Fabrication and characterization of an antibacterial chitosan/silk fibroin electrospun nanofiber loaded with a cationic peptide for wound-dressing application.

Wound infections are still problematic in many cases and demand new alternatives for current treatment strategies. In recent years, biomaterials-based wound dressings have received much attention due to their potentials and many studies have been performed based on them. Accordingly, in this study, we fabricated and optimized an antibacterial chitosan/silk fibroin (CS/SF) electrospun nanofiber bilayer containing different concentrations of a cationic antimicrobial peptide (AMP) for wound dressing applications. The fabricated CS/SF nanofiber was fully characterized and compared to the electrospun silk fibroin and electrospun chitosan alone in vitro. Then, the release rate of different concentrations of peptide (16, 32, and 64 µg/ml) from peptide-loaded CS/SF nanofiber was investigated. Finally, based on cytotoxic activity, the antibacterial activity of scaffolds containing 16 and 32 µg/ml of the peptide was evaluated against standard and multi-drug resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa isolated from burn patients. The peptide-loaded CS/SF nanofiber displayed appropriate mechanical properties, high water uptake, suitable biodegradation rate, a controlled release without cytotoxicity on Hu02 human foreskin fibroblast cells at the 16 and 32 µg/ml concentrations of peptide. The optimized CS/SF containing 32 µg/ml peptide showed strong antibacterial activity against all experimental strains from standard to resistance. The results showed that the fabricated antimicrobial nanofiber has the potential to be applied as a wound dressing for infected wound healing, although further studies are needed in vivo.

A Novel Anti-Infective Peptide BCCY-1 With Immunomodulatory Activities.

Antibiotic resistance has been considered to be a global threat which underscores the need to develop novel anti-infective therapeutics. Modulation of innate immunity by synthetic peptides is an attractive strategy to overcome this circumstance. We recently reported that BCCY-1, a human ß-casein-derived peptide displays regulatory activities on monocytes, thereby enhancing their actions in innate immune responses. However, the function of peptide BCCY-1 in host defense against infection remains unknown. In this study, we investigated the in vivo characteristics and effects of peptide BCCY-1 in mouse models of bacterial infection. Following intraperitoneal injection, the peptide BCCY-1 exhibited high level of cellular uptake by monocytes without obvious toxicities. Results revealed that peptide BCCY-1, but not the scrambled version, stimulated the chemokine production and monocyte recruitment in vivo. Treatment with BCCY-1 enhanced the pathogen clearance and protected mice against lethal infections. Because the anti-infective effects of BCCY-1 was abolished by in vivo depletion of monocytes/macrophages rather than lymphocytes and granulocytes, we conclude that monocytes/macrophages are key effector cells in BCCY-1-mediated anti-infective protection. Additionally, BCCY-1 lacks direct antimicrobial activity. To our knowledge, a human ß-casein-derived peptide that counters infection by selective regulation of innate immunity has not been reported previously. These results suggest peptide BCCY-1 as a promising alternative approach and a valuable complement to current anti-infective strategy.

Activity and Mechanism of Action of Antifungal Peptides from Microorganisms: A Review.

Harmful fungi in nature not only cause diseases in plants, but also fungal infection and poisoning when people and animals eat food derived from crops contaminated with them. Unfortunately, such fungi are becoming increasingly more resistant to traditional synthetic antifungal drugs, which can make prevention and control work increasingly more difficult to achieve. This means they are potentially very harmful to human health and lifestyle. Antifungal peptides are natural substances produced by organisms to defend themselves against harmful fungi. As a result, they have become an important research object to help deal with harmful fungi and overcome their drug resistance. Moreover, they are expected to be developed into new therapeutic drugs against drug-resistant fungi in clinical application. This review focuses on antifungal peptides that have been isolated from bacteria, fungi, and other microorganisms to date. Their antifungal activity and factors affecting it are outlined in terms of their antibacterial spectra and effects. The toxic effects of the antifungal peptides and their common solutions are mentioned. The mechanisms of action of the antifungal peptides are described according to their action pathways. The work provides a useful reference for further clinical research and the development of safe antifungal drugs that have high efficiencies and broad application spectra.

In Silico Evaluation of Human Cathelicidin LL-37 as a Novel Therapeutic Inhibitor of Panton-Valentine Leukocidin Toxin of Methicillin-Resistant Staphylococcus aureus.

