Growth and molecular characterization of dental plaque microcosms

McBain, Andrew J.; Bartolo, Robert G.; Catrenich, Carl E.; Charbonneau, Duane; Ledder, Ruth G.; Gilbert, Peter

doi:10.1046/j.1365-2672.2003.01876.x

Cited by 65 publications

(73 citation statements)

References 35 publications

(52 reference statements)

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“…In the first instance, this included a "by-eye" analysis as is common in the literature (27,48). It is relatively easy to comment on three or four patterns and select bands of interest on the basis of visual analysis.…”

Section: Discussionmentioning

confidence: 99%

“…DGGE has been applied in environmental microbiology (6,11,49) and food microbiology (2,5) and in the analysis of microbial communities in the human body (10,14,53,54). Recently DGGE has also been applied to analyze the bacterial diversity of human subgingival plaque (17,58) as well as laboratory-grown dental plaque microcosms (27). This paper presents a fingerprint analysis of dental plaque sampled from the gingival crevice of prepubertal children with and without gingivitis.…”

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confidence: 99%

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Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles

et al. 2005

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Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (n ‫؍‬ 30) and from those with gingivitis (n ‫؍‬ 30). Extracted DNA from gingival margin plaque was subjected to PCR targeting the 16S rRNA gene using universal primers. DGGE profiles were analyzed in three ways. (i) Bacterial diversity was compared between cohorts by using the Shannon-Wiener index (also known as the Shannon-Weaver index). (ii) A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. (iii) Individual DGGE bands and their intensities for both cohorts were compared using a logistic regression analysis. The Shannon-Wiener indices demonstrated a greater bacterial diversity associated with no-gingivitis plaque (P ‫؍‬ 0.009). Dendrograms demonstrated that seven clades associated with gingivitis and five clades associated with no gingivitis. The logistic regression demonstrated that one band was significantly associated with no gingivitis (P ‫؍‬ 0.001), while two bands were significantly associated with gingivitis (P ‫؍‬ 0.005 and P ‫؍‬ 0.042). In conclusion, this study demonstrates that the development of gingivitis might be accompanied by a decrease in bacterial diversity. Furthermore, we have demonstrated that logistic regression is a good statistical method for analyzing and characterizing DGGE profiles.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles

et al. 2005

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“…The gels were left to equilibrate at room temperature overnight in the tank containing 7 liters of 1ϫ Tris-acetate buffer solution. Gel electrophoresis was carried out at 140 V at a constant 60°C for 5.5 h (16,30). All gels were stained using SYBR Gold stain (Molecular Probes, Leiden, The Netherlands) for 30 min with occasional agitation, after which they were transferred to a UV transilluminator (UVP, Upland, CA), visualized under UV light at 312 nm, and photographed using a Canon D60 digital single lens reflex (DSLR) camera (Canon, Surrey, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

Molecular and Culture-Based Assessment of the Microbial Diversity of Diabetic Chronic Foot Wounds and Contralateral Skin Sites

et al. 2012

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Wound debridement samples and contralateral (healthy) skin swabs acquired from 26 patients attending a specialist foot clinic were analyzed by differential isolation and eubacterium-specific PCR-denaturing gradient gel electrophoresis (DGGE) in conjunction with DNA sequencing. Thirteen of 26 wounds harbored pathogens according to culture analyses, with Staphylococcus aureus being the most common (13/13). Candida (1/13), pseudomonas (1/13), and streptococcus (7/13) were less prevalent. Contralateral skin was associated with comparatively low densities of bacteria, and overt pathogens were not detected. According to DGGE analyses, all wounds contained significantly greater eubacterial diversity than contralateral skin (P < 0.05), although no significant difference in total eubacterial diversity was detected between wounds from which known pathogens had been isolated and those that were putatively uninfected. DGGE amplicons with homology to Staphylococcus sp. (8/13) and S. aureus (2/13) were detected in putatively infected wound samples, while Staphylococcus sp. amplicons were detected in 11/13 noninfected wounds; S. aureus was not detected in these samples. While a majority of skin-derived DGGE consortial fingerprints could be differentiated from wound profiles through principal component analysis (PCA), a large minority could not. Furthermore, wounds from which pathogens had been isolated could not be distinguished from putatively uninfected wounds on this basis. In conclusion, while chronic wounds generally harbored greater eubacterial diversity than healthy skin, the isolation of known pathogens was not associated with qualitatively distinct consortial profiles or otherwise altered diversity. The data generated support the utility of both culture and DGGE for the microbial characterization of chronic wounds.

