Calcium-Phosphate-Osteopontin Particles Reduce Biofilm Formation and pH Drops in in situ Grown Dental Biofilms

This 2-period crossover study investigated the effect of calcium-phosphate-osteopontin particles on biofilm formation and pH in 48-h biofilms grown in situ. Bovine milk osteopontin is a highly phosphorylated glycoprotein that has been shown to interfere with bacterial adhesion to salivary-coated surfaces. Calcium-phosphate-osteopontin particles have been shown to reduce biofilm formation and pH drops in a 5-species laboratory model of dental biofilm without affecting bacterial viability. Here, smooth surface biofilms from 10 individuals were treated ex vivo 6 times/day for 30 min with either calcium-phosphate-osteopontin particles or sterile saline. After growth, the amount of biofilm formed was determined by confocal microscopy, and pH drops upon exposure to glucose were monitored using confocal-microscopy-based pH ratiometry. A total of 160 biofilms were analysed. No adverse effects of repeated ex vivo treatment with calcium-phosphate-osteopontin particles were observed. Particle treatment resulted in a 32% lower amount of biofilm formed (p < 0.05), but large inter-individual differences could be observed. Biofilm pH was significantly higher upon particle treatment, both shortly after the addition of glucose and after 30 min of incubation with glucose (p < 0.05). Calcium-phosphate-osteopontin particles may represent a new therapeutic approach to caries control and aim at directly targeting virulence factors involved in the caries process. Further studies are required to determine the effect of particle treatment on more acidogenic/aciduric biofilms as well as the remineralizing potential of the particles.

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Effects of Experimental Agents Containing Tannic Acid or Chitosan on the Bacterial Biofilm Formation in Situ

Biomolecules ◽

10.3390/biom10091315 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1315

Author(s):

Anton Schestakow ◽

Matthias Hannig

Keyword(s):

Hydrochloric Acid ◽

Tannic Acid ◽

Biofilm Formation ◽

Antibacterial Properties ◽

Positive Control ◽

Bacterial Biofilm ◽

Dental Biofilm ◽

Acid Ph ◽

Bovine Enamel

Chitosan and tannic acid are known for their antibacterial properties. In the present in-situ study, their antibacterial and anti-adherent effects on biofilm formation on enamel were investigated. Six subjects carried upper jaw splints with bovine enamel specimens, allowing in-situ biofilm formation. During the two-day trial, subjects rinsed with experimental solutions that contained either chitosan, tannic acid (pH = 2.5), tannic acid (pH = 7) or hydrochloric acid. Water served as the negative and chlorhexidine as the positive control. Rinsing occurred four or five times following two different rinsing protocols to investigate both the immediate and long-lasting effects. After 48 h of intraoral exposure, the dental plaque was stained with LIVE/DEAD® BacLight, and fluorescence micrographs were evaluated by using the software ImageJ. The results were verified by scanning electron microscopy. Rinsing with chitosan resulted in little immediate antibacterial and anti-adherent effects but failed to show any long-lasting effect, while rinsing with tannic acid resulted in strong immediate and long-lasting effects. Except for a slightly lower antibacterial effect, the neutral solution of tannic acid was as good as the acidic solution. Hydrochloric acid showed neither an antibacterial nor an anti-adherent effect on dental biofilm formation. Experimental solutions containing tannic acid are promising anti-biofilm agents, irrespective of the pH values of the solutions. Chitosan, on the other hand, was not able to prevent biofilm formation.

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Tonsillolith: Not Just a Stone But a Living Biofilm

Otolaryngology ◽

10.1016/j.otohns.2008.05.504 ◽

2008 ◽

Vol 139 (2_suppl) ◽

pp. P94-P95 ◽

Cited By ~ 1

Author(s):

Yosef P Krespi ◽

Anil Arora ◽

Mark Longwell ◽

Laura Nistico ◽

Luanne Hall-Stoodley ◽

...

Keyword(s):

