scholarly journals Low Concentrations of Chlorhexidine Inhibit the Formation and Structural Integrity of Enzyme-Treated Multispecies Oral Biofilms

2021 ◽  
Vol 12 ◽  
Author(s):  
Kay Andrin Gränicher ◽  
Lamprini Karygianni ◽  
Thomas Attin ◽  
Thomas Thurnheer
Keyword(s):  
Structural Integrity ◽  
Fluorescent Dyes ◽  
Combined Treatment ◽  
Extracellular Proteins ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Anaerobic Growth ◽  
Low Concentration ◽  
Low Concentrations ◽  
Dental Hard Tissues

The self-produced matrix of biofilms, consisting of extracellular polymeric substances, plays an important role in biofilm adhesion to surfaces and the structural integrity of biofilms. In dentistry, biofilms cause multiple diseases such as caries, periodontitis, and pulpitis. Disruption of these biofilms adhering to dental hard tissues may pose a major challenge since biofilms show higher tolerance to antimicrobials and antibiotics than planktonic cells. In this study, the effect of low concentrations of chlorhexidine (CHX) on enzyme-treated multispecies oral biofilm was investigated in an in vitro model. Six-species biofilms were enzymatically treated by anaerobic growth in a medium containing DNase I and proteinase K. Biofilms were exposed to a low concentration of CHX at defined time points. After 64h, biofilms were either harvested and quantified by cultural analyses or stained for confocal laser scanning microscopy (CLSM) analyses using either Live/Dead kit or different fluorescent dyes. A mixture of YoPro1 and SYTOX™ Green, Fluorescent Brightener 28 (Calcofluor), and SYPRO™ Ruby Protein Gel Stain was used to stain total DNA, exopolysaccharides, and extracellular proteins, respectively. Extracellular DNA (eDNA) was visualized via an indirect immunofluorescence assay (Mouse anti-DNA IgG, Goat anti-Mouse IgG, Streptavidin-Cy3). Overall, the total colony-forming units significantly decreased after combined treatment with a low concentration of CHX and enzymes compared to the group treated with CHX alone (p<0.001). These findings also apply to five species individually (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Veillonella dispar, and Candida albicans) occurring in the biofilms, with Fusobacterium nucleatum being the only exception. Furthermore, CLSM images showed less dense biofilms and a reduction in cell numbers after combined treatment compared to the group without enzymes. The combination of enzymes capable of disturbing the matrix integrity with antimicrobial agents thus appears to be a promising approach for biofilm disruption and killing.

scholarly journals 1640. Toward New Anti-Biofilm Therapies: High Mobility Group Box 1 (HMGB1) Protein and Its Structural Variants Can Be Used to Disrupt Bacterial Biofilms (BBs)

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S46-S46
Author(s):  
Aspasia Katragkou ◽  
Lauren Warren ◽  
John Buzzo ◽  
Steven Goodman
Keyword(s):  
Laser Scanning ◽  
Structural Integrity ◽  
Public Health Problem ◽  
Full Length ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Major Public Health Problem ◽  
Average Thickness ◽  
Research Support

Abstract Background BB-related infections are a major public health problem, as they are notoriously refractory to current treatments. One of the defining characteristics of BBs is the extracellular polymeric substance (EPS). Extracellular DNA and the bacterial DNABII family of proteins are key components of EPS and are crucial for BBs structural integrity. It is known that targeting DNABII proteins disrupts BBs. We hypothesized that HMGB1, a DNA-binding eukaryotic protein, could affect BBs as it binds to the same DNA structures as the DNABII proteins. HMGB1 is comprised of 3 domains, A Box, B Box, and C tail, all of which have different functions. We aimed to determine in vitro the effects of HMGB1 and its individual domains against BBs. Methods Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for all BBs disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1–215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1–89), B Box (90–176), AB Boxes (1–176), B-linker Box (80–179), and B-linker Box C106S were expressed (in E. coli) and purified to >95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to preformed BBs at 24 hours. At 40 hours the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass. Results Exogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (P < 0.05) in average thickness (AT) and biomass (BM) of KP biofilms when compared with untreated KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15 and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively). Conclusion Full-length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections. Disclosures J. Buzzo, ProclaRx: Collaborator, Research support. S. Goodman, ProclaRx: Collaborator and Scientific Advisor, Research support.

scholarly journals Combined DNase and Proteinase Treatment Interferes with Composition and Structural Integrity of Multispecies Oral Biofilms

