Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

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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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The in vivo dynamics of Streptococcus spp., Actinomyces naeslundii, Fusobacterium nucleatum and Veillonella spp. in dental plaque biofilm as analysed by five-colour multiplex fluorescence in situ hybridization

Journal of Medical Microbiology ◽

10.1099/jmm.0.47094-0 ◽

2007 ◽

Vol 56 (5) ◽

pp. 681-687 ◽

Cited By ~ 97

Author(s):

Ali Al-Ahmad ◽

Axel Wunder ◽

Thorsten Mathias Auschill ◽

Marie Follo ◽

Gabriele Braun ◽

...

Keyword(s):

Dental Plaque ◽

Laser Scanning ◽

Fusobacterium Nucleatum ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Actinomyces Naeslundii ◽

Plaque Biofilm ◽

Bacterial Constituents

The formation and composition of dental plaque biofilm in vivo are important factors which influence the development of gingivitis, caries and periodontitis. Studying dental plaque biofilm in in vitro models can cause an oversimplification of the real conditions in the oral cavity. In this study, bovine enamel slabs were fixed in an individual acrylic appliance in situ to quantify dental plaque formation and composition using multiplex fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. Each of the five oligonucleotide probes used for FISH was specific for either eubacteria or one of four frequently isolated bacterial constituents belonging to early and late colonizers of tooth surfaces. The thickness of formed biofilm increased from 14.9±5.0 μm after 1 day to 49.3±11.6 μm after 7 days. Streptococcus spp. were predominant in 1-day-old dental plaque and decreased significantly after 7 days (P=0.0061). Compared to the first day, Fusobacterium nucleatum decreased after 2 days and increased significantly after 7 days (P=0.0006). The decreases of Actinomyces naeslundii content on day 2 and day 7 were significant (P=0.0028). Changes in Veillonella spp. were not significant during the study period (P >0.05). The results showed that an in vivo observation period of 7 days was required to detect significant changes in Streptococcus spp. and F. nucleatum. The multiplex FISH used is suitable for analysing the dynamics of four important bacterial constituents in the oral biofilm in epidemiological studies.

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Effect of Etching Procedures on the Adhesion of Biofilm-Coated Dentin

Materials ◽

10.3390/ma13122762 ◽

2020 ◽

Vol 13 (12) ◽

pp. 2762

Author(s):

Bo-Kyung Jeon ◽

Chang-Ha Lee ◽

A Reum Kim ◽

Seung Hyun Han ◽

Hyun-Jung Kim ◽

...

Keyword(s):

Bond Strength ◽

Biofilm Formation ◽

Laser Scanning ◽

Failure Modes ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Acid Etching ◽

Dentin Surface ◽

Biofilm Removal ◽

Oral Biofilms

Oral biofilms coat all surfaces in the oral cavity including the exposed dentin surface. This study aimed to investigate biofilm removal by acid etching procedures and the effects of the residual biofilm on dentin surfaces on composite–dentin adhesion. Dentin discs were assigned to five groups: no biofilm formation (C); biofilm formation and no surface treatment (BF); biofilm formation and acid etching (BF-E); biofilm formation and acid etching followed by chlorhexidine soaking (BF-EC); and biofilm formation and rubbing with pumice, followed by acid etching (BF-RE). Biofilms were formed on saliva-precoated dentin discs by soaking the discs in Streptococcus mutans (S. mutans) suspension. Biofilm removal from the dentin surface was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and scanning electron microscopy, respectively. To compare the bond strength of the biofilm-coated dentin discs with the surface treatments, specimens were assigned to four groups: no biofilm formation and acid etching (C-E); BF-E; BF-EC; and BF-RE. Assessments of the micro-shear bond strength and subsequent failure modes were performed. BF-E and BF-EC did not remove the biofilm, whereas BF-RE partially removed the biofilm attached to the dentin (p < 0.05). The bond strength of BF-RE was significantly higher than those of BF-E and BF-EC, but lower than that of C-E (p < 0.05). In conclusion, mechanical biofilm removal is recommended before etching procedures to enhance adhesion to the biofilm-coated dentin.