Confocal Microscopy ◽

Acid Production ◽

Ex Vivo ◽

Physiological Activity ◽

Anaerobic Respiration ◽

Acid Fermentation ◽

Chemical Gradients ◽

Dental Biofilm ◽

Acidophilic Bacteria ◽

Oxygen Nitrous Oxide

Problem To study the morphology and activity of tonsilloliths ex vivo, using confocal microscopy and microelectrodes. Understanding the similarities of tonsilloliths to dental biofilm by demonstrating oxygen respiration, de-nitrification, and acid production. Methods Tonsilloliths were harvested from several patients with cryptic pockets and sent to the laboratory under sterile conditions. The tonsilloliths were examined by confocal microscopy to determine the presence and distribution of bacteria. Microelectrodes (dissolved oxygen, nitrous oxide, and pH) were used to measure the rates of aerobic and anaerobic respiration, and acid production, when exposed to saliva (10%) and after sucrose and fluoride addition. Results Morphologically, the tonsilloliths were similar to dental-plaque biofilms, containing ‘corn-cob’ structures, filaments and cocci. The microorganisms respired both oxygen and nitrate in physiological concentrations. The oxygen concentration in the center of the tonsillolith (500 microns) was depleted to approximately 1/10th that of the overlying fluid. The addition of 10% sucrose resulted in the production of acid within the tonsillolith by dropping the pH from 7.3 to 5.8 at a depth of 200 microns. On addition of fluoride (1000 pm) the pH increased to 6.1, suggesting that fluoride suppressed the acid fermentation of sucrose. The profiles showed, aerobic respiration near the top, de-nitrification slightly lower and acidification towards the center. The tonsillolith therefore, had stratified layers, similar to dental (and other) biofilms. The depletion of oxygen and acid production following addition of sucrose may encourage the proliferation of anaerobic/acidophilic bacteria within the tonsillolith. Conclusion Tonsilloliths exhibit biofilm structure and the formation of chemical gradients through physiological activity. Significance While complete or intracapsular tonsillectomy is an option for treating chronic cryptic infections, understanding the morphology and biofilm characteristics of tonsilloliths may stimulate scientists in using limited or non-surgical remedies in treating chronic cryptic infections in the future. Support This study was funded by Valam and Philips Oral Healthcare.

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Actinomyces naeslundii in initial dental biofilm formation

Microbiology ◽

10.1099/mic.0.027706-0 ◽

2009 ◽

Vol 155 (7) ◽

pp. 2116-2126 ◽

Cited By ~ 73

Author(s):

I. Dige ◽

M. K. Raarup ◽

J. R. Nyengaard ◽

M. Kilian ◽

B. Nyvad

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Oral Appliances ◽

Dental Biofilm ◽

Actinomyces Naeslundii ◽

Combined Use ◽

Notable Increase

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics of Actinomyces naeslundii compared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes against A. naeslundii (ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences.

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Effects of Octenidine on the Formation and Disruption of Dental Biofilms: An Exploratory In Situ Study in Healthy Subjects

Journal of Dental Research ◽

10.1177/0022034521999044 ◽

2021 ◽

pp. 002203452199904

Author(s):

B. Reda ◽

J. Dudek ◽

M. Martínez-Hernández ◽

M. Hannig

Keyword(s):

Biofilm Formation ◽

Healthy Subjects ◽

Periodontal Diseases ◽

Oral Diseases ◽

Biofilm Thickness ◽

Dental Biofilm ◽

Octenidine Dihydrochloride ◽

Biofilm Disruption ◽

Low Sensitivity

Dental biofilms are highly structured, complex multispecies communities that, if left untreated, lead to severe oral complications such as caries and periodontal diseases. Therefore, antibiofilm agents are often recommended for both preventive and therapeutic measures. However, biofilm management can be challenging due to the low sensitivity of biofilms to antimicrobial treatments. Octenidine dihydrochloride (OCT) is a highly effective antibacterial agent. Because the OCT antibiofilm efficacy has not been studied in situ, this exploratory crossover study aimed to evaluate the effects of OCT mouth rinsing on biofilm formation and on the disruption of mature biofilms. Moreover, a comparison to the gold-standard chlorhexidine (CHX) was conducted. The biofilms were formed intraorally by 5 healthy volunteers on enamel specimens fixed to acrylic splints. For biofilm formation analysis, OCT, CHX, or water rinses were applied for 30 s every 12 h. The samples evaluation took place at 24-and 48-h time points. For biofilm disruption analysis, sample assessment was performed before and directly after the first OCT or CHX rinse on 48-h mature biofilms. A second rinse was carried out 12 h later. The last assessment was applied to 72-h mature biofilms. The biofilms were analyzed by fluorescence microscopy and transmission electron microscopy. The results showed OCT significantly reducing biofilm formation and bacterial vitality in situ. Simultaneously, the biofilm thickness was strongly decreased. Moreover, a single application of OCT to a 48-h mature biofilm induced substantial biofilm disruption. In addition, the efficacy of OCT compared favorably to CHX. These findings show that OCT rinses prevent biofilm formation and disrupt preexisting mature biofilms formed by healthy subjects. This work suggests that OCT might be used for dental biofilm management as a part of the medical treatment of oral diseases. Future studies with a larger subject heterogeneity and number are needed to confirm the observed OCT effects.

