Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50. μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10. μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Variables to be considered when assessing the photocatalytic destruction of bacterial pathogens

The current study sought to assess the importance of three common variables on the outcome of TiO₂ photocatalysis experiments with bacteria. Factors considered were (a) ability of test species to withstand osmotic pressure, (b) incubation period of agar plates used for colony counts following photocatalysis and (c) chemical nature of suspension medium used for bacteria and TiO₂. Staphylococcus aureus, Escherichia coli, Salmonella ser. Typhimurium and Pseudomonas aeruginosa were found to vary greatly in their ability to withstand osmotic pressure, raising the possibility that osmotic lysis may be contributing to loss of viability in some photocatalytic disinfection studies. Agar plate incubation time was also found to influence results, as bacteria treated with UV light only grew more slowly than those treated with a combination of UV and TiO_2. The chemical nature of the suspension medium used was found to have a particularly pronounced effect upon results. Greatest antibacterial activity was detected when aqueous sodium chloride solution was utilised, with ∼1 × 10⁶ CFU mL^-1 S. aureus being completely killed after 60 min. Moderate activity was observed when distilled water was employed with bacteria being killed after 2 h and 30 min, and no antibacterial activity at all was detected when aqueous tryptone solution was used. Interestingly, the antibacterial activity of UV light on its own appeared to be very much reduced in experiments where aqueous sodium chloride was employed instead of distilled water. 

Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens

The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The 'ESKAPE' pathogens (Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure nonthermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360 s, with the exception of A. baumannii where a >4log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens.

Efficacy of instant hand sanitizers against foodborne pathogens compared to hand washing with soap and water in food preparation settings: a systematic review

Hands can be a vector for transmitting pathogenic microorganisms to foodstuffs and drinks, and to the mouths of susceptible hosts. Hand washing is the primary barrier to prevent transmission of enteric pathogens via cross contamination from infected persons. Conventional hand washing involves the use of warm water, soap and friction to remove dirt and microorganisms. Over recent years there has been an increasing availability of hand sanitizing products for use when water and soap are unavailable. The aim of this systematic review was to collate scientific information on the efficacy of hand sanitizers compared to hand washing with soap and water for the removal of foodborne pathogens from the hands of food handlers. An extensive literature search was carried out using three electronic databases - Web of Science, Scopus and PubMed. Twenty-eight scientific publications were ultimately included in the review. Analysis of the literature showed various limitations in the scientific information due to the absence of a standardized protocol to evaluate efficacy of hand products, and variation in experimental conditions applied in different studies. Despite the existence of conflicting results, scientific evidence seems to support the historical scepticism about the use of water-less hand sanitizers in food preparation settings. Water and soap appear to achieve greater removal of soil and microorganisms than water-less products from hands. None of the hand sanitizers tested in the literature seemed to achieve complete inactivation or removal of all foodborne pathogens tested, and the presence of food debris significantly affected inactivation rates of hand products.

Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens

The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes).

Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied.

The association between subgingival periodontal pathogens and systemic inflammation

Aim To investigate associations between periodontal disease pathogens and levels of systemic inflammation measured by C-reactive protein (CRP). Methods A representative sample of dentate 60-70-year-old men in Northern Ireland had a comprehensive periodontal examination. Men taking statins were excluded. Subgingival plaque samples were analysed by quantitative real time PCR to identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. High-sensitivity CRP (mg/l) was measured from fasting blood samples. Multiple linear regression analysis was performed using log-transformed CRP concentration as the dependent variable, with the presence of each periodontal pathogen as predictor variables, with adjustment for various potential confounders. Results A total of 518 men (mean age 63.6 SD 3.0 years) were included in the analysis. Multiple regression analysis showed that body mass index (p < 0.001), current smoking (p < 0.01), the detectable presence of P. gingivalis (p < 0.01) and hypertension (p = 0.01), were independently associated with an increased CRP. The detectable presence of P. gingivalis was associated with a 20% (95% confidence interval 4-35%) increase in CRP (mg/l) after adjustment for all other predictor variables. Conclusion In these 60-70-year-old dentate men, the presence of P. gingivalis in subgingival plaque was significantly associated with a raised level of C-reactive protein.

