## 3 Discussion  
This is the first study to show that prophage-derived endolysins active on _Gardnerella_ could be used as an innovative treatment for bacterial vaginosis. Although no _Gardnerella_ phages were ever isolated in a lab, we were able to identify putative endolysins in silico. We expressed them as recombinant proteins, improved their potency by systematic domain shuffling, demonstrated their efficacy and selectivity in vitro, and, ultimately, on the native polymicrobial biofilms in vaginal swabs from BV patients. The currently available therapy of bacterial vaginosis is unsatisfactory. Antibiotic-centered \"standard-of-care\" treatment for BV is associated with a recurrence rate of \(\sim\)60% within 6 months, which may be explained by the low efficacy of antibiotics on bacteria growing as biofilms, but also by the selective destruction of beneficial lactobacillus, which are thereby prevented from re-establishing a protective environment [26]. Bacteriophages derived endolysins have been described as a potential alternative to antibiotics; they are both highly active on mucosal biofilms and selective for individual genera or even species of bacteria [35]. Neither endolysins nor whole phages specific to _Gardnerella_ have been isolated previously, although the existence of _Gardnerella_-specific phages has been postulated on the basis of genomic analyses [40]. We identified _Gardnerella_-specific endolysin genes in the prophage-like genome sequences of strains of _Gardnerella_. A similar approach was reported earlier, in which the phage lysin PlyCD was identified by searching prophage-like sequences in the genomes of _Clostridium difficile_ strains [45]. The fact that it is possible to predict and generate endolysins from prophage encoded sequences in the genomes of unrelated species (_C. difficile_ and _Gardnerella_) may indicate a wide application of this approach, which could easily be adapted to other pathogenic bacteria. We could show that the wild-type endolysins we identified and, even more so, our engineered constructs, are highly bactericidal and selective for the genus _Gardnerella_. Our engineered endolysin PM-477 is superior to the current standard of care antibiotics in that it is active in the low ug/mL range and effective against all 20 of the _Gardnerella_ strains we tested, covering all four named species. In total, 60% of the 20 strains were resistant to MDZ and 75% to TZD, reinforcing the notion that PM-477 may be more effective against BV than currently used standard antibiotics. The high resistance rate for MDZ, which was also reported elsewhere [31], does not support the finding that MDZ treatment temporarily relieves symptoms in a majority of cases [26]. However, long-term cure is mostly not achieved with MDZ, indicating that its effectiveness may be reduced due to the high resistance rate of _Gardnerella_ strains [46]. In our study, CLI was also effective against all the strains of different _Gardnerella_ genotypes we tested. Curiously, however, CLI has not shown superior efficacy when compared to MDZ in clinical trials [26], which may indicate that BV biofilms are more tolerant to CLI than they are of MDZ, thus compensating the higher rate of resistance against MDZ. In conclusion, PM-477 is superior to all tested antibiotics because, unlike nitroimidazoles, it is active on all _Gardnerella_ strains and, unlike CLI, it does not kill lactobacilli. Antibiotics may have the advantage over PM-477 of being active against a broader spectrum of pathogens. Yet, unlike _Gardnerella_, these pathogens may not be pivotal in vaginal biofilm formation as is the case in BV. Additionally, antibiotics typically have low activity on bacteria in biofilms [27] whereas our ex vivo data indicate that PM-477 works well on _Gardnerella_ in vaginal biofilms. Progress has been made in developing animal models for BV. For example, it was possible to model some of the clinical characteristics of BV, such as epithelial exfoliation and clue cells, upon infection of mice with _Gardnerella_[47].