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Action of food preservatives on 14-days dental biofilm formation, biofilm vitality and biofilm-derived enamel demineralisation in situ

Clinical Oral Investigations ◽

10.1007/s00784-013-1053-9 ◽

2013 ◽

Vol 18 (3) ◽

pp. 829-838 ◽

Cited By ~ 5

Author(s):

Nicole Birgit Arweiler ◽

Lutz Netuschil ◽

Daniel Beier ◽

Sebastian Grunert ◽

Christian Heumann ◽

...

Keyword(s):

Biofilm Formation ◽

Food Preservatives ◽

Dental Biofilm ◽

Biofilm Vitality

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Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces

Applied and Environmental Microbiology ◽

10.1128/aem.67.5.2319-2325.2001 ◽

2001 ◽

Vol 67 (5) ◽

pp. 2319-2325 ◽

Cited By ~ 89

Author(s):

Dorthe Bagge ◽

Mette Hjelm ◽

Charlotte Johansen ◽

Ingrid Huber ◽

Lone Gram

Keyword(s):

Biofilm Formation ◽

Shewanella Putrefaciens ◽

Model Systems ◽

Laboratory Model ◽

Flow Conditions ◽

Iron Starvation ◽

Culture Studies ◽

Microbially Induced Corrosion ◽

Steel Surfaces

ABSTRACT Laboratory model systems were developed for studyingShewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 108 CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 102 CFU/cm2) than in a batch system (reaching 107 CFU/cm2), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.

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Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate

Journal of Dairy Research ◽

10.1017/s0022029905001482 ◽

2006 ◽

Vol 73 (1) ◽

pp. 74-78 ◽

Cited By ~ 63

Author(s):

Glenn Walker ◽

Fan Cai ◽

Peiyan Shen ◽

Coralie Reynolds ◽

Brent Ward ◽

...

Keyword(s):

Calcium Phosphate ◽

Amorphous Calcium Phosphate ◽

Tooth Enamel ◽

Bovine Milk ◽

Double Blind ◽

Milk Samples ◽

Randomized Clinical Study ◽

Casein Phosphopeptide ◽

Control Milk

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.

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Tuning the Magnetic Alignment of Cellulose Nanocrystals from Perpendicular to Parallel Using Lepidocrocite Nanoparticles

10.26434/chemrxiv.8061839.v2 ◽

2019 ◽

Author(s):

Valentina Guccini ◽

Sugam Kumar ◽

Yulia Trushkina ◽

Gergely Nagy ◽

Christina Schütz ◽

...

Keyword(s):

Magnetic Field ◽

Magnetic Fields ◽

Cellulose Nanocrystals ◽

Small Angle Neutron Scattering ◽

Lower Amount ◽

Magnetic Alignment ◽

Polarized Optical Microscopy ◽

Parallel Orientation ◽

Weak Magnetic Fields

The magnetic alignment of cellulose nanocrystals (CNC) and lepidocrocite nanorods (LpN), pristine and in hybrid suspensions has been investigated using contrast-matched small-angle neutron scattering (SANS) under in situ magnetic fields (0 – 6.8 T) and polarized optical microscopy. The pristine CNC (diamagnetic) and pristine LpN (paramagnetic) align perpendicular and parallel to the direction of field, respectively. The alignment of both the nanoparticles in their hybrid suspensions depends on the relative amount of the two components (CNC and LpN) and strength of the applied magnetic field. In the presence of 10 wt% LpN and fields < 1.0 T, the CNC align parallel to the field. In the hybrid containing lower amount of LpN (1 wt%), the ordering of CNC is partially frustrated in all range of magnetic field. At the same time, the LpN shows both perpendicular and parallel orientation, in the presence of CNC. This study highlights that the natural perpendicular ordering of CNC can be switched to parallel by weak magnetic fields and the incorporation of paramagnetic nanoparticle as LpN, as well it gives a method to influence the orientation of LpN.<br>

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Capsazepine decreases corneal pain syndrome in severe dry eye disease

Journal of Neuroinflammation ◽

10.1186/s12974-021-02162-7 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Darine Fakih ◽

Adrian Guerrero-Moreno ◽

Christophe Baudouin ◽

Annabelle Réaux-Le Goazigo ◽

Stéphane Mélik Parsadaniantz

Keyword(s):

In Situ Hybridization ◽

Inflammatory Pain ◽

Transient Receptor Potential ◽

Ex Vivo ◽

Receptor Potential ◽

Eye Disease ◽

Ocular Pain ◽

Trpv1 Antagonist ◽

Transient Receptor

Abstract Background Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. Methods Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. Results First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. Conclusion These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

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