Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment.

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.

Rapid SARS-CoV-2 Detection Using Electrochemical Immunosensor.

The outbreak of the coronavirus disease (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been declared an international public health crisis. It is essential to develop diagnostic tests that can quickly identify infected individuals to limit the spread of the virus and assign treatment options. Herein, we report a proof-of-concept label-free electrochemical immunoassay for the rapid detection of SARS-CoV-2 virus via the spike surface protein. The assay consists of a graphene working electrode functionalized with anti-spike antibodies. The concept of the immunosensor is to detect the signal perturbation obtained from ferri/ferrocyanide measurements after binding of the antigen during 45 min of incubation with a sample. The absolute change in the [Fe(CN)6]3-/4- current upon increasing antigen concentrations on the immunosensor surface was used to determine the detection range of the spike protein. The sensor was able to detect a specific signal above 260 nM (20 µg/mL) of subunit 1 of recombinant spike protein. Additionally, it was able to detect SARS-CoV-2 at a concentration of 5.5 × 105 PFU/mL, which is within the physiologically relevant concentration range. The novel immunosensor has a significantly faster analysis time than the standard qPCR and is operated by a portable device which can enable on-site diagnosis of infection.

The Lantibiotic NAI-107 Efficiently Rescues Drosophila melanogaster from Infection with Methicillin-Resistant Staphylococcus aureus USA300.

We used the fruit fly Drosophila melanogaster as a cost-effective in vivo model to evaluate the efficacy of novel antibacterial peptides and peptoids for treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. A panel of peptides with known antibacterial activity in vitro and/or in vivo was tested in Drosophila Although most peptides and peptoids that were effective in vitro failed to rescue lethal effects of S. aureus infections in vivo, we found that two lantibiotics, nisin and NAI-107, rescued adult flies from fatal infections. Furthermore, NAI-107 rescued mortality of infection with the MRSA strain USA300 with an efficacy equivalent to that of vancomycin, a widely applied antibiotic for the treatment of serious MRSA infections. These results establish Drosophila as a useful model for in vivo drug evaluation of antibacterial peptides.

Antimicrobial activity of GN peptides and their mode of action.

Increasing prevalence of bacteria that carries resistance towards conventional antibiotics has prompted the investigation into new compounds for bacterial intervention to ensure efficient infection control in the future. One group of potential lead structures for antibiotics is antimicrobial peptides due to their characteristics as naturally derived compounds with antimicrobial activity. In this study, we aimed at characterizing the mechanism of action of a small set of in silico optimized peptides. Following determination of peptide activity against E. coli, S. aureus, and P. aeruginosa, toxicity was assessed revealing meaningful selectivity indexes for the majority of the peptides. Investigation of the peptides effect on bacteria demonstrated a rapid growth inhibition with signs of bacterial lysis together with increased bacterial size. Both visual and quantitative assays clearly demonstrated bacterial membrane disruption after 10 min for the most active peptides. The membrane disrupting effect was verified by measuring the release of calcein from bacterial mimicking liposomes. This revealed the most active peptides as inducers of immediate release, indicating the kinetics of membrane permeabilization as an important determinant of bacterial activity. No well-defined secondary structure of the peptides could be determined using CD-spectroscopy in the presence of different liposomes mixtures, implying that there is no correlation between peptide secondary structure and the observed anti-bacterial and cytotoxic activity for this set of peptides. In conjunction, these findings provide strong indications of membrane disruption as the primary mechanism of bacterial growth inhibition for the tested peptides. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 172-183, 2016.

Structure-activity relationship study of novel peptoids that mimic the structure of antimicrobial peptides.

The constant emergence of new bacterial strains that resist the effectiveness of marketed antimicrobials has led to an urgent demand for and intensive research on new classes of compounds to combat bacterial infections. Antimicrobial peptoids comprise one group of potential candidates for antimicrobial drug development. The present study highlights a library of 22 cationic amphipathic peptoids designed to target bacteria. All the peptoids share an overall net charge of +4 and are 8 to 9 residues long; however, the hydrophobicity and charge distribution along the abiotic backbone varied, thus allowing an examination of the structure-activity relationship within the library. In addition, the toxicity profiles of all peptoids were assessed in human red blood cells (hRBCs) and HeLa cells, revealing the low toxicity exerted by the majority of the peptoids. The structural optimization also identified two peptoid candidates, 3 and 4, with high selectivity ratios of 4 to 32 and 8 to 64, respectively, and a concentration-dependent bactericidal mode of action against Gram-negative Escherichia coli.

