Neutral natural deep eutectic solvents as anti-biofilm agents.

Natural deep eutectic solvents (NADES) are a class of liquids with promising properties as components in pharmaceutical formulations, such as a low toxicity profile, biodegradability and versatility. Recently, their potential use as anti-biofilm agents has been proposed, due to their ability to solubilize and stabilize biological macromolecules. In the current work, the ability to break down biofilm matrix and the biofilm killing activity of three NADES of neutral pH were investigated against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027 biofilms. The tested NADES were choline chloride:xylitol (ChX), choline chloride:glycerol (ChG) and betaine:sucrose (BS). Two of the NADES (ChX and ChG) significantly reduced the number of remaining viable cells of both bacterial species in pre-formed biofilm by 4-6 orders of magnitude, while the average biofilm biomass removal for all NADES was 27-67% (S. aureus) and 34-49% (P. aeruginosa). The tested NADES also inhibited biofilm formation of both bacterial species at concentrations at or below 0.5 x the minimal inhibitory concentration (MIC), possibly in part due to observed restrictions imposed by NADES on planktonic growth. These results demonstrate the potential value of neutral NADES as anti-biofilm agents in future antimicrobial preparations.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Photostability studies of GarKS peptides for topical formulation development.

There is a growing interest in the use of antimicrobial peptides (AMPs) as potent alternatives for conventional antibiotics, especially in chronic infected wounds. The development of a suitable topical formulation requires a thorough assessment of the photostability profiles of AMPs. In this study, we sought to investigate the photostability of novel Garvicin KS (GarKS; composed of three peptides GakA, GakB, and GakC) peptides either as an individual peptide or in combinations. The photostability of the aqueous peptide solution was determined using Suntest (indoor and outdoor conditions). Furthermore, the antimicrobial efficacy of the peptides was evaluated following UVA irradiations. Photodegradation of the peptides under indoor and outdoor conditions followed first-order kinetics. Individual peptides (GakA, GakB, and GakC) were more prone to photodegradation as compared to combination peptides (GakA+GakB, GakB+GakC, and GakA+GakC) both under indoor and outdoor conditions where the GakA+GakB combination was the most photostable. A combination of GakA+GakB+GakC enhanced photostability under indoor conditions but was reduced under outdoor conditions. A combination of three peptides with an antioxidant (glutathione) or superoxide/hydrogen peroxide scavenger (trehalose) enhanced the photostability of peptides with the highest stability achieved at a peptide:photostabilizer molar ratio of 1:0.8 for glutathione. A nominal increase in the MIC value for the peptide combinations as opposed to a larger increase for individual peptides further supports the photostability effects of combination peptides following UVA irradiations. These results suggest that the GakA+GakB or GakA+GakB+GakC combinations exhibited the highest photostability with excellent antimicrobial efficacy deemed suitable for the development of a potent AMP formulation for topical applications.

The Use of Extracellular Membrane Vesicles for Immunization against Francisellosis in Nile Tilapia (Oreochromis niloticus) and Atlantic Cod (Gadus morhua L.).

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Hybrid hydrogels for bacteriocin delivery to infected wounds.

Superficial infections in chronic wounds can prevent the wound healing process by the development of persistent infections and drug-resistant biofilms. Topically applied antimicrobial formulations with stabilized and controlled release offer significant benefits for the effective treatment of wound infections. Bacteriocins are the antimicrobial peptides (AMPs) produced by bacteria that are viable alternatives to antibiotics owing to their natural origin and low propensity for resistance development. Herein, we developed a hybrid hydrogel composed of Pluronic F127 (PF127), ethylenediaminetetraacetic acid (EDTA) loaded liposomes, glutathione (GSH), and the bacteriocin Garvicin KS (GarKS) referred to as "GarKS gel". The GarKS gel exhibited suitable viscosity and rheological properties along with controlled release behavior (up to 9 days) for effective peptide delivery following topical application. Potent in vitro antibacterial and anti-biofilm effects of GarKS gel were evident against the Gram-positive bacterium Staphylococcus aureus. The in vivo treatment of methicillin resistant S. aureus (MRSA) infected mouse wounds suggested potent antibacterial effects of the GarKS gel following multiple applications of once-a-day application for three consecutive days. Altogether, these results provide proof-of-concept for the successful development of AMP loaded topical formulation for effective treatment of wound infections.

Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane.

Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In Actinobacillus pleuropneumoniae, a Gram-negative porcine pathogen responsible for extensive economic losses worldwide, we identified a conserved general secretion pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA (ApfA stem: ApfAs). ApfAs was used as an outer membrane anchor, to which potential immunogens can be attached. To enable confirmation of correct positioning, ApfAs, was cloned in combination with the modified acyl carrier protein (ACP) fluorescent tag ACP mini (ACPm) and the putative immunogen VacJ. The chimeric construct was inserted in the pMK-express vector, subsequently transformed into A. pleuropneumoniae for expression. Flow cytometry, fluorescence imaging and mass spectrometry analysis were employed to demonstrate that the outer membrane of the transformed strain was enriched with the chimeric ApfAs-ACPm-VacJ antigen. Our results confirmed correct positioning of the chimeric ApfAs-ACPm-VacJ antigen and supported this system's potential as platform technology enabling antigenic enrichment of the outer membrane of A. pleuropneumoniae.

Preformulation studies on novel garvicin KS peptides for topical applications.

Antimicrobial peptides (AMPs) are emerging as a viable alternative to antibiotics attributable to their potent antimicrobial effects and low propensity for resistance development, especially in chronic infected wounds. The development of an optimized topical formulation of AMPs is thus warranted. Preformulation studies for determination of the suitability and optimization requirements of AMPs in topical formulation development are important. Therefore, we sought to investigate the preformulation studies for a novel bacteriocin garvicin KS (GarKS), which is composed of three peptides (GakA, GakB, and GakC). The effects of physiological fluids and varying temperatures on GarKS peptide stability were determined. The antimicrobial effects of the peptides and their combinations were evaluated in Staphylococcus aureus (methicillin sensitive and resistant strains). Furthermore, their effects on fibroblast viability and proliferation were determined. The GarKS peptides were stable in water and PBS at room and physiological temperatures, however, the peptides were significantly degraded in simulated wound fluid. The antimicrobial and fibroblast cell viability/proliferation effects of either individual GarKS peptides or their combinations varied. A careful consideration of the peptide stability, antimicrobial efficacy, and fibroblast viability/proliferation effects suggests GakA+GakB as a potent combination for the development of an optimized topical formulation of GarKS peptides.

Vesicles From Vibrio cholerae Contain AT-Rich DNA and Shorter mRNAs That Do Not Correlate With Their Protein Products.

Extracellular vesicles secreted by Gram-negative bacteria have proven to be important in bacterial defense, communication and host-pathogen relationships. They resemble smaller versions of the bacterial mother cell, with similar contents of proteins, LPS, DNA, and RNA. Vesicles can elicit a protective immune response in a range of hosts, and as vaccine candidates, it is of interest to properly characterize their cargo. Genetic sequencing data is already available for vesicles from several bacterial strains, but it is not yet clear how the genetic makeup of vesicles differ from that of their parent cells, and which properties may characterize enriched genetic material. The present study provides evidence for DNA inside vesicles from Vibrio cholerae O395, and key characteristics of their genetic and proteomic content are compared to that of whole cells. DNA analysis reveals enrichment of fragments containing ToxR binding sites, as well as a positive correlation between AT-content and enrichment. Some mRNAs were highly enriched in the vesicle fraction, such as membrane protein genes ompV, ompK, and ompU, DNA-binding protein genes hupA, hupB, ihfB, fis, and ssb, and a negative correlation was found between mRNA enrichment and transcript length, suggesting mRNA inclusion in vesicles may be a size-dependent process. Certain non-coding and functional RNAs were found to be enriched, such as VrrA, GcvB, tmRNA, RNase P, CsrB2, and CsrB3. Mass spectrometry revealed enrichment of outer membrane proteins, known virulence factors, phage components, flagella and extracellular proteins in the vesicle fraction, and a low, negative correlation was found between transcript-, and protein enrichment. This result opposes the hypothesis that a significant degree of protein translation occurs in vesicles after budding. The abundance of viral-, and flagellar proteins in the vesicle fraction underlines the importance of purification during vesicle isolation.

Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.

Antimicrobial Activity and Cytotoxicity of Ag(I) and Au(I) Pillarplexes.

The biological activity of four pillarplex compounds featuring different metals and anions was investigated. The toxicity of the compounds against four bacterial strains [Bacillus subtilis (ATCC6633), Staphylococcus aureus (ATCC6538), Escherichia coli (UVI isolate), Pseudomonas aeruginosa], one fungus (Candida albicans), and a human cell line (HepG2) was determined. Additionally, a UV-Vis titration study of the pillarplexes was carried out to check for stability depending on pH- and chloride concentration changes and evaluate the applicability in physiological media. All compounds are bioactive: the silver compounds showed higher activity against bacteria and fungi, and the corresponding gold pillarplexes were less toxic against human cells.

Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo.

The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org .

Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions.

BACKGROUND: Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species. RESULTS: Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene. CONCLUSION: Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.