Isolation and characterization of bacteriophages specific to Streptococcus equi subspecies zooepidemicus and evaluation of efficacy ex vivo

<i>Streptococcus (S.) equi</i> subspecies (subsp.) <i>zooepidemicus</i> is an important facultative pathogen in horses and can cause severe infections in other species including humans. Facing the post-antibiotic era, novel antimicrobials are needed for fighting bacterial infections. Bacteriophages (phages) are the natural predators of bacteria and discussed as a promising antimicrobial treatment option. The objective of this study was to isolate and characterize <i>S. equi</i> subsp. <i>zooepidemicus-</i>specific phages for the first time and to evaluate their efficacy <i>in vitro</i> and <i>ex vivo</i>. In total, 13 phages with lytic activity were isolated and host ranges were determined. Two phages with broad host ranges and high efficiency of plating (vB_SeqZP_LmqsRe26-2 (lytic activity: 30/37 bacterial isolates) and vB_SeqZP_LmqsRe26-3 (lytic activity: 29/37 bacterial isolates)) and one phage with relatively low efficiency of plating (vB_SeqZP_LmqsRe26-1) were selected for further characterization, including electron microscopy and whole genome sequencing. In <i>in vitro</i> planktonic killing assays at two tested multiplicities of infection (MOI 1 and MOI 10), significant bacterial growth reduction was observed when the phages vB_SeqZP_LmqsRe26-2 and vB_SeqZP_LmqsRe26-3 were added. These phages were subsequently co-incubated with clinical <i>S. equi</i> subsp. <i>zooepidemicus</i> isolates in an equine endometrial explant model but did not achieve bacterial growth reduction at MOI 1 and MOI 10. However, helium ion microscopy revealed presence of particles adherent to the bacteria on the explant after incubation (25 h), suggesting possible phage-bacteria interactions. In conclusion, phages against <i>S. equi</i> subsp. <i>zooepidemicus</i> were successfully isolated and characterized. Promising results were observed in <i>in vitro</i> but no significant reduction was detected in <i>ex vivo</i> experiments, requiring additional investigations. However, after further adaptations (e.g., optimization of MOIs and phage administration or use of phage-antibiotic combination), phages could be a potential antimicrobial tool for future therapeutic use in <i>S. equi</i> subsp. <i>zooepidemicus</i> infections, although the available results do not currently support the therapeutic usage.