Antibacterial Effect of the Natural Polymer ε-Polylysine Against Oral Pathogens Associated with Periodontitis and Caries

Antimicrobials are important adjuncts in the treatment of caries and periodontitis. However, increased bacterial resistance and hypersensitivity reactions to commonly used antimicrobials have led to an increasing demand for safe and natural substances. The objective of this study was to investigate the antibacterial effects of ε-polylysine against oral pathogens Streptococcus mutans and Porphyromonas gingivalis. Broth dilution assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses were performed to explore the antibacterial effect of ε-polylysine against S. mutans strain ATCC25175 and P. gingivalis strain ATCC332277. For the test solution, ε-polylysine was added to the bacterial suspension to prepare 0.125%, 0.25%, 0.5% and 1% ε-polylysine solutions diluted in broth medium. All four concentrations demonstrated complete inhibition of S. mutans and significantly reduced viable cell counts of P. gingivalis after 24 h. From starting inoculum of 9.15 log CFU/mL, P. gingivalis cell counts reduced to 4.01 log CFU/mL in the 0.125% ε-polylysine treatment group. SEM, CLSM, and the LIVE/DEAD bacterial assay of ε-polylysine application on P. gingivalis biofilm-dentin specimens revealed bacterial cell membrane disruption and irregular cell morphologies. The results indicated satisfactory antibacterial efficacy of ε-polylysine against P. gingivalis and S. mutans in liquid medium and as an application on biofilm-dentin specimens.

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Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Viruses ◽

10.3390/v10080438 ◽

2018 ◽

Vol 10 (8) ◽

pp. 438 ◽

Cited By ~ 13

Author(s):

Nanna Olsen ◽

Elowine Thiran ◽

Tobias Hasler ◽

Thomas Vanzieleghem ◽

Georgios Belibasakis ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Laser Scanning ◽

Combined Treatment ◽

Viable Cell ◽

Laser Scanning Microscopy ◽

Enzyme Treatment ◽

Confocal Laser ◽

Cell Counts ◽

Glass Surfaces ◽

Individual Enzyme

Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

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Assessing toxicity of titanium dioxide (TiO2) nanoparticles on Pseudomonas species biofilms

10.32920/ryerson.14663781 ◽

2021 ◽

Author(s):

Yevheniya Chabanyuk

Keyword(s):

Titanium Dioxide ◽

Biofilm Formation ◽

Laser Scanning ◽

Titanium Dioxide Nanoparticles ◽

Viable Cell ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Long Term Effects ◽

Software Analysis ◽

Cell Counts

Biofilms are essential to the aquatic environment. Recent advances in technology resulted in increased use of nanomaterials (such as titanium dioxide nanoparticles) and their release into aquatic environments with unknown long-term effects. Potential toxicity of titanium dioxide, known for its photocatalytic properties, on Pseudomonas aeruginosa (PAO1-gfp) and Pseudomonas sp. (CT07-gfp) biofilm formation and proliferation was assessed using flowcells, confocal laser scanning microscopy (CLSM), and total and viable cell release into effluent under different titanium dioxide concentrations (100 ppm, 10 ppm and 1 ppm). COMSTAT software analysis was used to obtain quantitative morphological biofilm data. Results showed that titanium dioxide had a concentration and media-dependent effect on biofilm formation, growth, proliferation and viability. Viable effluent cell counts remained within the same order of magnitude. Biofilm recovery was evident within 24-48 hours after exposure. At environmentally relevant concentration (1 ppm), there was no effect on formation, proliferation or growth of the biofilm.

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Assessing toxicity of titanium dioxide (TiO2) nanoparticles on Pseudomonas species biofilms

10.32920/ryerson.14663781.v1 ◽

2021 ◽

Author(s):

Yevheniya Chabanyuk

Keyword(s):

Titanium Dioxide ◽

Biofilm Formation ◽

Laser Scanning ◽

Titanium Dioxide Nanoparticles ◽

Viable Cell ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Long Term Effects ◽

Software Analysis ◽

Cell Counts

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The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

Microorganisms ◽

10.3390/microorganisms9020335 ◽

2021 ◽

Vol 9 (2) ◽

pp. 335

Author(s):

Novaria Sari Dewi Panjaitan ◽

Yu-Tze Horng ◽

Chih-Ching Chien ◽

Hung-Chi Yang ◽

Ren-In You ◽

...

