Immunomodulatory potential of Nisin A with application in wound healing.

Antimicrobial peptides can have a dual role with both antimicrobial activity against a broad range of bacteria and immunomodulatory effect, making them attractive as therapeutic treatment of difficult wounds. Nisin A is widely known for its antimicrobial activity, and a preliminary study demonstrated that it increased wound closure, but the mechanism behind its effect is unknown. The aim of this study is to elucidate the wound healing potential of Nisin A and the mechanism behind. First, an epithelial and endothelial cell line, human keratinocyte (HaCaT) and human umbilical vein endothelial cell, were used to demonstrate migration and proliferation effects in vitro. From HaCaT cells and peripheral blood mononuclear cell, changes in cytokine levels were shown by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Second, the ex vivo porcine wound healing model was used to investigate the re-epithelization potential of Nisin A. Finally, the model Galleria mellonella was used to confirm antimicrobial activity and to investigate potential immunomodulatory effects in vivo. Here, we demonstrated that Nisin A affected migration significantly of both human umbilical vein endothelial cell and HaCaT cells (p < 0.05) but not proliferation, potentially by decreasing the levels of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-8 (p < 0.001). Furthermore, Nisin A treatment diminished lipopolysaccharide-induced tumor necrosis factor-α levels from peripheral blood mononuclear cells and monocyte chemoattractant protein-1 from HaCaT cells (p < 0.001). Furthermore, Nisin A did not affect proliferation ex vivo either but increased re-epithelization of the porcine skin. Nisin A improved survival of G. mellonella significantly from Staphylococcus epidermidis (p < 0.001) but not from Escherichia coli, indicating that Nisin A did not help the larvae to survive the infection in a different than direct antimicrobial way. All together this makes Nisin A a potential treatment to use in wound healing, as it increases the mobility of skin cells, dampens the effect of lipopolysaccharide and proinflammatory cytokines, and decreases bacterial growth.

Whole-genome sequencing for antimicrobial surveillance: species-specific quality thresholds and data evaluation from the network of the European Union Reference Laboratory for Antimicrobial Resistance genomic proficiency tests of 2021 and 2022.

As antimicrobial resistance (AMR) surveillance shifts to genomics, ensuring the quality of whole-genome sequencing (WGS) data produced across laboratories is critical. Participation in genomic proficiency tests (GPTs) not only increases individual laboratories' WGS capacity but also provides a unique opportunity to improve species-specific thresholds for WGS quality control (QC) by repeated resequencing of distinct isolates. Here, we present the results of the EU Reference Laboratory for Antimicrobial Resistance (EURL-AR) network GPTs of 2021 and 2022, which included 25 EU national reference laboratories (NLRs). A total of 392 genomes from 12 AMR-bacteria were evaluated based on WGS QC metrics. Two percent (n = 9) of the data were excluded, due to contamination, and 11% (n = 41) of the remaining genomes were identified as outliers in at least one QC metric and excluded from computation of the adjusted QC thresholds (AQT). Two QC metric correlation groups were identified through linear regression. Eight percent (n = 28) of the submitted genomes, from 11 laboratories, failed one or more of the AQTs. However, only three laboratories (12%) were identified as underperformers, failing across AQTs for uncorrelated QC metrics in at least two genomes. Finally, new species-specific thresholds for "N50" and "number of contigs > 200 bp" are presented for guidance in routine laboratory QC. The continued participation of NRLs in GPTs will reveal WGS workflow flaws and improve AMR surveillance data. GPT data will continue to contribute to the development of reliable species-specific thresholds for routine WGS QC, standardizing sequencing data QC and ensure inter- and intranational laboratory comparability.IMPORTANCEIllumina next-generation sequencing is an integral part of antimicrobial resistance (AMR) surveillance and the most widely used whole-genome sequencing (WGS) platform. The high-throughput, relative low-cost, high discriminatory power, and rapid turnaround time of WGS compared to classical biochemical methods means the technology will likely remain a fundamental tool in AMR surveillance and public health. In this study, we present the current level of WGS capacity among national reference laboratories in the EU Reference Laboratory for AMR network, summarizing applied methodology and statistically evaluating the quality of the obtained sequence data. These findings provide the basis for setting new and revised thresholds for quality metrics used in routine WGS, which have previously been arbitrarily defined. In addition, underperforming participants are identified and encouraged to evaluate their workflows to produce reliable results.

Methods and Challenges of Using the Greater Wax Moth (Galleria mellonella) as a Model Organism in Antimicrobial Compound Discovery.

Among non-mammalian infection model organisms, the larvae of the greater wax moth Galleria mellonella have seen increasing popularity in recent years. Unlike other invertebrate models, these larvae can be incubated at 37 °C and can be dosed relatively precisely. Despite the increasing number of publications describing the use of this model organism, there is a high variability with regard to how the model is produced in different laboratories, with respect to larva size, age, origin, storage, and rest periods, as well as dosing for infection and treatment. Here, we provide suggestions regarding how some of these factors can be approached, to facilitate the comparability of studies between different laboratories. We introduce a linear regression curve correlating the total larva weight to the liquid volume in order to estimate the in vivo concentration of pathogens and the administered drug concentration. Finally, we discuss several other aspects, including in vivo antibiotic stability in larvae, the infection doses for different pathogens and suggest guidelines for larvae selection.

Antibiofilm Peptides and Peptidomimetics with Focus on Surface Immobilization.

Bacterial biofilms pose a major threat to public health, as they are associated with at least two thirds of all infections. They are highly resilient and render conventional antibiotics inefficient. As a part of the innate immune system, antimicrobial peptides have drawn attention within the last decades, as some of them are able to eradicate biofilms at sub-minimum inhibitory concentration (MIC) levels. However, peptides possess a number of disadvantages, such as susceptibility to proteolytic degradation, pH and/or salinity-dependent activity and loss of activity due to binding to serum proteins. Hence, proteolytically stable peptidomimetics were designed to overcome these drawbacks. This paper summarizes the current peptide and peptidomimetic strategies for combating bacteria-associated biofilm infections, both in respect to soluble and surface-functionalized solutions.

Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves.

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)-an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor-has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants.