*2.3. Microbiological Processing by Means of Quantitative Polymerase Chain Reaction*

## 2.3.1. Extraction of Total Genomic DNA

Total DNA was extracted from subgingival samples using a commercial kit (MolYsis Complete 5, Molzym Gmbh & Co. KG. Bremen, Germany) following manufacturer's instructions (the protocol for bacterial DNA extraction was followed from step 6, avoiding preliminary steps). The extracted DNA was eluded in 100 μL of sterile water (Roche Diagnostic GmbH, Mannheim, Germany) and frozen at −20 ◦C for further analysis.

## 2.3.2. Polymerase Chain Reaction

Multiplex quantitative PCR (qPCR) technology was used for detecting and quantifying the bacterial DNA [32]. The sequence of the primers and probes used for *Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans)*, *Porphyromonas gingivalis (P. gingivalis)* and *Tannerella forsythia (T. forsythia)*, targeting against 16S rRNA gene, have been previously reported [33,34]. PCR amplification was performed in a total reaction mixture volume of 10 μL, which included 5 μL of 2×TaqMan master mixture (LC 480 Probes Master, Roche Diagnostic GmbH), optimal concentrations of primers and hydrolysis probe (300, 300 and 200 nM for *A. actinomycetemcomitans*; 300, 300 and 300 nM for *P. gingivalis* and 300, 300 and 200 nM for *T. forsythia*) and 2.5 μL of DNA from the samples. The no-template control (NTC) consisted of 2.5 μL of sterile water. Samples were subjected to an initial amplification cycle of 95 ◦C for 10 min, followed by 40 cycles at 95 ◦C for 15 s and 60 ◦C for 1 min in LightCycler® 480 II thermocycler (Roche Diagnostic GmbH). Each DNA sample was analyzed in duplicate.

All assays were performed using calibration curves with a linear quantitative detection range established by the slope range of 3.3–3.6 cycles/log decade, r2 > 0.997 and an efficiency range of 1.9–2.0. Quantification was based on standard curves, which were constructed by plotting cross point cycle (Cp) values generated from qPCR against DNA extracted from serial 10-fold dilutions of purified genomic DNA from each bacterium (log of colony forming units (CFU)/mL).

#### *2.4. Microbiological Processing by Means of Culturing*

At the laboratory, aliquots of 0.1 mL from the vials were plated on two different culture media: the selective Dentaid-1 medium for the detection of *A. actinomycetemcomitans* [35] and a non-selective blood agar medium (Blood Agar Base, Oxoid, Basingstoke, England),

supplemented with hemin (5 mg/L) (Sigma, St. Louis, MO, USA), menadione (1 mg/L) (Merck, Darmstadt, Germany) and 5% of sterile horse blood (Oxoid), for detection of target periodontal pathogens (*Capnocytophaga* spp., *Campylobacter rectus (C. rectus), Eikenella corrodens (E. corrodens), Fusobacterium nucleatum (F. nucleatum), P. gingivalis, Prevotella intermedia (P. intermedia), Parvimonas micra (P. micra), T. forsythia, Actinomyces odontolyticus (A. odontolyticus)*) and for evaluating total anaerobic bacterial counts. After 2–5 days of capnophilic incubation (Dentaid-1 medium) or 7–14 days of anaerobic incubation (blood agar medium), total counts and counts of representative colonies were calculated in the most suitable plates. Suspected colonies were identified by microscopy, Gram-staining and enzyme activity (Table S1). Counts were transformed in CFU per mL of the original sample.