Incidence of drug resistance in clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) is attributed to its diverse repertoire of virulence factors. Of these virulence determinants, Panton-Valentine Leukocidin (PVL) has been experimentally validated as a prospective drug target due to its conspicuous and comprehensive role in nosocomial infections. This study encompassed an in silico approach to elucidate the antimicrobial potentiality of human cathelicidin LL-37 against PVL toxin of MRSA. Molecular docking studies of LL-37 and its segments with the PVL toxin subunits LukS and LukF were carried out using PatchDock server and the results were refined using FireDock server. The paramount ligand-receptor combination was selected and analyzed based on diverse parametric attributes and compared with the commercial inhibitors of PVL viz. Andrimid, Beclobrate, Beta-sitosterol, Diathymosulfone, and Probucol to determine the most potent inhibitor among them. Our results elucidated that the interaction of LL-37 with the LukS subunit of PVL toxin (minimum global energy of -61.82 kcal/mol) depicted 34 molecular interactions, while the commercial PVL inhibitors depicted fewer and insubstantial interactions. SWISS-ADME (Absorption, Distribution, Metabolism, and Excretion) and ToxinPred analysis of LL-37 further corroborated its null potency of toxicity in systemic milieu. The results obtained may credit this study as basis for the development of LL-37 as a potential inhibitor against virulent MRSA toxins, thereby exalting the treatment regimes for nosocomial infections in health care facilities worldwide.

Cyclic gomesin, a stable redesigned spider peptide able to enter cancer cells.

Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.

ABC Exporters in Pathogenesis: Role of Synthetic Anti-Microbial Peptides.

ABC exporters are involved in diverse cellular processes including lipid trafficking, drug resistance, pathogenesis etc. The greatest thrust has been in the area of drug resistance that explains the underlying well-crafted canonical architecture of its structure. Interestingly, ranging from structural organisation to subsequent design and delivery aspects lays the niche of antimicrobial peptides. One of the major highlight of this paper is the role of synthetic antimicrobial peptides in current scenario.

Antimicrobial Peptides and Ectosymbiotic Relationships: Involvement of a Novel Type IIa Crustin in the Life Cycle of a Deep-Sea Vent Shrimp.

The symbiotic shrimp Rimicaris exoculata dominates the macrofauna inhabiting the active smokers of the deep-sea mid Atlantic ridge vent fields. We investigated the nature of the host mechanisms controlling the vital and highly specialized ectosymbiotic community confined into its cephalothoracic cavity. R. exoculata belongs to the Pleocyemata, crustacean brooding eggs, usually producing Type I crustins. Unexpectedly, a novel anti-Gram-positive type II crustin was molecularly identified in R. exoculata. Re-crustin is mainly produced by the appendages and the inner surfaces of the cephalothoracic cavity, embedding target epibionts. Symbiosis acquisition and regulating mechanisms are still poorly understood. Yet, symbiotic communities were identified at different steps of the life cycle such as brooding stage, juvenile recruitment and molt cycle, all of which may be crucial for symbiotic acquisition and control. Here, we show a spatio-temporal correlation between the production of Re-crustin and the main ectosymbiosis-related life-cycle events. Overall, our results highlight (i) a novel and unusual AMP sequence from an extremophile organism and (ii) the potential role of AMPs in the establishment of vital ectosymbiosis along the life cycle of deep-sea invertebrates.

Interactions of "de novo" designed peptides with bacterial membranes: Implications in the antimicrobial activity.

Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level.

NP213 (Novexatin®): A unique therapy candidate for onychomycosis with a differentiated safety and efficacy profile.

NP213 (Novexatin®) is a novel antifungal peptide specifically designed for the topical treatment of onychomycosis. NP213 was designed using host defense peptides (HDP), essential components of the innate immune response to infection, as a template. NP213 is a water-soluble cyclic fungicidal peptide that effectively penetrates human nail. NP213 demonstrated a promising preclinical and clinical safety profile, with no evidence of systemic exposure following topical application to the skin and nails. NP213 was efficacious in two phase IIa human trials with 43.3% of patients having no fungi detectable by culture of fragments from NP213-treated nails after 180 days in the first study and likewise 56.5% of patients were culture negative for dermatophytes after 360 days in the second phase IIa study. In both trials, NP213 was applied daily for only 28 days in marked contrast to other topical onychomycosis treatments that require application for up to 52 weeks. Patient reported outcomes from the phase IIa studies were positive with participants recording an improved appearance of their nails after only 14 days of application. All fungi identified in these studies were Trichophyton spp. NP213 (Novexatin®) is a promising, highly differentiated peptide-based candidate for the topical treatment of onychomycosis, addressing the infectious cause and cosmetic issues of this very common condition.

Combined effect of lasioglossin LL-III derivative with azoles against Candida albicans virulence factors: biofilm formation, phospholipases, proteases and hemolytic activity.

