Orange Complex Species Research Articles

Peri-Implantitis-Associated Microbiota before and after Peri-Implantitis Treatment, the Biofilm “Competitive Balancing” Effect: A Systematic Review of Randomized Controlled Trials

This systematic review of RCTs aimed to characterize short- and long-term changes in peri-implantitis-associated microbiota (total biofilm microbial load and predominant pathogens’ counts) following (any) peri-implantitis treatment in systemically healthy, non-smoking, partially/totally edentulous adults. The study protocol, compliant with the PRISMA statement, was registered on PROSPERO (CRD42024514521) before the literature search. Data from 11 RCTs, assessed through the ROBINS-2 tool, were qualitatively synthesized. No data were retrieved on total edentulism, healthy peri-implant/periodontal sites, treated mucositis, gingivitis, and periodontitis sites. Shortly after treatment, Prevotella intermedia, Fusobacterium nucleatum, and Peptostreptococcus micros prevailed, indicating early colonization, as after implant placement. After both surgical and non-surgical approaches, although not eradicated, the peri-implant total biofilm load, red- and orange-complex species, and Aggregatibacter actinomycetemcomitans counts generally decreased for up to about three months. However, one month after treatment, red-complex species and Prevotella intermedia increased, likely due to persistent tissue-invasive bacteria, unresolved pathological conditions (high probing depth values) favoring anaerobiosis and dysbiosis, and a qualitatively and quantitatively decreased biofilm community, competing and balancing the predominant pathogens (biofilm “competitive balancing” effect), thus allowing recolonization by more virulent bacteria. Red-complex bacteria gradually leveled off to baseline at the six- and twelve-month follow-ups. Fusobacterium nucleatum remained almost unchanged after treatment.

Assessment of microbial contamination in removable orthodontic appliances with and without the use of antimicrobial agents by checkerboard DNA-DNA hybridization analysis

This clinical, crossover, double-blind trial evaluated the microbial contamination of removable orthodontic appliances used by children and the efficacy of 0.12% chlorhexidine gluconate spray use for disinfection. Twenty children aged 7-11 years were instructed to wear removable orthodontic appliances for 1 week. They were instructed to use a placebo solution (control) or 0.12% chlorhexidine gluconate (experimental) to clean the appliances on days 4 and 7 after installation. After this period, the microbial contamination on the surfaces of the appliance was analyzed using checkerboard DNA-DNA hybridization for 40 bacterial species. Data were analyzed by Fisher exact, t, and Wilcoxon tests (α= 0.05). Removable orthodontic appliances were heavily contaminated by the target microorganisms. Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, and Eikenella corrodens were found in 100% of the appliances. Among cariogenic microorganisms, Streptococcus mutans and Streptococcus sobrinus were more abundant than Lactobacillus acidophilus and Lactobacillus casei. Red complex pathogens were more abundant than orange complex species. Purple complex bacteria were the most prevalent among bacterial complexes not associated with specific pathologies, detected in 34% of the samples. After the use of chlorhexidine, the number of cariogenic microorganisms (S. mutans, S. sobrinus, and L. casei) decreased significantly (P<0.05), and the numbers of periodontal pathogenic species from the orange and red complex also decreased significantly (P<0.05). There was no reduction for Treponema socranskii. Removable orthodontic appliances were densely contaminated by several bacterial species. Twice-a-week application of chlorhexidine spray effectively reduced cariogenic and orange and red complex periodontal pathogens.

Full-mouth scaling and root planing in type 2 diabetic subjects: one-year microbiological outcomes.