Nano-topography and functionalization with the synthetic peptoid GN2-Npm9 as a strategy for antibacterial and biocompatible titanium implants.

In recent years, antimicrobial peptides (AMPs) have attracted great interest in scientific research, especially for biomedical applications such as drug delivery and orthopedic applications. Since they are readily degradable in the physiological environment, scientific research has recently been trying to make AMPs more stable. Peptoids are synthetic N-substituted glycine oligomers that mimic the structure of peptides. They have a structure that does not allow proteolytic degradation, which makes them more stable while maintaining microbial activity. This structure also brings many advantages to the molecule, such as greater diversity and specificity, making it more suitable for biological applications. For the first time, a synthesized peptoid (GN2-Npm9) was used to functionalize a nanometric chemically pre-treated (CT) titanium surface for bone-contact implant applications. A preliminary characterization of the functionalized surfaces was performed using the contact angle measurements and zeta potential titration curves. These preliminary analyses confirmed the presence of the peptoid and its adsorption on CT. The functionalized surface had a hydrophilic behaviour (contact angle = 30°) but the hydrophobic tryptophan-like residues were also exposed. An electrostatic interaction between the lysine residue of GN2-Npm9 and the surface allowed a chemisorption mechanism. The biological characterization of the CT_GN2-Nmp9 surfaces demonstrated the ability to prevent surface colonization and biofilm formation by the pathogens Escherichia coli and Staphylococcus epidermidis thus showing a broad-range activity. The cytocompatibility was confirmed by human mesenchymal stem cells. Finally, a bacteria-cells co-culture model was applied to demonstrate the selective bioactivity of the CT_GN2-Nmp9 surface that was able to preserve colonizing cells adhered to the device surface from bacterial infection.

Exploiting Saturation Regimes and Surface Effects to Tune Composite Design: Single Platelet Nanocomposites of Peptoid Nanosheets and CaCO3.

Mineral-polymer composites found in nature exhibit exceptional structural properties essential to their function, and transferring these attributes to the synthetic design of functional materials holds promise across various sectors. Biomimetic fabrication of nanocomposites introduces new pathways for advanced material design and explores biomineralization strategies. This study presents a novel approach for producing single platelet nanocomposites composed of CaCO3 and biomimetic peptoid (N-substituted glycines) polymers, akin to the bricks found in the brick-and-mortar structure of nacre, the inner layer of certain mollusc shells. The significant aspect of the proposed strategy is the use of organic peptoid nanosheets as the scaffolds for brick formation, along with their controlled mineralization in solution. Here, we employ the B28 peptoid nanosheet as a scaffold, which readily forms free-floating zwitterionic bilayers in aqueous solution. The peptoid nanosheets were mineralized under consistent initial conditions (σcalcite = 1.2, pH 9.00), with variations in mixing conditions and supersaturation profiles over time aimed at controlling the final product. Nanosheets were mineralized in both feedback control experiments, where supersaturation was continuously replenished by titrant addition and in batch experiments without a feedback loop. Complete coverage of the nanosheet surface by amorphous calcium carbonate was achieved under specific conditions with feedback control mineralization, whereas vaterite was the primary CaCO3 phase observed after batch experiments. Thermodynamic calculations suggest that time-dependent supersaturation profiles as well as the spatial distribution of supersaturation are effective controls for tuning the mineralization extent and product. We anticipate that the control strategies outlined in this work can serve as a foundation for the advanced and scalable fabrication of nanocomposites as building blocks for nacre-mimetic and functional materials.

Solid-phase synthesis of novel antimicrobial peptoids with α- and ß-chiral side chains.

