe-specific pathogen-associated molecular pattern (PAMP) molecules, such as lipoteichoic acid (LTA). PAMPs impede wound healing by lengthening the inflammatory phase of healing and contributing to the development of chronic wounds. Preventing PAMPs from triggering the release of inflammatory cytokines will counteract the dysregulation of inflammation. Here, we use ELISA to evaluate the use of cationic molecules branched polyethylenimine (BPEI), PEGylated BPEI (PEG-BPEI), and polymyxin-B to neutralize anionic LTA and lower levels of TNF-α cytokine release from human THP-1 monocytes in a concentration-dependent manner. Additional data collected with qPCR shows that BPEI and PEG-BPEI reduce the expression profile of the TNF-α gene. Similar effects are observed for the neutralization of whole-cell S. aureus bacteria. In vitro cytotoxicity data demonstrate that PEGylation lowers the toxicity of PEG-BPEI (IC50 = 2661 µm) compared to BPEI (IC50 = 853 µM) and that both compounds are orders of magnitude less toxic than the cationic antibiotic polymyxin-B (IC50 = 79 µM). Additionally, the LTA neutralization ability of polymyxin-B is less effective than BPEI or PEG-BPEI. These properties of BPEI and PEG-BPEI expand their utility beyond disabling antibiotic resistance mechanisms and disrupting S. aureus biofilms, providing additional justification for developing these agents as wound healing therapeutics. The multiple mechanisms of action for BPEI and PEG-BPEI are superior to current wound treatment strategies that have a single modality.

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.

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.

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.

Antibiofilm Activity of PEGylated Branched Polyethylenimine.

Biofilm formation is an adaptive resistance mechanism that pathogens employ to survive in the presence of antimicrobials. Pseudomonas aeruginosa is an infectious Gram-negative bacterium whose biofilm allows it to withstand antimicrobial attack and threaten human health. Chronic wound healing is often impeded by P. aeruginosa infections and the associated biofilms. Previous findings demonstrate that 600 Da branched polyethylenimine (BPEI) can restore ß-lactam potency against P. aeruginosa and disrupt its biofilms. Toxicity concerns of 600 Da BPEI are mitigated by covalent linkage with low-molecular-weight polyethylene glycol (PEG), and, in this study, PEGylated BPEI (PEG350-BPEI) was found exhibit superior antibiofilm activity against P. aeruginosa. The antibiofilm activity of both 600 Da BPEI and its PEG derivative was characterized with fluorescence studies and microscopy imaging. We also describe a variation of the colony biofilm model that was employed to evaluate the biofilm disruption activity of BPEI and PEG-BPEI.

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.

Expression level of chromodomain Y (CDY): potential marker for prediction of sperm recovery in non-obstructive azoospermia.

BACKGROUND: The availability of testis specific genes will be of help in choosing the most promising biomarkers for the detection of testicular sperm retrieval in patients with non-obstructive azoospermia (NOA). Testis specific chromodomain protein Y 1 (CDY1) is a histone acetyltransferase which concentrates in the round spermatid nucleus, where histone hyperacetylation occurs and causes the replacement of histones by the sperm-specific DNA packaging proteins, TNPs and PRMs. OBJECTIVE: The aim was to evaluate CDY1 gene as a marker for predicting of successful sperm retrieval in NOA patients. MATERIALS AND METHODS: This research was conducted on 29 patients with NOA who had undergone testicular sperm extraction (TESE) procedure. NOA patients were subdivided into patients with successful sperm retrieval (NOA+, n=12) and patients with unsuccessful sperm retrieval (NOA-, n=17). Relative expression of CDY1 gene and chromatin incorporation of CDY1 protein were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA assay, respectively. RESULTS: Quantification of mRNA relative expression and incorporation of CDY1 protein in chromatin showed significant lower expressions and protein levels of CDY1 in testis tissues of NOA- in comparison to NOA+ group. CONCLUSION: The findings in this study demonstrated a correlation between the low levels of CDY1 function and unsuccessful sperm recovery in the testicular tissues of NOA- compared to NOA+ patients. Therefore, it can be reasonable to consider CDY1 as a potential biomarker for predicting the presence of spermatozoa, although the claim needs more samples to be confirmed.