This study evaluated the microbiological effects of full-mouth (FM) and partial-mouth (PM) scaling and root planing (SRP) in type 2 diabetic subjects with chronic periodontitis (ChP), up to 12months. Thirty-four type 2 diabetic subjects with ChP received either FMSRP (n = 17), in two sessions within two consecutive days, or PMSRP (n = 17) in four sessions within 21days. Six subgingival biofilm samples per subject were analysed by checkerboard DNA-DNA hybridization for 40 bacterial species at baseline, 3 and 12months. Both therapies significantly reduced the levels of the red complex species up to 12months (p < 0.05). The levels of three putative pathogens from the orange complex were significantly reduced in the FMSRP group, whereas a single orange complex species was significantly decreased in the PMSRP group (p < 0.05). Furthermore, the proportions of the host-compatible Actinomyces species were significantly increased in both groups at 3 and 12months. No significant differences were observed between groups for the counts and proportions of the individual species and the proportions of microbial complexes at any time point (p > 0.05). There were no differences in the bacterial species evaluated after FMSRP and PMSRP in the treatment of type 2 diabetic subjects with ChP, up to 12months.

Transcriptional Responses of Treponema denticola to Other Oral Bacterial Species

The classic organization by Socransky and coworkers categorized the oral bacteria of the subgingival plaque into different complexes. Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia are grouped into the red complex that is highly correlated with periodontal disease. Socransky's work closely associates red with orange complex species such as Fusobacterium nucleatum and Prevotella intermedia but not with members of the other complexes. While the relationship between species contained by these complexes is in part supported by their ability to physically attach to each other, the physiological consequences of these interactions and associations are less clear. In this study, we employed T. denticola as a model organism to analyze contact-dependent responses to interactions with species belonging to the same complex (P. gingivalis and T. forsythia), the closely associated orange complex (using F. nucleatum and P. intermedia as representatives) and the unconnected yellow complex (using Streptococcus sanguinis and S. gordonii as representatives). RNA was extracted from T. denticola alone as well as after pairwise co-incubation for 5 hrs with representatives of the different complexes, and the respective gene expression profiles were determined using microarrays. Numerous genes related to motility, metabolism, transport, outer membrane and hypothetical proteins were differentially regulated in T. denticola in the presence of the tested partner species. Further analysis revealed a significant overlap in the affected genes and we identified a general response to the presence of other species, those specific to two of the three complexes as well as individual complexes. Most interestingly, many predicted major antigens (e.g. flagella, Msp, CTLP) were suppressed in responses that included red complex species indicating that the presence of the most closely associated species induces immune-evasive strategies. In summary, the data presented here provide an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket.

Microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease

To monitor microbial shifts during dental biofilm re-development. Supra- and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects at baseline and immediately after tooth cleaning. Samples were taken again from seven teeth in randomly selected quadrants during 1, 2, 4 and 7 days of no oral hygiene. Samples were analysed using checkerboard DNA-DNA hybridization. Species counts were averaged within subjects at each time point. Significant differences in the counts between healthy and periodontitis subjects were determined using the Mann-Whitney test. The total supra- and subgingival counts were significantly higher in periodontitis on entry and reached or exceeded the baseline values after day 2. Supragingival counts of Veillonella parvula, Fusobacterium nucleatum ss vincentii and Neisseria mucosa increased from 2 to 7 days. Subgingival counts were greater for Actinomyces, green and orange complex species. Significant differences between groups in supragingival counts occurred for 17 of 41 species at entry, 0 at day 7; for subgingival plaque, these values were 39/41 taxa at entry, 17/41 at day 7. Supragingival plaque re-development was similar in periodontitis and health, but subgingival species recolonization was more marked in periodontitis.