Bacterial resistance to conventional antibiotics poses an immense threat to human health and consequently many bacterial infections arise from multi-drug resistant pathogens. It is, therefore, necessary to continue to develop novel antimicrobials. Peptoids are a novel class of antimicrobial agents that mimic the structures of peptides. These N-substituted glycines differ from peptides in the side chain attachment site where in peptoids, the side chain functionality is introduced at the nitrogen rather than the α-carbon in peptides. During the process of design and development of antimicrobial peptoids, several key elements such as charge and amphiphilicity appear to guide peptoid bioactivity against bacterial pathogens. In this study, we report a quick synthesis of three novel cationic antimicrobial peptoids that contain charged, hydrophobic, and chiral monomers. This study is the first to use a monomer that contains both charge and chiral center for the design of the novel peptoid sequences.

Immunomodulatory Properties of Host Defence Peptides in Skin Wound Healing.

Cutaneous wound healing is a vital biological process that aids skin regeneration upon injury. Wound healing failure results from persistent inflammatory conditions observed in diabetes, or autoimmune diseases like psoriasis. Chronic wounds are incurable due to factors like poor oxygenation, aberrant function of peripheral sensory nervature, inadequate nutrients and blood tissue supply. The most significant hallmark of chronic wounds is heavily aberrant immune skin function. The immune response in humans relies on a large network of signalling molecules and their interactions. Research studies have reported on the dual role of host defence peptides (HDPs), which are also often called antimicrobial peptides (AMPs). Their duality reflects their potential for acting as antibacterial peptides, and as immunodulators that assist in modulating several biological signalling pathways related to processes such as wound healing, autoimmune disease, and others. HDPs may differentially control gene regulation and alter the behaviour of epithelial and immune cells, resulting in modulation of immune responses. In this review, we shed light on the understanding and most recent advances related to molecular mechanisms and immune modulatory features of host defence peptides in human skin wound healing. Understanding their functional role in skin immunity may further inspire topical treatments for chronic wounds.

Current State of SLC and ABC Transporters in the Skin and Their Relation to Sweat Metabolites and Skin Diseases.

With a relatively large surface area (2 m2) and 15% of total body mass, the skin forms the largest organ of the human body. The main functions of the skin include regulation of body temperature by insulation or sweating, regulation of the nervous system, regulation of water content, and protection against external injury. To perform these critical functions, the skin encodes genes for transporters responsible for the cellular trafficking of essential nutrients and metabolites to maintain cellular hemostasis. However, the knowledge on the expression, regulation, and function of these transporters is very limited and needs more work to elucidate how these transporters play a role both in disease progression and in healing. Furthermore, SLC and ABC transporters are understudied, and even less studied in skin. There are sparse reports on relation between transporters in skin and sweat metabolites. This mini review focuses on the current state of SLC and ABC transporters in the skin and their relation to sweat metabolites and skin diseases.

Changes in toxin production of environmental Pseudomonas aeruginosa isolates exposed to sub-inhibitory concentrations of three common antibiotics.

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

The Role of Proteomics in Bacterial Response to Antibiotics.

For many years, we have tried to use antibiotics to eliminate the persistence of pathogenic bacteria. However, these infectious agents can recover from antibiotic challenges through various mechanisms, including drug resistance and antibiotic tolerance, and continue to pose a global threat to human health. To design more efficient treatments against bacterial infections, detailed knowledge about the bacterial response to the commonly used antibiotics is required. Proteomics is a well-suited and powerful tool to study molecular response to antimicrobial compounds. Bacterial response profiling from system-level investigations could increase our understanding of bacterial adaptation, the mechanisms behind antibiotic resistance and tolerance development. In this review, we aim to provide an overview of bacterial response to the most common antibiotics with a focus on the identification of dynamic proteome responses, and through published studies, to elucidate the formation mechanism of resistant and tolerant bacterial phenotypes.

Synthesis of Peptoids Containing Multiple Nhtrp and Ntrp Residues: A Comparative Study of Resin, Cleavage Conditions and Submonomer Protection.

