Ez1 automate

The DNA extraction was performed on an EZ1 automate (Qiagen, Courtaboeuf, France) using a commercial extraction kit, the QIAamp Tissue Kit^® (Qiagen), following the manufacturer’s instructions. The DNA was extracted from 200 µL of vaginal sample digested with 200 μL of G2 buffer and 10 μL of proteinase K at 56 °C for 20 min, then eluted in 100 µL of distilled water.

Bacterial colonies that were not identified at the species level using MALDI-TOF MS were further tested using 16S rRNA sequencing. Genomic DNA was extracted using an EZ1 automate and the DNA tissue kit (Qiagen, Hilden, Germany). The complete 16S rRNA gene amplification and sequencing was performed using eight primers on an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Bio systems, Bedford, MA, USA). The primers used were Fd1 (5′-AGAGTTTGATCCTGGCTCAG-3′), Rp2 (5′-ACGGCTACCTTGTTACGACTT-3′), F536 (5′-CAGCAGCCGCGGTAATAC-3′), R536 (5′-GTATTACCGCGGCTGCTG-3′), F800 (5′-ATTAGATACCCTGGTAG-3′), R800 (5′-CTACCAGGGTATCTAAT-3′), F1050 (5′-TGTCGTCAGCTCGTG-3′) and R1050 (5′-CACGAGCTGACGACA-3′) (Eurogentec, Angers, France). The CodonCode Aligner software was used for sequence alignment, assembly and correction (https://www.codoncode.com/). For taxonomic assignation, a BLASTn search was performed against the nr database^{16 (link)}. A sequence similarity threshold of 98.65% by comparison with the phylogenetically closest species with standing in nomenclature was used to delineate a putative new species^{17 (link)}. Phylogenetic relationships were inferred from the comparison of 16S rRNA sequences using the MEGA7 software^{18 (link)}.

Specific primers were designed against Rpob and Gyrase subunit A genes based on Klenkia spp available in NCBI database and with our Klenkia genome sequencing using Primer-blast^{35 (link)}. DNA extraction was performed using EZ1 DNA tissue kit (Qiagen) with Bacterial card and EZ1 automate.
For the qPCR detection, we used the same primers. The DNA was amplified with the LightCycler 480 SYBR green I Master (Roche). The reaction mixture (20 µL) per sample was prepared as follows: DNA template (5 µL), forward primer (1 µL, 10 µM), reverse primer (1 µL, 10 µM), master mix (2×, 10 µL) and DEPC-treated water (3 µL). The amplification program includes an initial step of denaturation at 95 °C for 5 minutes followed by 45 cycles; each cycle consisted of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 20 seconds and extension at 72 °C for 30 seconds, then single cycle of melting curve step followed by cooling.
The real time amplification was performed using CFX96 real time (Bio-Rad®, France).
The results of real time amplification were analysed by Bio-Rad CFX Manager (Bio-Rad).