It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.

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Objectives to explore relationships among serum adipokines, vitamin D, clinical and microbial parameters of chronic periodontitis before and after treatment. Methods weight, height and smoking status were recorded for 56 patients with chronic periodontitis. Plaque, gingivitis, bleeding on probing (BOP), suppuration, pocket depth (PD) and attachment level (AL) were measured at all teeth present. Subgingival biofilm samples from each tooth were analyzed for levels of 40 bacterial species using checkerboard DNA-DNA hybridization. Serum levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), adiponectin, leptin, resistin and vitamin D were measured at baseline. Sample collection was then performed in a subset of the population 6 months post-therapy (n=17). Serum samples were analyzed using ELISA and immunoassays. Differences in clinical, microbial and serum factors among groups were sought using the Mann-Whitney test. Correlations among factors were evaluated using regression analysis. Effects of therapy were sought using the Wilcoxon signed ranks test Results There were positive correlations between adiponectin/vitamin D and between IL-6/leptin; negative correlations between IL-6/vitamin D, and leptin/vitamin D, but no associations between serum analytes and clinical or microbial parameters. Gender and BMI were associated with levels of adipokines. Periodontal therapy improved clinical and microbiological parameters, but did not influence the levels of serum analytes. Conclusions Adipokines and IL-6 levels were affected by gender and BMI. Serum analytes were not influenced by periodontal therapy.

Porphyromonas gingivalis has been isolated from periodontitis lesions in subjects from many geographical locations. The purpose of this investigation was to determine whether similar ribotypes of P. gingivalis could be detected among strains isolated in different countries. A total of 198 isolates of P. gingivalis were obtained from 52 periodontitis patients in Boston (130 isolates), Bergen, Norway (17 isolates), Khartoum, Sudan (26 isolates), and Bucharest, Romania (25 isolates). DNA was isolated from each strain, cut separately by the restriction endonucleases KpnI and PstI. The resulting preparations were subjected to electrophoresis in a 0.8% agarose gel using a Tris-acetate EDTA buffer. Uncut lambda and a 1000-bp fragment of 16S rRNA were included as internal standards in each lane. In addition, a HindIII digest of lambda was present in a separate lane in each run. The DNA fragments were transferred to a nylon membrane by downward capillary transfer. 16S rRNA bands were detected using a 1000-kb digoxigenin-labelled probe generated by a polymerase chain reaction. At the same time, a digoxigenin-labelled probe to lambda was employed to detect the internal and molecular weight standards. The bands were detected using antibody to digoxigenin conjugated to alkaline phosphatase and chemiluminescence. The positions of the bands relative to the internal standards were determined and normalized to correct for run-to-run variations, and the molecular weight of each band was determined by comparison with standards within each gel. The resulting data for the 2 enzymes were combined and subjected to cluster analysis using an average unweighted linkage sort. In some instances, isolates that appeared to be of identical ribotype using one endonuclease gave different ribotypes using the other. Strains of P. gingivalis within a subject were usually identical, except for 3 patients who harbored 2 different ribotypes/individual. All subsequent analyses employed a single ribotype strain for each subject. A total of 32 ribotypes were observed for isolates from distant countries. A total of 11.5% of the patients had isolates exhibiting the same ribotype: ribotype 7a. Identical ribotypes of P. gingivalis can be recovered from subgingival plaque samples of periodontitis patients in different countries.

dChronic periodontitis is one of the most prevalent human diseases and is caused by dysbiosis of the subgingival microbiota. Treatment involves primarily mechanical disruption of subgingival biofilms and, in certain cases, adjunctive use of systemic antibiotic therapy. In vitro biofilm models have been developed to study antimicrobial agents targeting subgingival species. However, these models accommodate a limited number of taxa, lack reproducibility, and have low throughput. We aimed to develop an in vitro multispecies biofilm model that mimics subgingival plaque, to test antimicrobial agents. Biofilms were cultivated using the Calgary Biofilm Device and were exposed to amoxicillin (AMX), metronidazole (MTZ), azithromycin (AZM), and AMX-MTZ at four different concentrations for 12, 24, or 36 h. Chlorhexidine (CHX) (0.12%) was used as the positive control. The compositions of the biofilms were analyzed by checkerboard DNA-DNA hybridization, and the percent reduction in biofilm metabolic activity was determined using 2,3,5-triphenyltetrazolium chloride and spectrophotometry. Thirty-five of the 40 species used in the inoculum were consistently recovered from the resulting in vitro biofilms. After 36 h of exposure at the 1:27 dilution, AMX-MTZ reduced metabolic activity 11% less than CHX (q ‫؍‬ 0.0207) but 54% more than AMX (q ‫؍‬ 0.0031), 72% more than MTZ (q ‫؍‬ 0.0031), and 67% more than AZM (q ‫؍‬ 0.0008). Preliminary evidence of a synergistic interaction between AMX and MTZ was also observed. In summary, we developed reproducible biofilms with 35 subgingival bacterial species, and our results suggested that the combination of AMX and MTZ had greater antimicrobial effects on these in vitro multispecies biofilms than expected on the basis of the independent effects of the drugs. P eriodontitis is a persistent health problem that affects the U.S. population in epidemic proportions. The most recent data from the National Health and Nutrition Examination Survey (NHANES) (2009 to 2010) suggest that the prevalence of chronic periodontitis among U.S. adults is over 47%, representing 64.7 million adults (1). Chronic periodontitis is the main cause of tooth loss in adults. Treatment costs U.S. taxpayers $4.4 billion each year (2), but existing therapies do not eradicate the disease. Frequent posttreatment follow-up visits are needed and are costly, which may be one reason why the prevalence is higher in populations of low socioeconomic status (3).A single periodontal pocket harbors a complex polymicrobial community of up to hundreds of taxa, and periodontal diseases are triggered by dysbiosis of subgingival organisms. Organization of the subgingival microbiota in biofilms makes it challenging to control periodontal infections, since biofilms help protect resident organisms from both antimicrobial agents and immune mechanisms (4). Still, the use of systemic and local antibiotics as an adjunct to mechanical treatment provides clinical benefit beyond that achieved by scaling and root planing alone (5-15).However, the threa...