Candida albicans has several virulence factors at its disposal, including yeast-hyphal transition associated with biofilm formation, phospholipases, proteases and hemolytic activity, all of which contribute to its pathogenesis. We used synthetic derivative LL-III/43 of antimicrobial peptide lasioglossin LL-III to enhance effect of azoles on attenuation of C. albicans virulence factors. LL-III/43 was able to inhibit initial adhesion or biofilm formation of C. albicans strains at 50 µM. Azoles, however, were ineffective at this concentration. Using fluorescently labeled LL-III/43, we observed that peptide covered C. albicans cells, partially penetrated through their membranes and then accumulated inside cells. LL-III/43 (25 µM) in combination with clotrimazole prevented biofilm formation already at 3.1 µM clotrimazole. Neither LL-III/43 nor azoles were able to significantly inhibit phospholipases, proteases, or hemolytic activity of C. albicans. LL-III/43 (25 µM) and clotrimazole (50 µM) in combination decreased production of these virulence factors, and it completely attenuated its hemolytic activity. Scanning electron microscopy showed that LL-III/43 (50 µM) prevented C. albicans biofilm formation on Ti-6Al-4 V alloy used in orthopedic surgeries and combination of LL-III/43 (25 µM) with clotrimazole (3.1 µM) prevented biofilm formation on urinary catheters. Therefore, mixture of LL-III/43 and clotrimazole is suitable candidate for future pharmaceutical research.

Omiganan Enhances Imiquimod-Induced Inflammatory Responses in Skin of Healthy Volunteers.

Omiganan (OMN; a synthetic cationic peptide) and imiquimod (IMQ; a TLR7 agonist) have synergistic effects on interferon responses in vitro. The objective of this study was to translate this to a human model for proof-of-concept, and to explore the potential of OMN add-on treatment for viral skin diseases. Sixteen healthy volunteers received topical IMQ, OMN, or a combination of both for up to 4 days on tape-stripped skin. Skin inflammation was quantified by laser speckle contrast imaging and 2D photography, and molecular and cellular responses were analyzed in biopsies. IMQ treatment induced an inflammatory response of the skin. Co-treatment with OMN enhanced this inflammatory response to IMQ, with increases in perfusion (+17.1%; 95% confidence interval (CI) 5.6%-30%; P < 0.01) and erythema (+1.5; 95% CI 0.25%-2.83; P = 0.02). Interferon regulatory factor-driven and NFκB-driven responses following TLR7 stimulation were enhanced by OMN (increases in IL-6, IL-10, MXA, and IFNÉ£), and more immune cell infiltration was observed (in particular CD4+, CD8+, and CD14+ cells). These findings are in line with the earlier mechanistic in vitro data, and support evaluation of imiquimod/OMN combination therapy in human papillomavirus-induced skin diseases.

LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies.

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 × 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.

Inhalation Delivery of Host Defense Peptides (HDP) using Nano- Formulation Strategies: A Pragmatic Approach for Therapy of Pulmonary Ailments.

Host defense peptides (HDP) are small cationic molecules released by the immune systems of the body, having multidimensional properties including anti-inflammatory, anticancer, antimicrobial and immune-modulatory activity. These molecules gained importance due to their broad-spectrum pharmacological activities, and hence being actively investigated. Presently, respiratory infections represent a major global health problem, and HDP has an enormous potential to be used as an alternative therapeutics against respiratory infections and related inflammatory ailments. Because of their short half-life, protease sensitivity, poor pharmacokinetics, and first-pass metabolism, it is challenging to deliver HDP as such inside the physiological system in a controlled way by conventional delivery systems. Many HDPs are efficacious only at practically high molar-concentrations, which is not convincing for the development of drug regimen due to their intrinsic detrimental effects. To avail the efficacy of HDP in pulmonary diseases, it is essential to deliver an appropriate payload into the targeted site of lungs. Inhalable HDP can be a potentially suitable alternative for various lung disorders including tuberculosis, Cystic fibrosis, Pneumonia, Lung cancer, and others as they are active against resistant microbes and cells and exhibit improved targeting with reduced adverse effects. In this review, we give an overview of the pharmacological efficacy of HDP and deliberate strategies for designing inhalable formulations for enhanced activity and issues related to their clinical implications.

Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37.

PURPOSE: Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. METHODS: In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. RESULTS: The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. CONCLUSION: This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.

Epinecidin-1 Protects against Methicillin Resistant Staphylococcus aureus Infection and Sepsis in Pyemia Pigs.

