## 2 Results
### Efficacy of PM-477 against Biofilms of Gardenella in Human BV Samples  
To analyze the efficacy of PM-477 in a physiological environment that closely resembles the in vivo situation, we treated vaginal swabs from 15 BV patients, and analyzed them by fluorescence in situ hybridization (FISH). Areas of strong yellow fluorescence in the untreated (baseline) and buffer control-treated samples indicated the presence of a _Gardnerella_-dominated biofilm on exfoliated vaginal epithelial cells (visible due to their pale autofluorescence and clearly corresponding to DAPI counterstain), called clue cells. At baseline and after buffer treatment, the yellow fluorescence of the _Gardnerella_ probe outshines the red fluorescence of the probe for all bacteria, indicating that the biofilm is composed, in large part, of _Gardnerella_ cells (Figure 5a). Upon treatment with PM-477 a total resolution of the dense yellow staining on the epithelial cells is seen. Therefore, PM 477 proved strong bacteriolytic activity on _Gardnerella_ also in this ex vivo setting. The disappearance of intense yellow fluorescent _Gardnerella_ bacteria unmasks the red fluorescence of still intact bacterial mass, indicating mostly non-_Gardnerella_ cells. Only very few individual _Gardnerella_ cells seemed to remain associated with or in between the epithelial cells. However, the comparison of bacteriolytic and bactericidal effect in Figure 4b-e would indicate that these might be dead cell-like structures with sufficient DNA associated to them to hybridize the FISH probes. In contrast to PM-477, Octenisept killed and lysed both the _Gardnerella_ and other bacterial cells non-specifically, as indicated by the visible reduction in both types of fluorescence. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & \multicolumn{6}{c}{**MIC of Antimicrobials [ug/mL]**} \\ \hline
**Species** & **Identification** & **Clade** & **PM-477** & **CLI** & **MDZ** & **TDZ** \\ \hline _G. vaginalis_ & ATCC 14018T & I & 1 & 0.25 & 8 & 128 \\ _G. vaginalis_ & UGent 09.07 & I & 2 & 0.25 & \textgreater{}128 & \textgreater{}128 \\ _G. vaginalis_ & UGent 09.01 & I & 0.25 & 0.13 & 8 & 4 \\ _G. vaginalis_ & UGent 25.49 & I & 0.13 & \textless{}0.06 & 8 & 4 \\ _G. vaginalis_ & BV50.1 & n.d. & 8 & 0.25 & 32 & 64 \\ _[PERSON]_ & BV111.5.1 & n.d. & 2 & 0.125 & 8 & 4 \\ _[PERSON]_ & FB049-01 & n.d. & 4 & 0.5 & 16 & 8 \\ _[PERSON]_ & FB061-03 & n.d. & 8 & 0.25 & 8 & 4 \\ _[PERSON]_ & UGent 09.48 & IV & 4 & 0.5 & 128 & 128 \\ _[PERSON]_ & BV13.2 & n.d. & 4 & 0.5 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]_ & BV86.5 & n.d. & 1 & 0.25 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]_ & UGent 18.01T & II & 8 & 0.5 & 32 & 64 \\ _[PERSON]_ & UGent 21.28 & II & 2 & 0.25 & 64 & \textgreater{}128 \\ _[PERSON]_ & P80275 & n.d. & 1 & 0.5 & 16 & 32 \\ _[PERSON]_ & FB041 & n.d. & 4 & 1 & 32 & 64 \\ _[PERSON]_ & VMF1800 & n.d. & 8 & 1 & 32 & 64 \\ _[PERSON]_ & GS 10234 & IV & 0.5 & 0.25 & 64 & \textgreater{}128 \\ _[PERSON]_ & GS 9838-1T & IV & 0.25 & \textless{}0.06 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]_ & BV7.1 & n.d. & 1 & 0.5 & \textgreater{}128 & 128 \\ _[PERSON]_ & BV112.4 & n.d. & 1 & 0.06 & 64 & 64 \\ _[PERSON]_ & DSM 20584 & \textgreater{}128 & 4 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]_ & DSM 20077 & \textgreater{}128 & 64 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]_ & DSM 20243 & \textgreater{}128 & 32 & \textgreater{}128 & \textgreater{}128 \\ _[PERSON]. [PERSON]_ & PB2003-073-T2-2 & \textgreater{}128 & 0.25 & \textgreater{}128 & \textgreater{}128 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Minimum inhibitory concentration (MIC) of PM-477 and the antibiotics CLI, MDZ, and TDZ for various _Gardnerella_ spp. and _Lactobacillus_ spp. (R) Resistance, defined as \(\geq\)32 ug/mL for MDZ and TDZ, and \(\geq\)8 ug/mL for CLI, according to international standards [44]. The effect of treatment was also quantified by counting the number of bacterial cells per 10 epithelial cells in representative fields of vision (Figure 5b).