Phylloseptin-1 (PSN-1) from Phyllomedusa sauvagei skin secretion: a novel broad-spectrum antimicrobial peptide with antibiofilm activity

Amphibian skin secretions have proven to be rich sources of antimicrobial peptides that are proposed to be fundamental components of the innate immune system. As amphibian skin is a multi-functional organ playing, among other things, a crucial role in respiration, it has been deemed that a core biological role for such peptides is control of microbial flora on this surface. To date, however, antimicrobial efficacy has been universally determined by means of establishing minimum inhibitory concentrations (MICs) using planktonic organisms rather than those within a biofilm such as would occur on this exposed surface. Here we describe the identification and structural characterisation of a novel 19 amino acid residue antimicrobial peptide of the phylloseptin family, named PSN-1, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. PSN-1 displayed broad-spectrum activity against a range of planktonic organisms with a high potency (MIC 5 µM) against Staphylococcus aureus. In a specific bioassay with the same organism grown as a biofilm, the minimal biofilm eradication concentration (MBEC) was found to be of the same high potency (5 µM). The present data would suggest that evaluation of actions and potency of amphibian skin secretion antimicrobial peptides might best be achieved by evaluating MBEC rather than MIC using planktonic organisms and that data arising from such studies may have more biological relevance in reflecting the purpose for which they have evolved through natural selection.

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.

Antibiofilm activity, pH and solubility of endodontic sealers

Aim: To evaluate antibiofilm activity against Enterococcus faecalis, pH and solubility of AH Plus, Sealer 26, Epiphany SE, Sealapex, Activ GP, MTA Fillapex (MTA-F) and an experimental MTA-based Sealer (MTA-S). Methodology: Sealer samples were manipulated and stored for 2 or 7 days. Prepared sealers were evaluated by a modified direct contact test (DCT) for 5 h, 10 h or 15 h with biofilm previously induced on bovine dentine for 14 days. In the control group, the biofilm was not exposed to the sealers. The number of colony-forming units (CFU mL-1) in the remaining biofilm was determined. Sealer solubility was assessed by the percentage of mass loss after 15 h of immersion in distilled water. Sealer pH was measured at 5 h, 10 h and 15 h. Statistical analysis was performed using Kruskal-Wallis and Dunn or anova and Tamhane's T2 tests, at 5% significance. Results: At 2 days post-manipulation, the DCT showed that Sealapex and MTA-F were associated with a reduction in the number of bacteria in all 3 contact periods evaluated, compared with the control group (P < 0.05). At 7 days, Sealapex had the greatest antibiofilm action at 10 h and 15 h. Sealapex had the highest pH values 2 and 7 days post-manipulation. Regarding the solubility, at 2 days the highest values were observed for MTA-F, MTA-S, Sealapex and Activ GP (P < 0.05). At 7 days, MTA-S and MTA-F had greater solubility than the other materials (P < 0.05). AH Plus had the lowest solubility for both post-manipulation periods (P < 0.05). Conclusion: Sealapex and MTA-F were associated with a reduction in the number of bacteria in biofilms and had greater solubility. The high solubility and pH may be related to the antibacterial activity of these materials. © 2012 International Endodontic Journal.

Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy

AimTo evaluate the antibiofilm activity of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) solutions associated with cetrimide (CTR), and QMiX using confocal laser scanning microscopy.MethodologyEnterococcus faecalis (ATCC- 29212) biofilms were induced on bovine dentine blocks for 14days. The dentine blocks containing biofilm were immersed for 1min in the following solutions: 2.5% NaOCl; 2.5% NaOCl+0.2% CTR; 2% CHX; 2% CHX+0.2% CTR; 0.2% CTR; QMiX. After contact with the solutions, the dentine blocks were stained with Live/Dead((R)) BacLight for analysis of the remaining biofilm using confocal laser scanning microscope. Images were evaluated using the BioImage_L software to determine the total biovolume (m(3)), the green biovolume (live cells) (m(3)) and the percentage of substrate coverage (%). The data were subjected to nonparametric statistical test using Kruskal-Wallis and Dunn's tests at 5% significance level.ResultsAfter exposure to irrigants, the total biovolume observed for CHX, CHX+CTR, CTR, QMiX was similar to distilled water (P>0.05). NaOCl and NaOCl+CTR had the lowest total and green biovolume. The CTR and QMiX had intermediate green biovolume, with greater antibacterial activity than CHX and CHX+CTR (P<0.05). The NaOCl and NaOCl+CTR solutions were associated with microorganism removal and substrate cleaning ability.ConclusionsNaOCl and NaOCl+CTR solutions were effective on microorganism viability and were able to eliminate biofilm. The addition of cetrimide did not influence antibacterial activity.