Methicillin resistant Staphylococcus aureus (MRSA) may be found on the skin, nose, and throats of long-term hospitalized patients. While MRSA infections are usually minor, serious infections and death may occur in immunocompromised or diabetic patients, or after exposure of MRSA to blood. This report demonstrates that the antimicrobial peptide (AMP) epinecidin-1 (Epi-1) efficiently protects against MRSA infection in a pyemia pig model. We first found that Epi-1 exhibits bactericidal activity against MRSA. Next, pharmacokinetic analysis revealed that Epi-1 was stable in serum for 4 h after injection, followed by a gradual decrease. This pharmacokinetic profile suggested Epi-1 may bind serum albumin, which was confirmed in vitro. Harmful effects were not observed for doses up to 100 mg/kg body weight in pigs. When Epi-1 was supplied as a curative agent 30 min post-infection, MRSA-induced abnormalities in blood uric acid (UA), blood urea nitrogen (BUN), creatine (CRE), GOT, and GPT levels were restored to normal levels. We further showed that the bactericidal activity of Epi-1 was higher than that of the antibiotic drug vancomycin. Epi-1 significantly decreased MRSA counts in the blood, liver, kidney, heart, and lungs of infected pigs. Elevated levels of serum C reactive protein (CRP), proinflammatory cytokine IL6, IL1ß, and TNFα were also attenuated by Epi-1 treatment. Moreover, the MRSA genes, enterotoxin (et)-A, et-B, intrinsic methicillin resistance A (mecA), and methicillin resistance factor A (femA), were significantly reduced or abolished in MRSA-infected pigs after treatment with Epi-1. Hematoxylin and eosin staining of heart, liver, lung, and kidney sections indicated that Epi-1 attenuated MRSA toxicity in infected pigs. A survival study showed that the pyemia pigs infected with MRSA alone died within a week, whereas the pigs post-treated with 2.5 mg/kg Epi-1 were completely protected against death. The present investigation, thus, demonstrates that Epi-1 effectively protects pyemia pigs against pathogenic MRSA without major toxic side effects.

Electrospun acid-neutralizing fibers for the amelioration of inflammatory response.

Biodegradable aliphatic polyesters, especially polylactide (PLA), polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), are the most representative and widely used synthetic polymers in the field of tissue engineering and regenerative medicine. However, these polyesters often give rise to aseptic inflammation because of their acidic degradation products after implantation. Here, unidirectional shell-core structured fibers of chitosan/poly(lactide-co-glycolide) (i.e., CTS/PLGA) with acid-neutralizing capability were developed for addressing the noted issue by coating the PLGA fiber surfaces with a layer of the alkaline chitosan by coaxial electrospinning. Our results showed that during a period of 8-week degradation, the shell-layer of chitosan with its unique alkaline nature for acid-neutralization obviously hindered the pH decrease as a result of the degradation of PLGA-core. In a mocked acidic environment testing of the human dermal fibroblasts, chitosan-enabled acidity neutralization could significantly reduce in vitro the secretion of inflammatory factors and downregulate the expression of related inflammatory genes. Thereafter, biocompatibility assessment in vitro showed that the CTS/PLGA fibers had poorer cell adhesion capacity than the PLGA fibers but were cytocompatible and promoted cell migration and secretion of collagen. Moreover, subcutaneous embedding for two and four weeks in vivo revealed that the CTS/PLGA fibers significantly reduced the recruitment of inflammatory cells and the formation of foreign body giant cells (FBGCs). This study thereby demonstrated the evident acid-neutralizing effect of the chitosan-coating layer on alleviating the inflammatory responses caused by the acidic degradation products of the PLGA-core. Our highly aligned CTS/PLGA fibers, as a kind of quasi "pH-neutral fibers" with the acid-neutralizing capability, could be potentially applied for engineering those architecturally anisotropic tissues (e.g., tendon/ligament) toward improved efficacy of regeneration. STATEMENT OF SIGNIFICANCE: It is well known that acidic degradation products from representative aliphatic polyesters (e.g., PLA, PGA, and PLGA) give rise to the problem of aseptic inflammation. Various alkaline components acting as neutralizing agents have been used to address the noted issue. However, rather less attention has been paid to engineer these polyesters into a fibrous form with acid-neutralizing functionality. The present study proposes the concept of "pH-neutral fibers" and develops shell-core structured unidirectional fibers of chitosan/poly(lactide-co-glycolide) with acid-neutralizing capability for ameliorating inflammatory responses caused by the acidic degradation products of PLGA. It provides a comprehensive study encompassing fiber characterization and in vitro and in vivo evaluation, which would pave the way for developing sophisticated pH-neutral fibers for functional tissue regeneration.