Antibiofilm Activity of the Brown Alga Halidrys siliquosa against Clinically Relevant Human Pathogens

The marine brown alga Halidrys siliquosa is known to produce compounds with antifouling activity against several marine bacteria. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of organic extracts obtained from the marine brown alga H. siliquosa against a focused panel of clinically relevant human pathogens commonly associated with biofilm-related infections. The partially fractionated methanolic extract obtained from H. siliquosa collected along the shores of Co. Donegal; Ireland; displayed antimicrobial activity against bacteria of the genus Staphylococcus; Streptococcus; Enterococcus; Pseudomonas; Stenotrophomonas; and Chromobacterium with MIC and MBC values ranging from 0.0391 to 5 mg/mL. Biofilms of S. aureus MRSA were found to be susceptible to the algal methanolic extract with MBEC values ranging from 1.25 mg/mL to 5 mg/mL respectively. Confocal laser scanning microscopy using LIVE/DEAD staining confirmed the antimicrobial nature of the antibiofilm activity observed using the MBEC assay. A bioassay-guided fractionation method was developed yielding 10 active fractions from which to perform purification and structural elucidation of clinically-relevant antibiofilm compounds.

Biocontrol agents promote growth of potato pathogens, depending on environmental conditions

There is a pressing need to understand and optimize biological control so as to avoid over-reliance on the synthetic chemical pesticides that can damage environmental and human health. This study focused on interactions between a novel biocontrol-strain, Bacillus sp. JC12GB43, and potato-pathogenic Phytophthora and Fusarium species. In assays carried out in vitro and on the potato tuber, the bacterium was capable of near-complete inhibition of pathogens. This Bacillus was sufficiently xerotolerant (water activity limit for growth = 0.928) to out-perform Phytophthora infestans (~0.960) and challenge Fusarium coeruleum (~0.847) and Fusarium sambucinum (~0.860) towards the lower limits of their growth windows. Under some conditions, however, strain JC12GB43 stimulated proliferation of the pathogens: for instance, Fusarium coeruleum growth-rate was increased under chaotropic conditions in vitro (132 mM urea) by >100% and on tubers (2-M glycerol) by up to 570%. Culture-based assays involving macromolecule-stabilizing (kosmotropic) compatible solutes provided proof-of-principle that the Bacillus may provide kosmotropic metabolites to the plant pathogen under conditions that destabilize macromolecular systems of the fungal cell. Whilst unprecedented, this finding is consistent with earlier reports that fungi can utilize metabolites derived from bacterial cells. Unless the antimicrobial activities of candidate biocontrol strains are assayed over a full range of field-relevant parameters, biocontrol agents may promote plant pathogen infections and thereby reduce crop yields. These findings indicate that biocontrol activity, therefore, ought to be regarded as a mode-of-behaviour (dependent on prevailing conditions) rather than an inherent property of a bacterial strain.

Engineered alpha-defensin peptides display antimicrobial activity against oral pathogens

