*3.2. Clinical Outcome Variables*

PD showed significant differences among groups for both, sampling sites and Ramfjord index teeth (*p* < 0.01), with deeper pockets in periodontitis patients (3.28 mm and 2.76 mm, respectively), than in healthy subjects (2.03 and 2.09, respectively; *p* < 0.01) and gingivitis patients (2.20 and 2.19, respectively; *p* < 0.01). Similar differences were observed for proximal PD at Ramfjord teeth and for CAL. BOP also showed significant differences among groups, for both sampling sites and Ramfjord teeth (*p* < 0.01) but differences corresponded to significant higher values in gingivitis (38.2% and 29.1%, respectively; *p* < 0.01) and periodontitis patients (50.9% and 35.8%, respectively; *p* < 0.01), when compared with healthy subjects (7.8% and 5.7%, respectively). Similar results were observed for PlI, with lower values in the periodontal health group and significantly higher levels in gingivitis and periodontitis patients, with no differences between them (Table 2 and Table S3).

## *3.3. Subgingival Microbiota as Evaluated by Means of qPCR*

Differences in PCR counts were significantly different among groups (*p* = 0.01), with significantly higher counts in periodontitis patients, when compared with the periodontal health (*p* = 0.01) or gingivitis (*p* = 0.02) groups (Table 3 and Table S4).

Frequency of detection and counts of *A. actinomycetemcomitans* were low in all groups, although with increasing frequencies and counts in periodontitis subjects but differences were not statistically significant. *P. gingivalis*, also showed increasing frequencies and counts in periodontitis but, again, differences were not statistically significant. *T. forsythia*, showed high frequencies of detection (prevalence) in all groups (67.9% in periodontitis, 47.0% in gingivitis and 48.8% in periodontal health), with no statistically significant differences among groups, while PCR counts demonstrated significant differences (*p* = 0.01), with higher percentages in periodontitis when compared with gingivitis (*p* = 0.02) or periodontally healthy (*p* = 0.01) subjects.

#### *3.4. Subgingival Microbiota as Evaluated by Means of Culture*

Total anaerobic counts were significantly different among groups (*p* < 0.01), with significantly higher counts in periodontitis, as compared with periodontal health (*p* < 0.01).

Only four target bacterial species (*P. gingivalis, P. intermedia, T. forsythia* and *E. corrodens*) showed an overall increase in frequency of detection, counts and proportions, as the periodontal status worsened. *P. micra* and *A. odontolyticus* demonstrated higher counts, proportions and frequencies in healthy subjects. *C. rectus* and *Capnocytophaga* spp. presented the highest counts, proportions and frequencies in gingivitis patients. Finally, *A. actinomycetemcomitans* was not detected with culture methods.

Statistically significant differences among groups were only detected for *E. corrodens* (*p* = 0.01), corresponding to higher frequencies of detection (*p* = 0.01), proportions (*p* = 0.01) and counts (*p* = 0.01) in periodontitis patients, when compared with periodontal health subjects (Tables 4 and 5, Tables S5 and S6).


*Appl. Sci.* **2021**, *11*, 778


**Table 3.** Microbiological findings (quantitative polymerase chain reaction, qPCR), with PCR counts expressed as means and standard deviations (SD) or as medians and interquartile ranks(IQR) and frequencies of detection as percentages, for the complete study population and for each study group, with the appropriate comparisons.


n, number of samples; (+), samples with pathogen detection; prev., frequency of detection of the target pathogen; A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans; P. gingivalis:Porphyromonas gingivalis; T. forsythia: Tannerella forsythia.\* Statistically significant differences.



ˆ Counts (Kruskal-Wallis *p* value). For comparison between groups, they were available just for anaerobic counts (H-G, 0.25; G-P, 0.43; H-P, 0.00 \*) and *E. corrodens* (H-G, 1.00; G-P, 0.11; H-P, 0.01 \*). Statistically significant differences. n, number of samples; (+), samples with pathogen detection; prev., frequency of detection of the target pathogen; *A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans; P gingivalis: Porphyromonas gingivalis; T. forsythia: Tannerella forsythia; P. micra: Parvimonas micra; F. nucleatum: Fusobacterium nucleatum; C. rectus: Campylobacter rectus; E. corrodens: Eikenella corrodens; A. odontolyticus: Actinomycesodontolyticus.*

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**Table 5.** Microbiological findings (culture), with proportions of total anaerobic microbiota in percentage, expressed as medians and interquartile ranks (IQR), for each study group, with the appropriate comparisons.

ˆ Proportions of total anaerobic microbiota (Kruskal-Wallis p value); for comparison between groups, they were available just for *E. corrodens* (H-G, 1.00; G-P, 0.13; H-P, 0.01 \*). \* Statistically significant differences. *A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans; P. gingivalis: Porphyromonas gingivalis; T. forsythia: Tannerella forsythia; P. micra: Parvimonas micra; F. nucletum: Fusobacterium nucleatum; C. rectus: Campylobacter rectus; E. corrodens: Eikenella corrodens; A. odontolyticus: Actinomyces odontolyticus.*