Relationships between subgingival microbiota and GCF biomarkers in generalized aggressive periodontitis

To examine relationships between subgingival biofilm composition and levels of gingival crevicular fluid (GCF) cytokines in periodontal health and generalized aggressive periodontitis (GAP). Periodontal parameters were measured in 25 periodontally healthy and 31 GAP subjects. Subgingival plaque and GCF samples were obtained from 14 sites from each subject. Forty subgingival taxa were quantified using checkerboard DNA-DNA hybridization and the concentrations of eight GCF cytokines were measured using Luminex. Cluster analysis was used to define sites with similar subgingival microbiotas in each clinical group. Significance of differences in clinical, microbiological and immunological parameters among clusters was determined using the Kruskal-Wallis test. GAP subjects had statistically significantly higher GCF levels of interleukin-1beta (IL-1beta) (p<0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (p<0.01) and IL-1beta/IL-10 ratio (p<0.001) and higher proportions of Red and Orange complex species than periodontally healthy subjects. There were no statistically significant differences in the mean proportion of cytokines among clusters in the periodontally healthy subjects, while the ratio IL-1beta/IL-10 (p<0.05) differed significantly among clusters in the aggressive periodontitis group. Different subgingival biofilm profiles are associated with distinct patterns of GCF cytokine expression. Aggressive periodontitis subjects were characterized by a higher IL-1beta/IL-10 ratio than periodontally healthy subjects, suggesting an imbalance between pro- and anti-inflammatory cytokines in aggressive periodontitis.

Pro‐inflammatory biomarkers during experimental gingivitis in patients with type 1 diabetes mellitus: a proof‐of‐concept study

To compare gingival crevicular fluid (GCF) biomarker levels and microbial distribution in plaque biofilm (SP) samples for subjects with type 1 diabetes (T1DM) versus healthy subjects without diabetes during experimental gingivitis (EG). A total of nine T1DM patients and nine healthy controls of age and gender similar to the T1DM patients were monitored for 35 days during EG. Hygiene practices were stopped for 3 weeks, and GCF, SP, plaque index (PI) and gingival index were determined. IL-1beta, IL-8, MMP-8 and MMP-9 were quantified by enzyme-linked immunosorbent assay, and SP samples were assessed by DNA-DNA hybridization for a panel of 40 subgingival microbial species. IL-1beta levels in T1DM patients were elevated compared with healthy individuals, and showed differences between groups at 7-21 days while healthy patients showed IL-1beta increases from baseline to 14-21 days (p<0.05). Differences were observed in MMP-9 levels between patients with and without T1DM at 7-14 days (p<0.05). Orange complex species and PI measurements displayed a superior correlation with biomarker levels when compared with other complexes or clinical measurements during EG. The mean GCF biomarker levels for IL-1beta and MMP-8 were most significantly elevated in T1DM subjects compared with healthy individuals during EG, not resulting from differences in the mean PI or microbial composition.

Microbiological profile of untreated subjects with localized aggressive periodontitis

The microbial profile of localized aggressive periodontitis (LAgP) has not yet been determined. Therefore, the aim of this study was to evaluate the subgingival microbial composition of LAgP. One hundred and twenty subjects with LAgP (n=15), generalized aggressive periodontitis (GAgP, n=25), chronic periodontitis (ChP, n=30) or periodontal health (PH, n=50) underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analysed for their content of 38 bacterial species using checkerboard DNA-DNA hybridization. Red complex and some orange complex species are the most numerous and prevalent periodontal pathogens in LAgP. The proportions of Aggregatibacter actinomycetemcomitans were elevated in shallow and intermediate pockets of LAgP subjects in comparison with those with GAgP or ChP, but not in deep sites. This species also showed a negative correlation with age and with the proportions of red complex pathogens. The host-compatible Actinomyces species were reduced in LAgP. A. actinomycetemcomitans seems to be associated with the onset of LAgP, and Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter gracilis, Eubacterium nodatum and Prevotella intermedia play an important role in disease progression. Successful treatment of LAgP would involve a reduction in these pathogens and an increase in the Actinomyces species.