2020 ◽  
Vol 9 (4) ◽  
pp. 983 ◽  
Author(s):  
Lamprini Karygianni ◽  
Thomas Attin ◽  
Thomas Thurnheer
Keyword(s):  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Structural Integrity ◽  
Dnase I ◽  
Laser Scanning Microscopy ◽  
Proteinase K ◽  
Confocal Laser ◽  
Dental Hard Tissues ◽  
Oral Biofilms ◽  
The Impact

Modification of oral biofilms adhering to dental hard tissues could lead to new treatment approaches in cariology and periodontology. In this study the impact of DNase I and/or proteinase K on the formation of a simulated supragingival biofilm was investigated in vitro. Six-species biofilms were grown anaerobically in the presence of DNase I and proteinase K. After 64 h biofilms were either harvested and quantified by culture analysis or proceeded to staining followed by confocal laser scanning microscopy. Microbial cells were stained using DNA-dyes or fluorescent in situ hybridization. Exopolysaccharides, eDNA and exoproteins were stained with Calcofluor, anti-DNA-antibody, and SyproTM Ruby, respectively. Overall, results showed that neither DNase I nor proteinase K had an impact on total colony-forming units (CFUs) compared to the control without enzymes. However, DNase I significantly suppressed the growth of Actinomyces oris, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus oralis and Candida albicans. Proteinase K treatment induced significant increase in S. mutans and S. oralis CFUs (p < 0.001), whereas C. albicans and V. dispar showed lower CFUs compared to the control. Interestingly, confocal images visualized the biofilm degradation caused by DNase I and proteinase K. Thus, enzymatic treatment should be combined with conventional antimicrobial agents aiming at both bactericidal effectiveness and biofilm dispersal.

scholarly journals Effect of silver nanoparticles on human mesenchymal stem cell differentiation

2014 ◽  
Vol 5 ◽  
pp. 2058-2069 ◽  
Author(s):  
Christina Sengstock ◽  
Jörg Diendorf ◽  
Matthias Epple ◽  
Thomas A Schildhauer ◽  
Manfred Köller
Keyword(s):  
Silver Nanoparticles ◽  
Cell Differentiation ◽  
Osteogenic Differentiation ◽  
Stem Cell Differentiation ◽  
Laser Scanning Microscopy ◽  
Surgical Instruments ◽  
Dependent Manner ◽  
Low Concentration ◽  
Long Term Treatment

Background: Silver nanoparticles (Ag-NP) are one of the fastest growing products in nano-medicine due to their enhanced antibacterial activity at the nanoscale level. In biomedicine, hundreds of products have been coated with Ag-NP. For example, various medical devices include silver, such as surgical instruments, bone implants and wound dressings. After the degradation of these materials, or depending on the coating technique, silver in nanoparticle or ion form can be released and may come into close contact with tissues and cells. Despite incorporation of Ag-NP as an antibacterial agent in different products, the toxicological and biological effects of silver in the human body after long-term and low-concentration exposure are not well understood. In the current study, we investigated the effects of both ionic and nanoparticulate silver on the differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, osteogenic and chondrogenic lineages and on the secretion of the respective differentiation markers adiponectin, osteocalcin and aggrecan. Results: As shown through laser scanning microscopy, Ag-NP with a size of 80 nm (hydrodynamic diameter) were taken up into hMSCs as nanoparticulate material. After 24 h of incubation, these Ag-NP were mainly found in the endo-lysosomal cell compartment as agglomerated material. Cytotoxicity was observed for differentiated or undifferentiated hMSCs treated with high silver concentrations (≥20 µg·mL−1 Ag-NP; ≥1.5 µg·mL−1 Ag+ ions) but not with low-concentration treatments (≤10 µg·mL−1 Ag-NP; ≤1.0 µg·mL−1 Ag+ ions). Subtoxic concentrations of Ag-NP and Ag+ ions impaired the adipogenic and osteogenic differentiation of hMSCs in a concentration-dependent manner, whereas chondrogenic differentiation was unaffected after 21 d of incubation. In contrast to aggrecan, the inhibitory effect of adipogenic and osteogenic differentiation was confirmed by a decrease in the secretion of specific biomarkers, including adiponectin (adipocytes) and osteocalcin (osteoblasts). Conclusion: Aside from the well-studied antibacterial effect of silver, little is known about the influence of nano-silver on cell differentiation processes. Our results demonstrate that ionic or nanoparticulate silver attenuates the adipogenic and osteogenic differentiation of hMSCs even at non-toxic concentrations. Therefore, more studies are needed to investigate the effects of silver species on cells at low concentrations during long-term treatment.