Effect of zirconium oxide and zinc oxide nanoparticles on physicochemical properties and antibiofilm activity of a calcium silicate-based material

The aim of the present study was to evaluate the antibiofilm activity against Enterococcus faecalis, compressive strength. and radiopacity of Portland cement (PC) added to zirconium oxide (ZrO2), as radiopacifier, with or without nanoparticulated zinc oxide (ZnO).The following experimental materials were evaluated: PC, PC + ZrO2, PC + ZrO2 + ZnO (5%), and PC + ZrO2 + ZnO (10%). Antibiofilm activity was analyzed by using direct contact test (DCT) on Enterococcus faecalis biofilm, for 5 h or 15 h. The analysis was conducted by using the number of colony-forming units (CFU/mL). The compressive strength was performed in a mechanical testing machine. For the radiopacity tests, the specimens were radiographed together with an aluminium stepwedge. The results were submitted to ANOVA and Tukey tests, with level of significance at 5%. The results showed that all materials presented similar antibiofilm activity (

Antibiofilm activity of Brasilian propolis extracts against Staphylococcus aureus producers isolated from goat and sheep mastitic milk

The aim of this study was to investigate the action of inhibiting S. aureus biofilm formation, and the ability to eliminate formed biofilm, by alcoholic extracts of green, red and brown propolis from Brazil. Ten isolates of S. aureus have been tested, 8 field isolates, 1 MRSA and 1 ATCC 25923, by microplate quantitative method. For the evaluation of inhibitory action, the isolates were inoculated, in triplicate, in TSB 1% glucose in the presence of green (1), red (2) and brown (4) propolis extracts. Biofilm formation was evaluated by optical reading, compared to a negative control consisting of a mixture of TSB and extract. For biofilm elimination assay, extracts were added to plates with 24h cultures of the same isolates. Assays were repeated three times on three different days. Eight out of the 10 isolates produced less biofilm in the presence of the green propolis extracts, so the inhibitory effect is 80%. Brown propolis extracts inhibited the formation of biofilm in 10% to 70% of the isolates and the red extracts in 30% to 80%. Regarding the biofilm elimination activity, green propolis extract was positive for 9 out of the 10 isolates (90%), the brown propolis extracts were positive for 0% to 100% isolates and red extracts for 0% to 10% isolates.

Propolis components accountable for bactericidal accomplishment and antibiofilm activity

This study aimed at evaluating antimicrobial and antibiofilm activity of phenolic compounds present in propolis ethanol extracts (PEE). Seventy per cent ethanol extracts from seven types of propolis, one Green, two Red and four Brown collected in four Brazilian States were prepared and total phenolics, flavonoids, tannins and anthocyanins were assessed by high-performance liquid chromatography (HPLC). Minimum bactericidal concentration (MBC) and inhibitor effect on Staphylococcus aureus biofilm formation and capacity to disrupt established biofilms were assessed towards eight S. aureus isolates from milk of small ruminants with mastitis, one methicillin-resistant S. aureus (MRSA) and S. aureus ATCC 25923. To evaluate different propolis components accountability for bactericidal accomplishment and antibiofilm activity, the results were analysed by the non-parametric Spearman coefficient. Results of phenolic compounds were 216,21 to 312,08 gallic acid milligram equivalent per extract gram (mg EGA/g) of total phenolics, 55,08 to 140,6 quercetin milligram equivalent per extract gram (mg EQ/g) of flavonoids, 118,51 to 3766,16 catechin milligram equivalent per extract gram (mg EC/g) of tannins and 1,03 to 8,39 milligram per extract gram (mg/g) of anthocyanins. Red1 and Red2 showed higher tannin contents, while Red2 exhibited superior amount of anthocyanins and total phenolics. Brown3 presented higher flavonoid quantity. Green, Red1 and Red2 PEE showed the lowest levels of flavonoids, but the higher antimicrobial activity. Most PEE exhibit bactericidal activity at a concentration of 1.6 mg/mL. Brown4 PEE showed the worst capacity to inhibit S. aureus. Green PEE showed to be the most efficient in both preventing and disrupting biofilm. All PEE studied exhibited a better inhibitory activity prior-to than post-biofilm formation. According to non-parametric Spearman correlation analysis, there seems to be a significant negative correlation between the ability to disrupt biofilm and both tannins and anthocyanins contents.