Introduction: Human alpha defensins are a family of neutrophil-derived antimicrobial peptides also known as human neutrophil peptides (HNPs). The defensin family of peptides are characterised by six invariant cysteine residues forming three disulphide bridges. The formation of the correct disulphide pairs complicates the synthesis of full length human alpha defensin and limits its therapeutic potential as an antimicrobial peptide. Objectives: The aim of this study was to determine whether truncated alpha defensins displayed antimicrobial activity against a range of micro-organisms including oral pathogens. Methods: Engineered peptides were synthesised by solid-phase methods using standard Fmoc chemistry. Antibacterial assays were performed using a previously described ultra sensitive radial diffusion method. A total of five engineered defensin peptides and full length alpha defensin were tested for their sensitivity against eight micro-organisms, including Gram negative bacteria, Gram positive bacteria and fungal pathogens Results: Antimicrobial activity was identified as clear zones around peptide-containing wells. Zone diameters were used to calculate minimum inhibitory concentrations (MICs) for each peptide. There was considerable variability in the susceptibility of the micro-organisms to the truncated analogues. Bacillus subtilis and Enterococcus faecalis were sensitive to the majority of the engineered peptides whereas Staphylococcus aureus, Escherichia coli and Candida albicans displayed resistance (defined as an MIC of greater than 250 ug/ml) to the truncated defensins. Of the five engineered peptides synthesised, the 2-aminobenzoic acid (Abz)-containing analogues based on the C-terminal sequence of alpha defensin displayed MIC values closest to that of the full length defensin in 5 out of 8 micro-organisms studied. Conclusion: This study demonstrates that truncated alpha defensins display variable antimicrobial activity against a range of micro-organisms, including oral pathogens. The generation of truncated defensins without disulphide bridges simplifies their synthesis and increases their therapeutic potential.

Eppin: A Novel Protein Possessing Antimicrobial Properties Against Oral Pathogens

Background: Epididymal protease inhibitor (eppin) is a dual motif protein belonging to the whey acidic protein (WAP) family. Although expressed in numerous different tissues, to date, its functional characterisation is limited. It has been shown to exhibit antibacterial activity against Gram-negative bacteria (Escherichia coli) and antiprotease activity against some proteases of the serine protease family. We are interested in determining the role of eppin in innate immune defence. Objectives: This study aims to determine eppin's potential function in the innate immune response in the oral cavity by investigating the antimicrobial activity of eppin against relevant oral pathogens. Methods: Eppin was recombinantly expressed in E. coli cells and purified by immobilised metal affinity chromatography (IMAC). The antimicrobial effects of the protein were then assessed against two oral pathogens, Fusobacterium nucleatum and Candida albicans, using a double layer radial diffusion assay. Results: Eppin displayed antimicrobial activities against both oral pathogens tested and these activities were shown to be comparable to the well characterised antimicrobial peptide, LL-37. The antifungal effects of eppin were shown to be more potent than those of the human cathelicidin, LL-37. Conclusions: Eppin has been shown to possess both antibacterial and antifungal properties against oral pathogens, suggesting an important role for this protein in the innate immune response in the oral cavity. This study furthers our knowledge of the physiological role exerted by eppin and its possible role in the modulation of chronic diseases such as periodontitis and oral candidiasis.

Validation of a high-throughput immunobead array technique for multiplex detection of three foodborne pathogens in chicken products

This study rigorously evaluated a previously developed immunobead array method to simultaneously detect three important foodborne pathogens, Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp., for its actual application in routine food testing. Due to the limitation of the detection limit of the developed method, an enrichment step was included in this study by using Campylobacter Enrichment Broth for C. jejuni and Universal Pre-enrichment Broth for L. monocytogenes and Salmonella spp.. The findings show that the immunobead array method was capable of detecting as low as 1 CFU of the pathogens spiked in the culture media after being cultured for 24 hours for all three pathogens. The immunobead array method was further evaluated for its pathogen detection capabilities in ready-to-eat (RTE) and ready-to-cook (RTC) chicken samples and proven to be able to detect as low as 1 CFU of the pathogens spiked in the food samples after being cultured for 24 hours in the case of Salmonella spp., and L. monocytogenes and 48 hours in the case of C. jejuni. The method was subsequently validated with three types of chicken products (RTE, n=30; RTC, n=20; raw chicken, n=20) and was found to give the same results as the conventional plating method. Our findings demonstrated that the previously developed immunobead array method could be used for actual food testing with minimal enrichment period of only 52 hours, whereas the conventional ISO protocols for the same pathogens take 90-144 hours. The immunobead array was therefore an inexpensive, rapid and simple method for the food testing.