Isolation and characterization of bacteriophages specific to selected equine genital pathogens

Abstract

Chronic bacterial endometritis is among the major problems in equine reproduction. Streptococcus equi ssp. zooepidemicus (SEZ), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA) are among the most important pathogens for the condition. Bacteriophages (phages) are the viruses of bacteria, having a species- and strain-specific lytic activity. Due to their bactericidal capacities, phages can be used as an alternative treatment approach for bacterial infections. The study aimed to isolate and characterize phages with lytic activity against Streptococcus equi ssp. zooepidemicus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phage samples were obtained from manure, uterine lavage fluid and drain water of horse husbandries (n=14). For isolation of phages, bacterial isolates of the equine genital tract (SEZ (n=1), KP (n=1), and PA (n=1)) were inoculated with the phage samples. For purification, bacteriophage plaques were repeatedly cultivated, harvested and filtered. For determining the host-range, the obtained phages were inoculated with bacterial isolates (SEZ (n=37), KP (n=26), and PA (n=33)). In total, 28 bacteriophages were isolated (against SEZ (n=13), KP (n=6), and PA (n=9)). Bacteriolysis was achieved in 32/37 Streptococcus-, 16/26 Klebsiella-, and 14/33 Pseudomonas-isolates. The bacteriophage showing the broadest host range covered 81% of all tested Streptococcus-isolates (30/37). Host ranges of Klebsiella pneumoniae- (9/26) and Pseudomonas aeruginosa-specific bacteriophages (14/33) covered a lower proportion of tested isolates. Seven bacteriophages (SEZ (n=3), KP (n=3), PA (n=1)) were selected according to theirhost ranges and submitted to electron microscopy for further characterization. Electron micrographs revealed that the selected bacteriophages belonged either to the family Demerecviridae (n=1), Myoviridae (n=1), Podoviridae (n=3) or Siphoviridae (n=2), which was confirmed by sequencing. Next, planktonic killing assay was conducted, incubating liquid cultures of Streptococcus equi ssp. zooepidemicus-, Klebsiella pneumoniae- and Pseudomonas aeruginosa-isolates in presence and absence of a specific bacteriophage and monitoring growth by optical density measurements for 24h. Significantly decreased bacterial growth, as determined by comparing the area under the curve (AUC), was observed in presence of 0/3 Streptococcus equi ssp. zooepidemicus-, 1/1 Pseudomonas aeruginosa- and 3/3 Klebsiella pneumoniae-specific phages, using a multiplicity of infection of 1 (MOI1; one phage per host cell). At MOI10 the most effective Streptococcus-phage reduced the AUC by 61%; the most effective Klebsiella-phage resulted in 95% reduction using MOI1 and for the Pseudomonas-phage AUC was reduced by 14% at MOI100. In conclusion, isolation of phages specific to equine uterine pathogens resulted in 28 purified phages. Seven phages were selected and further characterized by efficacy testing, sequencing and electron microscopy. Results of the in vitro host range determination and planktonic killing assay are promising, suggesting further investigations and the development of an alternative treatment approach for uterine infections in mares.