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“…and F. nucleatum, indicating that most species grown in this model could be detected by culture or that more species could not be detected due to the inherent biases of cloning techniques. Similarly, by using denaturing gradient gel electrophoresis to create genetic fingerprints of microcosm communities developed in the CDFF model (17), it was shown that few dominant bands were present, which corresponds to a low diversity of species. Therefore, for this study, to characterize the changes in the communities associated with environmental conditions emulating health and gingivitis, a combination of culture and QPCR for specific species was chosen.…”

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confidence: 99%

“…was identified by 16S rRNA gene sequencing under conditions emulating gingivitis, including A. naeslundii and A. viscosus, which are associated with gingivitis (19,20,26,31). The increased species richness under conditions emulating gingivitis involved the emergence of species not typical of the oral cavity, such as Citrobacter spp., which have been isolated from other models of supragingival plaque (17).…”

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confidence: 99%

Use of Quantitative PCR and Culture Methods To Characterize Ecological Flux in Bacterial Biofilms

2007

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An in vitro model of supragingival plaque associated with gingivitis was characterized by traditional culture techniques, comparative 16S rRNA gene sequencing of isolates, and quantitative PCR (QPCR). Actinomyces naeslundii, Prevotella spp., and Porphyromonas gingivalis increased under conditions emulating gingivitis. Gram-negative species and total bacteria were dramatically underestimated by culture compared to the estimates obtained by QPCR.Inadequate oral hygiene leads to dental plaque accumulation around the gingival margin, resulting in inflammation of the gingiva. The environmental changes which occur as a result drive the ecological changes in the oral microbiota at this site, such as the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. (19,20,26,31). With the advance of molecular techniques such as cloning and sequencing of the bacterial 16S rRNA gene, a greater proportion of species present in the oral cavity than the proportion that can be detected by traditional culture techniques has been detected, and the number of bacterial species present in the dental plaque is now estimated to be upwards of 630 (8). Quantitative PCR (QPCR) has the potential to account for the uncultivable portion of the oral microbial community as well as species which are more difficult to culture, such as Porphyromonas gingivalis (14,22), Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans) (3), and oral treponemes (2).Using the constant-depth film fermentor (CDFF) model for oral biofilms, we have previously demonstrated reproducible population shifts in the microbial community associated with gingivitis by altering the environmental conditions (4). The aim of this study was to identify the range of cultivable species present in this in vitro system and to monitor changes in specific species and genera as a response to changing environmental conditions by QPCR.Dental plaque biofilms associated with health and gingivitis were produced in the CDFF, removed aseptically at various time points, and analyzed by selective cultural analysis by using methods described previously (4). Total anaerobic counts were obtained on fastidious anaerobe agar (FAA; Bioconnections, Leeds, United Kingdom), Actinomyces spp. were selected for on cadmium fluoride acriflavine tellurite plates (CFAT) (32), Streptococcus spp. were selected for on mitis-salivarius agar (MS; BD Biosciences, Oxford, United Kingdom), and gramnegative species were selected for on Columbia blood agar with a supplement for gram-negative organisms (GN; Oxoid).The identification of cultivable species was carried out by comparative 16S rRNA gene sequencing. At various sampling time points, all the different colony morphotypes were subcultured on selective media (MS, CFAT, and GN) and 15 random isolates were subcultured from the nonselective media (FAA and Columbia blood agar) for identification. The primers used for 16S rRNA PCR are described in Table 1 and were used under the conditions described previousl...

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Growth and molecular characterization of dental plaque microcosms

Cited by 65 publications

References 35 publications

Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles

Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles

Molecular and Culture-Based Assessment of the Microbial Diversity of Diabetic Chronic Foot Wounds and Contralateral Skin Sites

Use of Quantitative PCR and Culture Methods To Characterize Ecological Flux in Bacterial Biofilms

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