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Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a phagolysosome. The results indicate that an intracellular phase may be one way S. zooepidemicus survives in the host, and could in part explain how S. zooepidemicus can cause recurrent/persistent infections. Future studies should reveal the ability of S. zooepidemicus to internalize and survive in primary equine endometrial cells and during in vivo conditions.

We present the draft genome sequence of Streptococcus equi subsp. zooepidemicus S31A1, a strain isolated from equine infectious endometritis in Denmark. Comparative analyses of this genome were done with four published reference genomes: S. zooepidemicus strains MGCS10565, ATCC 35246, and H70 and S. equi subsp. equi strain 4047.

Infections with enterococci are challenging to treat due to intrinsic resistance to several antibiotics. Especially vancomycin-resistant Enterococcus faecium and Enterococcus faecalis are of considerable concern with a limited number of efficacious therapeutics available. From an initial screening of 20 peptidomimetics, 11 stable peptide/β-peptoid hybrids were found to have antibacterial activity against eight E. faecium and E. faecalis isolates. Microbiological characterization comprised determination of minimal inhibitory concentrations (MICs), probing of synergy with antibiotics in a checkerboard assay, time–kill studies, as well as assessment of membrane integrity. E. faecium isolates proved more susceptible than E. faecalis isolates, and no differences in susceptibility between the vancomycin-resistant (VRE) and -susceptible E. faecium isolates were observed. A test of three peptidomimetics (Ac-[hArg-βNsce]6-NH2, Ac-[hArg-βNsce-Lys-βNspe]3-NH2 and Oct-[Lys-βNspe]6-NH2) in combination with conventional antibiotics (vancomycin, gentamicin, ciprofloxacin, linezolid, rifampicin or azithromycin) revealed no synergy. The same three potent analogues were found to have a bactericidal effect with a membrane-disruptive mode of action. Peptidomimetics Ac-[hArg-βNsce-Lys-βNspe]3-NH2 and Oct-[Lys-βNspe]6-NH2 with low MIC values (in the ranges 2–8 µg/mL and 4–16 µg/mL against E. faecium and E. faecalis, respectively) and displaying weak cytotoxic properties (i.e., <10% hemolysis at a ~100-fold higher concentration than their MICs; IC50 values of 73 and 41 µg/mL, respectively, against HepG2 cells) were identified as promising starting points for further optimization studies.

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