Keyword(s):

Survival Rate ◽

Klebsiella Pneumoniae ◽

Laser Scanning ◽

Bacterial Resistance ◽

Capsular Polysaccharide ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

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Silver Nanoparticles Formation by Jatropha integerrima and LC/MS-QTOF-Based Metabolite Profiling

Nanomaterials ◽

10.3390/nano11092400 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2400

Author(s):

Afrah E. Mohammed ◽

Lamya Ahmed Al-Keridis ◽

Ishrat Rahman ◽

Modhi O. Alotaibi ◽

Rasha Saad Suliman ◽

...

Keyword(s):

Silver Nanoparticles ◽

Laser Scanning ◽

Antibacterial Effect ◽

Therapeutic Potential ◽

Laser Scanning Microscopy ◽

Ultrastructural Changes ◽

Confocal Laser ◽

Synthesis Methods ◽

Tem Analysis ◽

Average Size

The broad application of metal nanoparticles in different fields encourages scientists to find alternatives to conventional synthesis methods to reduce negative environmental impacts. Herein, we described a safe method for preparing silver nanoparticles (J-AgNPs) using Jatropha integerrima leaves extract as a reducing agent and further characterize its physiochemical and pharmacological properties to identify its therapeutic potential as a cytotoxic and antimicrobial agent. The biogenic synthesized J-AgNPs were physiochemically characterized by ultraviolet-visible spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and energy-dispersive X-ray spectroscopy. HPLC-DAD, followed by LC/MS and the Fourier-transform infrared spectroscopy (FTIR), was applied to detect the biomolecules of J. integerrima involved in the fabrication of NPs. Furthermore, J-AgNPs and the ampicillin-nanocomposite conjugate were investigated for their potential antibacterial effects against four clinical isolates. Finally, cytotoxic effects were also investigated against cancer and normal cell lines, and their mechanism was assessed using TEM analysis and confocal laser scanning microscopy (LSM). Ag ions were reduced to spherical J-AgNPs, with a zeta potential of −34.7 mV as well as an average size of 91.2 and 22.8 nm as detected by DLS and TEM, respectively. HPLC GC/MC analysis identified five biomolecules, and FTIR suggested the presence of proteins besides polyphenolic molecules; together, these molecules could be responsible for the reduction and capping processes during NP formation. Additionally, J-AgNPs displayed a strong antibacterial effect, although the ampicillin conjugated form had a very weak antibacterial effect. Furthermore, the NPs caused a reduction in cell viability of all the treated cells by initiating ultrastructural changes and apoptosis, as identified by TEM and LSM analysis. Therefore, J-AgNPs can be formed using the leaf extract from the J. integerrima plant. Furthermore, J-AgNPs may serve as a candidate for further biochemical and pharmacological testing to identify its therapeutic value.

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Evaluation of the Bactericidal Activity of a Hyaluronic Acid-Vehicled Clarithromycin Antibiotic Mixture by Confocal Laser Scanning Microscopy

Applied Sciences ◽

10.3390/app10080761 ◽

2020 ◽

Vol 10 (8) ◽

pp. 761

Author(s):

Narcisa Mandras ◽

Mario Alovisi ◽

Janira Roana ◽

Paola Crosasso ◽

Anna Luganini ◽

...

Keyword(s):

Hyaluronic Acid ◽

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

Antibacterial Effect ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Scanning Microscopy ◽

Confocal Laser ◽

Dentinal Tubules ◽

The Mean

Confocal laser scanning microscopy (CLSM) was used to evaluate the antibacterial effect and depth of action of a novel clarithromycin-containing triple antibiotic mixture, which was proposed for root canal disinfection in dental pulp regeneration. A previous study reported that this mixture had no tooth discoloration effects in vitro. After infection with Enterococcus faecalis for 3 weeks, the dentinal tubules in the cylindrical root specimens were exposed to different antibiotic mixtures: ciprofloxacin, metronidazole and minocycline (3-MIX); ciprofloxacin, metronidazole and clarithromycin (3-MIXC) and ciprofloxacin and metronidazole (2-MIX). Each antibiotic formulation was mixed with macrogol (MG) or hyaluronic acid (HA) vehicles. CLSM and viability staining were used to quantitatively analyze the mean depth of the antibacterial effect and the proportions of dead and live bacteria inside the dentinal tubules. The 3-MIX and 3-MIXC demonstrated a similar depth of action. The mean proportion of dead bacteria was similar in the 3-MIX and 3-MIXC groups, and both were statistically higher than that of 2-MIX (p = 0.014). Each antibiotic mixture showed a higher bactericidal efficacy if conveyed with HA, compared to MG (3-MIX, p = 0.019; 3-MIXC, p = 0.013 and 2-MIX, p = 0.0125). The depth of action and the antibacterial efficacy of 3-MIXC seemed comparable with 3-MIX.