Peptoids hold status as peptide-mimetics with versatile biological applications due to their proteolytic stability and structural diversity. Among those that have been studied in different biological systems, are peptoids with dominant balanced hydrophobic and charge distribution along the backbone. Tryptophan is an important amino acid found in many biologically active peptides. Tryptophan-like side chains in peptoids allow H-bonding, which is absent from the parent backbone, due to the unique indole ring. Furthermore, the rigid hydrophobic core and flat aromatic system influence the positioning in the hydrocarbon core and allows accommodating tryptophan-like side chains into the interfacial regions of bacterial membranes and causing bacterial membrane damage. Incorporating multiple tryptophan-like side chains in peptoids can be tricky and there is a lack of suitable, synthetic routes established. In this paper, we investigate the synthesis of peptoids rich in Nhtrp and Ntrp residues using different resins, cleavage conditions, and unprotected as well as tert-butyloxycarbonyl-protected amines suitable for automated solid-phase submonomer peptoid synthesis protocols.

Ultra-Short Antimicrobial Peptoids Show Propensity for Membrane Activity Against Multi-Drug Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.

Peptoids successfully inhibit the growth of gram negative E. coli causing substantial membrane damage.

Peptoids are an alternative approach to antimicrobial peptides that offer higher stability towards enzymatic degradation. It is essential when developing new types of peptoids, that mimic the function of antimicrobial peptides, to understand their mechanism of action. Few studies on the specific mechanism of action of antimicrobial peptoids have been described in the literature, despite the plethora of studies on the mode of action of antimicrobial peptides. Here, we investigate the mechanism of action of two short cationic peptoids, rich in lysine and tryptophan side chain functionalities. We demonstrate that both peptoids are able to cause loss of viability in E. coli susceptible cells at their MIC (16-32 µg/ml) concentrations. Dye leakage assays demonstrate slow and low membrane permeabilization for peptoid 1, that is still higher for lipid compositions mimicking bacterial membranes than lipid compositions containing Cholesterol. At concentrations of 4 × MIC (64-128 µg/ml), pore formation, leakage of cytoplasmic content and filamentation were the most commonly observed morphological changes seen by SEM in E. coli treated with both peptoids. Flow cytometry data supports the increase of cell size as observed in the quantification analysis from the SEM images and suggests overall decrease of DNA per cell mass over time.

Peptides and Peptidomimetics for Antimicrobial Drug Design.

The purpose of this paper is to introduce and highlight a few classes of traditional antimicrobial peptides with a focus on structure-activity relationship studies. After first dissecting the important physiochemical properties that influence the antimicrobial and toxic properties of antimicrobial peptides, the contributions of individual amino acids with respect to the peptides antibacterial properties are presented. A brief discussion of the mechanisms of action of different antimicrobials as well as the development of bacterial resistance towards antimicrobial peptides follows. Finally, current efforts on novel design strategies and peptidomimetics are introduced to illustrate the importance of antimicrobial peptide research in the development of future antibiotics.

The immunomodulation effect of Aronia extract lacks association with its antioxidant anthocyanins.

Polyphenols comprise a diverse group of molecules with antioxidative and anti-inflammatory activities. To compare the antioxidative and anti-inflammatory capacity of Aronia melanocarpa berries (chokeberries), recognized for their high content of anthocyanins, a noncytotoxic isolation method was developed to obtain high-purity anthocyanins in the extract. The antioxidative activity of the extract, the anthocyanin-rich fraction (AF) was determined by 1,1-diphenyl-2-picrylhydrazyl radical and ferric-reducing ability of plasma along with resveratrol as a reference. The immunomodulation properties were assessed in lipopolysaccharide (LPS)-stimulated human monocytes mono mac 6. The isolated AF, containing six different anthocyanins, exhibited a stronger antioxidative capacity compared to resveratrol. Resveratrol enhanced tumor necrosis factor-α and reduced interleukin-10 (IL-10) production by LPS, whereas AF only had a slight effect in reducing IL-10. These results demonstrated that there was no major relationship between the antioxidative effect and immunomodulation capacities of AF and resveratrol. The immunomodulatory activity of the extract is associated with bioactive compounds in Aronia other than its anthocyanins.