Replacement of l-Amino Acids by d-Amino Acids in the Antimicrobial Peptide Ranalexin and Its Consequences for Antimicrobial Activity and Biodistribution.

Infections caused by multidrug-resistant bacteria are a global emerging problem. New antibiotics that rely on innovative modes of action are urgently needed. Ranalexin is a potent antimicrobial peptide (AMP) produced in the skin of the American bullfrog Rana catesbeiana. Despite strong antimicrobial activity against Gram-positive bacteria, ranalexin shows disadvantages such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting exclusively of d-amino acids (named danalexin) was synthesized and compared to the original ranalexin for its antimicrobial potential and its biodistribution properties in a rat model. Danalexin showed improved biodistribution with an extended retention in the organisms of Wistar rats when compared to ranalexin. While ranalexin is rapidly cleared from the body, danalexin is retained primarily in the kidneys. Remarkably, both peptides showed strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria of the genus Acinetobacter with minimum inhibitory concentrations (MICs) between 4 and 16 mg/L (1.9-7.6 µM). Moreover, both peptides showed lower antimicrobial activities with MICs ≥32 mg/L (≥15.2 µM) against further Gram-negative bacteria. The preservation of antimicrobial activity proves that the configuration of the amino acids does not affect the anticipated mechanism of action, namely pore formation.

Safety, Tolerability, Pharmacokinetics, and Drug Interaction Potential of SPR741, an Intravenous Potentiator, after Single and Multiple Ascending Doses and When Combined with ß-Lactam Antibiotics in Healthy Subjects.

SPR741 is a novel polymyxin B derivative, with minimal intrinsic antibacterial activity and reduced nonclinical nephrotoxicity compared to levels with polymyxin B, that interacts with the outer membrane of Gram-negative bacteria, enhancing penetration of coadministered antibiotics. The safety, tolerability, and pharmacokinetics (PK) of SPR741 were evaluated in two studies, after single and multiple intravenous (i.v.) doses in healthy adult subjects and after coadministration with partner antibiotics. In the single and multiple ascending-dose study, SPR741 or placebo was administered as a 1-h infusion at single doses of 5 to 800 mg and in multiple doses of 50 to 600 mg every 8 h (q8h) for 14 days. In the drug-drug interaction study, a single 400-mg i.v. dose of SPR741 was administered alone and in combination with piperacillin-tazobactam, ceftazidime, and aztreonam. PK parameters for SPR741 and partner antibiotics were determined using noncompartmental analysis. After single doses, a dose-linear and proportional increase in mean maximum concentration in plasma (Cmax) and area under the concentration-time curve (AUC) was observed. At doses of 100 to 800 mg, >50% of the dose was excreted in the urine in the first 4 h postdose. After multiple doses, the mean half-life was 2.2 h on day 1 and up to 14.0 h on day 14, with no evidence of accumulation after 14 days of dosing up to 400 mg. The PK profile of SPR741 and partner antibiotics was unchanged with coadministration. SPR741 was generally well tolerated at doses up to 1,800 mg/day. These data support further clinical development of SPR741 for treating serious infections due to resistant bacteria. (These studies have been registered at ClinicalTrials.gov under identifiers NCT03022175 and NCT03376529.).

Mode of action of the antimicrobial peptide Mel4 is independent of Staphylococcus aureus cell membrane permeability.

Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus lysodeikticus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (p<0.001). Mel4 resulted in staphylococcal cells taking up PI with 3.9% cells predominantly stained after 150 min exposure, whereas melimine showed 34% staining. Unlike melimine, Mel4 did not release DNA/RNA. Cell-free media from Mel4 treated cells hydrolysed peptidoglycan and produced greater zones of inhibition against M. lysodeikticus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for staphylococci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarization, pore formation, release of cellular contents leading to cell death.

Dead bacterial absorption of antimicrobial peptides underlies collective tolerance.

The collective tolerance towards antimicrobial peptides (APs) is thought to occur primarily through mechanisms associated with live bacterial cells. In contrast to the focus on live cells, we discover that the LL37 antimicrobial peptide kills a subpopulation of Escherichia coli, forming dead cells that absorb the remaining LL37 from the environment. Combining mathematical modelling with population and single-cell experiments, we show that bacteria absorb LL37 at a timing that coincides with the permeabilization of their cytoplasmic membranes. Furthermore, we show that one bacterial strain can absorb LL37 and protect another strain from killing by LL37. Finally, we demonstrate that the absorption of LL37 by dead bacteria can be reduced using a peptide adjuvant. In contrast to the known collective tolerance mechanisms, we show that the absorption of APs by dead bacteria is a dynamic process that leads to emergent population behaviour.