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Acyl-Substituted Dermaseptin S4 Derivatives with Improved Bactericidal Properties, Including on Oral Microflora

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00750-06 ◽

2006 ◽

Vol 50 (12) ◽

pp. 4153-4160 ◽

Cited By ~ 22

Author(s):

Y. Porat ◽

K. Marynka ◽

A. Tam ◽

D. Steinberg ◽

A. Mor

Keyword(s):

Amino Acids ◽

Fatty Acids ◽

Laser Scanning ◽

Culture Media ◽

Antibacterial Properties ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Oral Diseases ◽

Bactericidal Properties ◽

Oral Pathogens

ABSTRACT The 15-mer dermaseptin S4 derivative S4(1-15) was recently shown to exhibit potent activity against oral pathogens associated with caries and periodontitis. Here, we investigated possible modes for improving the peptide's properties through systematic replacement of an N-terminal amino acid(s) with various fatty acids that modulate the peptide's hydrophobicity and/or charge. Deletion of 1 to 3 residues led to progressive loss of potency as assessed by MIC experiments performed on four test bacteria. Replacing the deleted amino acids with fatty acids most often resulted in potency recovery or improvement, as evidenced by lower MICs and faster bactericidal kinetics in culture media. Best results were obtained after replacement of the N-terminal dipeptide alanine-leucine with heptanoic (C7) or aminododecanoic (NC12) acid. Circular dichroism analysis correlated antibacterial properties to the peptide's secondary structure. MIC experiments and confocal laser scanning microscopy results indicated that C7-S4(3-15) and NC12-S4(3-15) were bactericidal to various oral pathogens, including those which are immobilized in a biofilm. C7-S4(3-15) performed similarly to or better than (depending on growth medium) IB-367, a peptide assessed in clinical trials for treatment of oral mucositis, reducing CFU counts by >3 log units within 2 min of incubation. Collectively, the data indicate that substitution of fatty acids for amino acids may be a useful strategy in revealing improved derivatives of known antimicrobial peptides and suggest the suitability of such compounds for controlling pathogens associated with oral diseases.

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Characterization of Binary Biofilms of Listeria monocytogenes and Lactobacillus and Their Response to Chlorine Treatment

Frontiers in Microbiology ◽

10.3389/fmicb.2021.638933 ◽

2021 ◽

Vol 12 ◽

Author(s):

Magdalena A. Olszewska ◽

Francisco Diez-Gonzalez

Keyword(s):

Listeria Monocytogenes ◽

Laser Scanning ◽

Spatial Organization ◽

Volume Ratio ◽

Single Species ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Interspecies Interactions ◽

Biofilm Thickness ◽

Cell Counts

In nature, Listeria may interact competitively and cooperatively with other organisms, resulting in unique spatial organization and functions for cells within the community. This study was undertaken to characterize the biofilm architecture of binary biofilms of Listeria monocytogenes and Lactobacillus species and to assess their effect on the survival of Listeria during exposure to hypochlorite. Three L. monocytogenes strains, ATCC 19115 (Lm5), ATCC 19117 (Lm7), and Coleslaw (LmC), were selected and combined individually with three Lactobacillus strains: L. fermentum (Lf), L. bavaricus (Lb), and L. plantarum (Lp). In binary Lm-Lp biofilms, the Lm cell counts were similar to single-species biofilms (8.5 log CFU/well), and the Lp cell numbers declined by 1.0 log CFU/well. In the presence of Lb, the Lm cell counts were reduced by 1.5 log CFU/well (p < 0.05), whereas the Lf cell counts increased at least by 3.5 log CFU/well. Confocal laser scanning microscopy (CLSM) determined that interspecies interactions significantly affected the spatial organization of three binary biofilms. Biofilm surface-to-volume ratio increased from 0.8 μm2/μm3 for Lm5 in the monoculture to 2.1 μm2/μm3 for Lm5-Lp in the dual-species model (p < 0.05), and was characterized by a thicker structure with a largely increased surface area. Biofilm roughness increased from 0.2 for Lm7 to 1.0 for Lm7-Lb biofilms (p < 0.05), which appeared as interspecific segregation. Biofilm thickness increased from 34.2 μm for LmC to 46.3 μm for LmC–Lf (p < 0.05), which produced flat and compact structures that covered the entire surface available. The biomass of the extracellular matrix was higher in the case of some binary biofilms (p < 0.05); however, this effect was dependent upon the species pair. When treated with hypochlorite, Lm5 in binary biofilms had an approximately 1.5 log CFU/well greater survival than individually. The unique spatial organization and greater protein production may explain the protective effect of Lp after hypochlorite exposure.

