## 4 Materials and Methods
### Phase Contrast Microscopy  
Dense bacterial suspensions were prepared in NYCIII+ 10 % HS either as _Gardnerella_ monocultures or as a co-cultures of _Gardnerella_ and _Lactobacillus_ strains at final OD\({}_{600}\)= 4 for each strain. Bacterial mixtures were centrifuged at 3800 \(\times\)\(g\) for 7 min at room temperature in a benchtop centrifuge (Heraeus Instruments, Biofuge Pico). Supernatants were removed and cell pellets were resuspended in 50 uL of PM-477 (460 ug/mL) or in MES buffer alone as a negative control and incubated for 5 h at 37 \({}^{\circ}\)C under anaerobic conditions. Subsequently, 5 uL of each bacterial suspension was placed on a glass slide, covered with a glass coverslip, and observed by phase contrast microscopy (Olympus BX41 microscope, magnification 1000\(\times\)). For the comparison of bacterolytic and bactericidal effects, bacterial suspensions of _Gardnerella swidsinskii_ and _Gardnerella vaginalis_ were prepared in NYCIII+ 10% HS at a final OD\({}_{600}\) = 1. Bacterial suspensions of 50 uL were centrifuged at 3800\(\times\)\(g\) for 7 min at room temperature in a benchtop centrifuge (Heraeus Instruments, Biofuge Pico). Supernatants were removed and pellets were resuspended in 50 uL of MES buffer containing PM-477 at 400, 200, 100, 50, or 25 ug/mL, or in MES buffer alone as a negative control, and incubated for 5 h at 37 \({}^{\circ}\)C in anaerobic conditions. Subsequently, 5 uL of each bacterial suspension was observed by phase contrast microscopy as above. To determine viability, 10 uL of the reaction mixture was serially diluted and spotted onto Choc agar plates. After incubation at 37 \({}^{\circ}\)C for 48 h, the colonies were counted, and CFU/ml was calculated. Lytic Effects of PM-477 and Octenisepet(r) on Ex Vivo BV Patient Samples as Detected by Fluorescence in situ Hybridization (FISH) Microscopy  
Vaginal samples were collected from randomly selected 24-49-year-old Caucasian women who were previously diagnosed with BV according to the Amsel criteria ([PERSON], 2018). In addition, the presence of clue cells (epithelial cells covered with cohesive _Gardnerella_ bacteria) was determined by FISH microscopy (Fresh vaginal smears were collected with a swab and put in ESwab(tm) 493C02 pre-filled vials (COPAN Diagnostics, Murrieta, CA, USA). The vials were vortexed gently, resulting in a suspension that contained epithelial cells and vaginal bacteria. Four consecutive vaginal smears from each patient were pooled. The effects of 0.2, 2, 20, and 200 ug/mL of PM-477 after 2, 6, and 24 h, respectively, were tested either at room temperature or 36 \({}^{\circ}\)C. Since the most pronounced lytic effect on _Gardneella_ was observed after 24 h exposure to 200 ug/mL. PM-477 at 36 \({}^{\circ}\)C (Table 3), this condition was used for further studies. Subsequently, vaginal swabs from 15 BV patients were collected, aliquoted, and mixed 1:1 (_v/v_) with 400 ug/mL PM-477 (final concentration of 200 ug/mL) or with MES buffer as a negative control or mixed 1:20 (_v/v_) with the disinfect Otenisept(r) ([PERSON], Vienna, Austria) and incubated for 24 h at 37 \({}^{\circ}\)C. Untreated samples were also included in the analyses as baseline controls. The samples were fixed with Carnoy solution (alcohol/chloroform/acetic acid 6/3/1 by volume) [55]. Fields of 10 \(\times\) 10 mm were marked on SuperFrost slides (Langenbrinck, Emmendingen, Germany) with a PAP pen (Kisker-Biotech, Steinfurt, Germany). Five-microliter aliquots of vortexed, fixed vaginal smear sample were dropped onto the marked field. The slides were dried for 60 min at 50 \({}^{\circ}\)C before FISH analysis. For multicolor FISH analyses, we used the Gard662 _Gardneella_-specific DNA hybridization probe [56] and the universal bacterial probe Eub338 [57]. Oligonucleotide probes were synthesized with a fluorescent dye (Cy3 or Cy5). Hybridization was performed at 50 \({}^{\circ}\)C as previously described [55]. The probe Ato291 was used for _Atopobium_, Liner23-2 for _L. iners_, and Lcrisp16-1 for _L. crispatus_[56].