scholarly journals Propidium iodide staining underestimates viability of adherent bacterial cells

2018 ◽  
Author(s):  
Merilin Rosenberg ◽  
Nuno F. Azevedo ◽  
Angela Ivask
Keyword(s):  
Dna Binding ◽  
Propidium Iodide ◽  
Laser Scanning Microscopy ◽  
Red Cells ◽  
Extracellular Dna ◽  
Dead Layer ◽  
Confocal Laser ◽  
Adherent Cells ◽  
Bacterial Viability ◽  
Viability Staining

AbstractCombining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids. We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at extracellular DNA (eDNA) being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Morphological Observation of the Cashmere Goat Fetal Fibroblasts after mTOR Kinase Inhibition with Combination of Fluorescent Dyes and Confocal Cell Imaging

2011 ◽  
Vol 343-344 ◽  
pp. 590-593
Author(s):  
Yan Liang ◽  
Xiao Jing Wang ◽  
Shu Yu Li ◽  
Cheberi ◽  
Zhi Gang Wang ◽  
...  
Keyword(s):  
Inner Mongolia ◽  
Mammalian Cells ◽  
Fluorescent Dyes ◽  
Cell Structure ◽  
Critical Role ◽  
Laser Scanning Microscopy ◽  
Morphological Observation ◽  
Cashmere Goat ◽  
Mtor Kinase ◽  
Fetal Fibroblasts

The mammalian target of rapamycin（mTOR）is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from nutrients, growth factors, energy and environmental stress to regulate cell growth and proliferation via different cellular processes. This study uses the fetal fibroblasts of Inner Mongolia Cashmere goat (Capra hircas) to prove that the mTOR plays a critical role in formation of the cell cytoskeleton structure. The mTOR kinase activity was inhibited in Inner Mongolia Cashmere goat fetal fibroblasts (GFb) after treatment with CCI-779 (temsirolimus), an mTOR specific inhibitor for 48 h. The results showed that GFb cells were sensitive to CCI-779. GFb cells morphology and its cytoskeleton structure changed under confocal laser scanning microscopy stained with the Fluorescent phalloidin (50µg/ml, Phalloidin-FITC5282) which combines with F-actin. In summary, mTOR signaling pathway was proved to be functional in GFb cells and acts as a key regulator to form cell structure. The Morphological results indicated that perhaps the synthesis of microfilament or organization of cytoskeleton was disrupted in GFb cells when mTOR was inhibited.

scholarly journals Low concentrations of commercial alcohol hand rubs facilitate growth of and secretion of extracellular proteins by multidrug-resistant strains of Acinetobacter baumannii

2007 ◽  
Vol 56 (12) ◽  
pp. 1595-1599 ◽  
Author(s):  
Justin Edwards ◽  
Geeta Patel ◽  
David W. Wareham
Keyword(s):  
Acinetobacter Baumannii ◽  
Culture Supernatant ◽  
Multidrug Resistant ◽  
Extracellular Proteins ◽  
Nosocomial Pathogen ◽  
Emulsifying Activity ◽  
Health Care Settings ◽  
Low Concentrations ◽  
Resistant Strains ◽  
Minimal Media

Acinetobacter baumannii is increasingly recognized as an important multidrug-resistant nosocomial pathogen. Recent work has highlighted enhanced growth and heightened virulence in the presence of ethyl alcohols. As alcohol-based hand rubs (ABHRs) are extensively used in health care settings, the authors set out to determine whether the hand rubs could also influence the growth of prevalent multidrug-resistant strains circulating in UK hospitals. A significant increase in growth was observed when minimal media were supplemented with concentrations of 1 % and lower of four commercially available hand rubs. In addition, growth in ABHR-supplemented media resulted in secretion of proteins into the culture supernatant. One of these was identified as OmpA, which is recognized as having emulsifying activity, which could potentially confer enhanced pathogenicity to A. baumannii.