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Effects of Bleaching Associated with Er:YAG and Nd:YAG Laser on Enamel Structure and Bacterial Biofilm Formation

Scanning ◽

10.1155/2021/6400605 ◽

2021 ◽

Vol 2021 ◽

pp. 1-7

Author(s):

Xiuxiu Hou ◽

Keyong Yuan ◽

Zhengwei Huang ◽

Rui Ma

Keyword(s):

Nd:Yag Laser ◽

Biofilm Formation ◽

Laser Scanning ◽

Fusobacterium Nucleatum ◽

Bacterial Biofilm ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Streptococcus Sanguis ◽

Enamel Structure ◽

Significant Difference

Objective. To compare the effects of bleaching associated with Er:YAG and Nd:YAG laser on enamel structure and mixed biofilm formation on teeth surfaces. Materials and Methods. Sixty-eight enamel samples were randomly divided into four groups ( n = 17 ), control, Opalescence Boost only, Opalescence Boost plus Er: YAG laser, and Opalescence Boost plus Nd:YAG laser. The structure was observed using SEM after bleaching. Subsequently, the treated enamel samples were also cultured in suspensions of Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus, and Fusobacterium nucleatum (Fn) for 24 and 48 h. Biofilm formation was quantified by crystal violet staining, and the structure was visualized by confocal laser scanning microscopy. The data were analyzed using the Kruskal-Wallis method. Results. The enamel structure significantly changed after bleaching. There was no obvious difference in the biofilm formation after 24 h; however, after 48 hours, the amount of biofilm increased significantly. Remarkably, the amount was significantly higher on enamel bleached only, however, there was no significant difference between samples bleached with Er:YAG or Nd:YAG laser compared to the control. Conclusions. Bleaching only appeared to markedly promote biofilm formation after 48 h, and the biofilms on samples bleached with Er:YAG or Nd:YAG laser did not change significantly, showing that bleaching with Er:YAG or Nd:YAG laser can be safely applied in clinical practice.

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Impact of Hydrodynamics on Oral Biofilm Strength

Journal of Dental Research ◽

10.1177/0022034509344569 ◽

2009 ◽

Vol 88 (10) ◽

pp. 922-926 ◽

Cited By ~ 41

Author(s):

E. Paramonova ◽

O.J. Kalmykowa ◽

H.C. van der Mei ◽

H.J. Busscher ◽

P.K. Sharma

Keyword(s):

Laser Scanning ◽

Periodontal Diseases ◽

Single Species ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Oral Biofilm ◽

Actinomyces Naeslundii ◽

Mechanical Removal ◽

Hydrodynamic Shear ◽

Oral Biofilms

Mechanical removal of oral biofilms is ubiquitously accepted as the best way to prevent caries and periodontal diseases. Removal effectiveness strongly depends on biofilm strength. To investigate the influence of hydrodynamics on oral biofilm strength, we grew single- and multi-species biofilms of Streptococcus oralis J22, Actinomyces naeslundii TV14-J1, and full dental plaque at shear rates ranging from 0.1 to 50 1/sec and measured their compressive strength. Subsequently, biofilm architecture was evaluated by confocal laser scanning microscopy. Multi-species biofilms were stronger than single-species biofilms, with strength values ranging from 6 to 51 Pa and from 5 to 17 Pa, respectively. In response to increased hydrodynamic shear, biofilm strength decreased, and architecture changed from uniform carpet-like to more “fluffy” with higher thickness. S. oralis biofilms grown under variable shear of 7 and 50 1/sec possessed properties intermediate of those measured at the respective single shears.

