In the peri-implantitis group, one additional subgingival plaque sample was obtained from partially edentulous patients with a history of periodontitis (n = 10) and obtained at a tooth exhibiting the highest PD but no signs of acute periodontal disease (i.e., BOP/no suppuration). None of these teeth were located adjacent to the sampled implant sites. The control samples were also prepared for PCR analysis.

At the Institute of Medical Microbiology and Hospital Hygiene, the specimens were re-suspended in the buffer by vortexing. After the addition of 10 μl Proteinase K solution (100 μg/ml Proteinase K), the samples were incubated for 30 min at 56°C. Total genomic DNA was isolated from 200 μl of the Proteinase K-digested samples by semiautomatic DNA preparation on an EZ1 biorobot machine (Qiagen) and the eluted 100 μl DNA samples stored at −20°C until use.

In house TaqMan PCRs for the quantification of Mycoplasma salivarium (M. salivarium) [12], Veillonella parvula (V. parvula) [13], S. aureus [14], P. gingivalis [15], Parvimonas micra (P. micra) [16], and T. forsythia [15] (Table 1) were carried out in a total volume of 25 μl consisting of 1× Eurogentec qPCR MasterMix (Eurogentec, Seraing, Belgium) without ROX (containing buffer, dNTPs (including dUTP), HotGoldStar DNA polymerase, 5 mM MgCl₂, uracil-N-glycosylase and stabilizers (RT-QP2X-03NR, Eurogentec)), 300 nM each forward and reverse primer, 200 nM labeled probe, and 2.5 μl of template DNA (primer and probes are listed in Tables 1 and 2). Amplicon carrying plasmids were used in concentrations of 10⁵ and 10² copies/μl as quantification standards. Thermal cycling conditions were as follows: 1 cycle at 50°C for 10 min, 1 cycle at 95°C for 10 min followed by 45 cycles at 95°C for 15 s, and 60°C for 1 min. Cycling and fluorescent data collection and analysis were carried out with an iCycler from BioRad (BioRad Laboratories, Munich, Germany) according to the manufacturer’s instructions.

Real-time PCR assays for the detection of fungal DNA (Table 2) were carried out in a total volume of 25 μl consisting of 1× MesaGreen qPCR MasterMix Plus for SYBR Assay (containing Buffer, dNTPs (including dUTP), Meteor Taq DNA polymerase, 4 mM MgCl2, uracil-N-glycosylase, SYBR Green I, stabilizers and passive reference (RT-SY2X-06 + WOU); Eurogentec, Seraing, Belgium), 300 nM each forward and reverse primer and 2.5 μl of template DNA. In multiplex assays with three forward primers, each primer was adjusted to 100 mM. Positive detection was verified by sequencing [17] and BLAST analysis [18]. Thermal cycling conditions were as follows: 10 min at 50°C, 10 min at 95°C followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. Subsequent melting point analysis followed after 15 s at 95°C and 1 min at 60°C from 65°C to 95°C with an increment of 0.5°C for 15 s and plate read.