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Functionalising antibiotics with nitroxides as an effective broad-spectrum biofilm eradication strategy.

10.5194/biofilms9-12 ◽

2020 ◽

Author(s):

Anthony Verderosa ◽

Kathryn Fairfull-Smith ◽

Makrina Totsika

Keyword(s):

Laser Scanning ◽

Antimicrobial Agents ◽

Bacterial Species ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Specific Test ◽

E Coli ◽

Cell Counts ◽

Eradication Strategy ◽

New Generation

Background: The adhesion of planktonic bacteria to a surface (biotic or abiotic), and their subsequent ability to aggregate into multicellular communities called biofilms, is a major driving force of failing antibiotic therapy and persistence in chronic infections caused by a variety of pathogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus)&#160;and plaguing healthcare systems worldwide. Biofilms are estimated to be involved in over 80% of all microbial infections in humans, and commonly exhibit extreme resistance to conventional antimicrobial treatments. Consequently, there is an urgent need for novel antimicrobial agents, which target biofilm residing cells. Here, we present the development and evaluation of a new generation of dual-acting nitroxide functionalised antibiotics with potent biofilm eradication activity. Methodology: Synthetic organic chemistry was utilised to produce a new generation of nitroxide functionalised antibiotics with targeted biofilm eradication capabilities. These compounds were tested for biofilm eradication and/or dispersal of several bacterial species using the MBECTM device, a reproducible high-throughput static biofilm formation system. Mature biofilms were treated with serial dilutions of the specific test agent(s) and recovered bacterial numbers were quantified by absorbance spectroscopy at 600 nm or plating for viable cell counts. Treated biofilms were also stained with Live/Dead (SYTO-9/PI) bacterial viability kit and analysed by fluorescence and confocal laser scanning microscopy. Results: Nitroxide functionalised antibiotics exhibit potent biofilm-eradication activity against a variety of medically important pathogens, including P. aeruginosa, uropathogenic E. coli, and S. aureus. In Minimal Biofilm Eradication Concentration (MBEC) assays nitroxide functionalised antibiotics were 64-fold more potent against S. aureus biofilms, and at least 2-fold more potent against uropathogenic E. coli biofilms than the parent antibiotic ciprofloxacin. Conclusions: Currently, antibiotics are often entirely ineffective against biofilm infections. Nitroxide functionalised antibiotics represent a promising new strategy, which could circumvent the resistance of Gram-positive and Gram-negative biofilms to conventional treatments.

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Effect of Propolis Nanoparticles against Enterococcus faecalis Biofilm in the Root Canal

Molecules ◽

10.3390/molecules26030715 ◽

2021 ◽

Vol 26 (3) ◽

pp. 715

Author(s):

Abhishek Parolia ◽

Haresh Kumar ◽

Srinivasan Ramamurthy ◽

Thiagarajan Madheswaran ◽

Fabian Davamani ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Bioactive Compounds ◽

Root Canal ◽

Laser Scanning ◽

Antibacterial Effect ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Internal Diameter ◽

Group I ◽

Significant Difference

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I—saline; group II—propolis 100 µg/mL; group III—propolis 300 µg/mL; group IV—propolis nanoparticle 100 µg/mL; group V—propolis nanoparticle 300µg/mL; group VI—6% sodium hypochlorite; group VII—2% chlorhexidine. Dentin shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal–Wallis and Mann–Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and β-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm−1 band and an increase in the 870 cm−1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

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