Transepithelial movement of calcium in crustaceans

1993 ◽  
Vol 184 (1) ◽  
pp. 1-16 ◽  
Author(s):  
D. S. Neufeld ◽  
J. N. Cameron
Keyword(s):  
Active Transport ◽  
Calcium Concentration ◽  
Cellular Mechanism ◽  
The Body ◽  
High Rate ◽  
Calcium Flux ◽  
Moult Cycle ◽  
Electrochemical Gradient ◽  
Low Concentration ◽  
Low Concentrations

The regulation of calcium in most crustaceans is especially challenging owing to the highly mineralized cuticle that must be recalcified after each moult, a process that often occurs in environments with low concentrations of calcium. The gill and carapace epithelia separate the major calcium-containing compartments of the body and therefore see large changes in the rate of calcium flux through the moult cycle. Large changes in the ultrastructure of these cells do not, however, correlate well with the periods of calcium movement and probably reflect other physiological events. Despite the challenges to regulating calcium levels at various acclimation salinities and moult stages, the calcium concentration in the blood is maintained relatively constant. There is a rapid increase to a high rate of calcium flux across both the gill and carapace epithelium shortly after the moult; on an area-specific basis these fluxes are among the highest reported for calcium-transporting epithelia. When in water with a very low concentration of calcium, the electrochemical gradient for calcium is directed outwards and net influx must occur by active transport. Evidence suggests that changes in the electrochemical gradient, permeability and active transport are all important in the ability of crustaceans to take up calcium from water with a low concentration of this ion. Although an enzyme transporter is presumably involved in the active transport of calcium across epithelia, very little is known about the cellular mechanism of the transepithelial movement of calcium in crustaceans.

scholarly journals Effect of Sodium Hypochlorite and Benzalkonium Chloride on the Structural Parameters of the Biofilms Formed by Ten Salmonella enterica Serotypes

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 154 ◽  
Author(s):  
Rosa Capita ◽  
Silvia Fernández-Pérez ◽  
Laura Buzón-Durán ◽  
Carlos Alonso-Calleja
Keyword(s):  
Sodium Hypochlorite ◽  
Salmonella Enterica ◽  
Benzalkonium Chloride ◽  
Laser Scanning ◽  
Surface Coverage ◽  
Structural Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Low Concentrations ◽  
Practical Field

The influence of the strain on the ability of Salmonella enterica to form biofilms on polystyrene was investigated by confocal laser scanning microscopy. The effects of sodium hypochlorite with 10% active chlorine (SHY; 25,000, 50,000, or 100,000 ppm), and benzalkonium chloride (BZK; 1000, 5000, or 10,000 ppm) on twenty-four-hour-old biofilms was also determined. The biofilms of ten Salmonella enterica isolates from poultry (S. Agona, S. Anatum, S. Enteritidis, S. Hadar, S. Infantis, S. Kentucky, S. Thompson, S. Typhimurium, monophasic variant of S. Typhimurium 1,4,(5),12:i:-, and S. Virchow) were studied. Biofilms produced by S. Anatum, S. Hadar, S. Kentucky, and S. Typhimurium showed a trend to have the largest biovolume and the greatest surface coverage and thickness. The smallest biofilms (P < 0.01) in the observation field (14.2 × 103 µm2) were produced by S. Enteritidis and S. 1,4,(5),12:i:- (average 12.9 × 103 ± 9.3 × 103 µm3) compared to the rest of the serotypes (44.4 × 103 ± 24.7 × 103 µm3). Biovolume and surface coverage decreased after exposure for ten minutes to SHY at 50,000 or 100,000 ppm and to BZK at 5000 or 10,000 ppm. However, the lowest concentrations of disinfectants increased biovolume and surface coverage in biofilms of several strains (markedly so in the case of BZK). The results from this study suggest that the use of biocides at low concentrations could represent a public health risk. Further research studies under practical field conditions should be appropriate to confirm these findings.

Confocal Laser Scanning Microscopy of Hamster Cerebellum using FM4-64 as Intracellular Staining

1998 ◽  
Vol 4 (S2) ◽  
pp. 1126-1127
Author(s):  
O. Castejón ◽  
P. Sims
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Granular Layer ◽  
Fluorescent Dyes ◽  
Laser Scanning Microscopy ◽  
Climbing Fibers ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Fluorescence Signal

Confocal laser scanning microscopy is an excellent method to study nerve cell morphology and the three-dimensional distribution and interrelationship of dentrites and axons in the central nervous system. The cerebellum has been taking as a model of a gray center.The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum,5 mm thick, were incubated in 10,30 and 100 μm solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances .They exhibited high fluorescence signal at the level of myelin sheath.Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fibers bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies.

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