Factors affecting human supragingival biofilm composition. I. Plaque mass

Little is known about the factors that affect the microbial composition of supragingival biofilms. This study was designed to examine the relationship between total DNA probe counts of supragingival biofilm samples, clinical parameters and supragingival biofilm composition. Supragingival plaque samples were taken from 187 systemically healthy adult subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. The relationship between total DNA probe counts and microbial composition was examined by subsetting the data into 10 groups based on 10 percentile increments of the total DNA probe counts. Differences among groups in terms of species counts and proportions were sought, as well as relationships of total plaque DNA probe count and clinical parameters. There was a wide distribution in mean total DNA probe counts among the 187 subjects. With increasing total plaque levels there was a change in the proportions of individual species and microbial complexes. 'Small plaques' were characterized by high proportions of species in the yellow, orange, purple and 'other' complexes; plaques of moderate mass were characterized by high proportions of Actinomyces and purple complex species, while 'large plaques' exhibited increased proportions of green and orange complex species. Measures of gingival inflammation, pocket depth and recession were significantly positively associated with total DNA probe counts. Increased plaque numbers were related to increased pocket depth irrespective of presence or absence of gingival inflammation. The proportions of individual species and microbial complexes in supragingival biofilms are influenced by the total numbers of organisms in the biofilm.

Microbiological changes associated with four different periodontal therapies for the treatment of chronic periodontitis

To examine subgingival microbiological changes in chronic periodontitis subjects receiving scaling and root planing (SRP) alone or with systemically administered azithromycin, metronidazole or a sub-antimicrobial dose of doxycycline. Ninety-two periodontitis subjects were randomly assigned to receive SRP alone or combined with azithromycin, metronidazole or sub-antimicrobial dose doxycycline. Subgingival plaque samples taken at baseline, 2 weeks, and 3, 6, and 12 months were analyzed for 40 bacterial species using checkerboard DNA-DNA hybridization. Percentage of resistant species and percentage of sites harboring species resistant to the test antibiotics were determined at each time-point. All treatments reduced counts of red complex species at 12 months, although no significant differences were detected among treatment groups for most species at all time-points. Both antibiotics significantly reduced counts of red complex species by 2 weeks. Percentage of resistant isolates increased in plaque samples in all adjunctive treatment groups, peaking at the end of administration, but returned to pretreatment levels by 12 months. The significant reduction of red and orange complex species at 2 weeks in the subjects receiving SRP plus azithromycin or metronidazole may have contributed to a better clinical response in these treatment groups. Therapy did not appear to create lasting changes in the percentage of resistant isolates or sites harboring resistant species.

Experimental gingivitis in type 1 diabetics: a controlled clinical and microbiological study

To monitor clinical and microbiological changes during experimental gingivitis in type 1 diabetics and non-diabetics. Nine type 1 diabetics with good/moderate metabolic control and nine age-gender matched non-diabetics were recruited. Probing pocket depths in all subjects did not exceed 4 mm and none were affected by attachment loss. According to the original model, an experimental 3-week plaque accumulation resulting in experimental gingivitis development and a subsequent 2-week period of optimal plaque control were staged. Subgingival plaque samples were collected at days 0, 21 and 35 from one site per quadrant, pooled and analysed using checkerboard DNA-DNA hybridization. Diabetics (mean age 25.6+/-5.8 standard deviation (SD), range 16-35 years) had a mean HbA1c level of 8.1+/-0.7% (SD), while non-diabetics (mean age 24.8+/-5.7 (SD), range 15-36 years) were metabolically controlled (HbA1c< or =6.5%). Between Days 0, 21 and 35, no statistically significant differences in mean plaque and gingival index scores were observed between diabetics and non-diabetics. At days 7 and 21, however, diabetics showed statistically significantly higher percentages of sites with gingival index scores > or =2 compared with non-diabetics. Mean DNA probe counts of the red and orange complex species increased significantly (p<0.05) between days 0 and 21 and decreased significantly (p<0.05) between days 21 and 35 in both groups. Both diabetics and non-diabetics react to experimental plaque accumulation with gingival inflammation. Type 1 diabetics, however, develop an earlier and higher inflammatory response to a comparable bacterial challenge.