Antimicrobial Peptides with Potential for Biofilm Eradication: Synthesis and Structure Activity Relationship Studies of Battacin Peptides

We report on the first chemical syntheses and structureactivity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methyl-hexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.

Antibiofilm activities of 1-alkyl-3-methylimidazolium chloride ionic liquids

Microbial biofilms are ubiquitous in nature and represent the predominant mode of growth of microorganisms. A general characteristic of biofilm communities is that they tend to exhibit significant tolerance to antimicrobial challenge compared with planktonic bacteria of the same species The antibiofilm activity of a series of 1-alkyl-3-methylimidazolium chloride ionic liquids has been evaluated against a panel of clinically significant microbial pathogens, including MRSA. A comparison of antimicrobial activity against planktonic bacteria and established biofilms is presented. In general, these ionic liquids possess potent, broad spectrum antibiofilm activity.

Antimicrobial and antibiofilm activities of 1-alkylquinolinium bromide ionic liquids

Quinoline derivatives are known to possess a range of bioactive and medicinal activities, which have been exploited in the design of antibacterial, antifungal and antimalarial compounds. In this study, we report on the microbiological toxicity of a series of 1-alkylquinolinium bromides against a range of clinically relevant microorganisms, in both planktonic and sessile (biofilm) cultures. A comparison of antimicrobial activity against planktonic bacteria and established biofilms is presented. In general, 1-alkylquinolinium ionic liquids possess excellent, broad spectrum antimicrobial activity against microorganisms grown in both the planktonic and sessile, or biofilm, mode of growth. Importantly, these compounds are potent against Gram positive and Gram negative bacteria, as well as fungi, with a clear dependency on length of the alkyl substituent for activity, with compounds containing twelve and fourteen carbons in the alkyl group exhibiting highest antimicrobial and antibiofilm activity. © 2010 The Royal Society of Chemistry.

Halophilic microorganisms as a source of novel antibiofilm compounds

Objectives: There is great urgency for alternate sources of antibiotics to be identified. One relatively untapped source of novel bioproducts, including antimicrobials, is organisms derived from extreme environments. Halophiles (which require high salt concentrations) are one such group which is being increasingly explored for their biotechnological potential. The aim of this study was to identify halophilic environmental isolates which possessed in vitro and in vivo antimicrobial and antibiofilm activities. Methods: 73 halophilic bacteria and archaea were isolated from Kilroot salt mine in Northern Ireland. Culture extracts of each isolate were screened for antimicrobial and antibiofilm activity against numerous pathogenic bacteria, including Staphylococcus species and Pseudomonas aeruginosa, both model strains and clinical isolates. The methods used included disc diffusion assays of crude extracts, MIC screening, the MBEC assay, and an in vivo model based on the Greater Wax Moth (Galleria mellonella). Results: The assays indicated >50% of extracts displayed antimicrobial and antibiofilm activity against at least one pathogen, the majority being Staphylococcus species, but also E. coli and P. aeruginosa. Biofilms were either reduced or eradicated by halophile extracts when tested with the MBEC device. Further experiments demonstrated that these effects could be replicated in vivo, with extracts reducing the severity of infections and enhancing the survival of infected G. mellonella. Conclusions: The importance of extremophiles to pharmaceutical research should not be underestimated. While not yet fully characterised, based on the data obtained, the halophiles isolated during this study may provide a promising reservoir of novel antimicrobial and antibiofilm compounds.

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.