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Spatial Arrangements and Associative Behavior of Species in an In Vitro Oral Biofilm Model

Applied and Environmental Microbiology ◽

10.1128/aem.67.3.1343-1350.2001 ◽

2001 ◽

Vol 67 (3) ◽

pp. 1343-1350 ◽

Cited By ~ 56

Author(s):

M. Guggenheim ◽

S. Shapiro ◽

R. Gmür ◽

B. Guggenheim

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Fusobacterium Nucleatum ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Streptococcus Sobrinus ◽

Growth Phenomenon ◽

Associative Behavior ◽

Species Specific

ABSTRACT The spatial arrangements and associative behavior ofActinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, and Streptococcus oralis strains in an in vitro model of supragingival plaque were determined. Using species-specific fluorescence-labeled antibodies in conjunction with confocal laser scanning microscopy, the volumes and distribution of the five strains were assessed during biofilm formation. The volume-derived cell numbers of each strain correlated well with respective culture data. Between 15 min and 64 h, populations of each strain increased in a manner reminiscent of batch growth. The microcolony morphologies of all members of the consortium and their distributions within the biofilm were characterized, as were interspecies associations. Biofilms formed 15 min after inoculation consisted principally of single nonaggregated cells. All five strains adhered strongly to the saliva-conditioned substratum, and therefore, coadhesion played no role during the initial phase of biofilm formation. This observation does not reflect the results of in vitro coaggregation of the five strains, which depended upon the nature of the suspension medium. While the possibility cannot be excluded that some interspecies associations observed at later stages of biofilm formation were initiated by coadhesion, increase in bacterial numbers appeared to be largely a growth phenomenon regulated by the prevailing cultivation conditions.

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Staphylococcus aureus Interferes with Streptococci Spatial Distribution and with Protein Expression of Species within a Polymicrobial Oral Biofilm

Antibiotics ◽

10.3390/antibiotics10020116 ◽

2021 ◽

Vol 10 (2) ◽

pp. 116

Author(s):

Etyene Schnurr ◽

Pune N. Paqué ◽

Thomas Attin ◽

Paolo Nanni ◽

Jonas Grossmann ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Laser Scanning ◽

Bacterial Species ◽

Fusobacterium Nucleatum ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Oral Diseases ◽

Streptococcus Oralis ◽

Oral Environment ◽

Associated Species

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases.

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Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

Applied and Environmental Microbiology ◽

10.1128/aem.00039-11 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3413-3421 ◽

Cited By ~ 21

Author(s):

Rebecca Peyyala ◽

Sreenatha S. Kirakodu ◽

Jeffrey L. Ebersole ◽

Karen F. Novak

Keyword(s):

Laser Scanning ◽

Bacterial Species ◽

Disease Process ◽

Laser Scanning Microscopy ◽

Host Cells ◽

Confocal Laser ◽

Content Type ◽

Oral Biofilms ◽

Multispecies Biofilms

ABSTRACTOral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novelin vitromodel system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprisingStreptococcus gordonii,Streptococcus oralis, andStreptococcus sanguinis;S. gordonii,Actinomyces naeslundii, andFusobacterium nucleatum; orS. gordonii,F. nucleatum, andPorphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

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Novel Broad-Spectrum Antimicrobial Photoinactivation ofIn SituOral Biofilms by Visible Light plus Water-Filtered Infrared A

Applied and Environmental Microbiology ◽

10.1128/aem.02490-14 ◽

2014 ◽

Vol 80 (23) ◽

pp. 7324-7336 ◽

Cited By ~ 34

Author(s):

L. Karygianni ◽

S. Ruf ◽

M. Follo ◽

E. Hellwig ◽

M. Bucher ◽

...

Keyword(s):

Visible Light ◽

Laser Scanning ◽

Treatment Approach ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Pharmaceutical Treatment ◽

Oral Biofilms ◽

Biofilm Bacteria ◽

Bovine Enamel

ABSTRACTAntimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtainedin situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 μg ml−1toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm−2for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.

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Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.02416-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8703-8711 ◽

Cited By ~ 21

Author(s):

L. Karygianni ◽

M. Follo ◽

E. Hellwig ◽

D. Burghardt ◽

M. Wolkewitz ◽

...

Keyword(s):

Laser Scanning ◽

Large Scale ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wide Field ◽

Bacterial Composition ◽

Entire Area ◽

Content Type ◽

Oral Biofilms

ABSTRACTA microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grownin situon BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria,Streptococcusspp., andFusobacterium nucleatumwere stained using specific fluorescencein situhybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.

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