Isolation of Rhodococcus equi bacteriophage ReT1 and investigation of its bactericidal effect

In recent years, multidrug-resistant Rhodococcus equi has emerged globally. Phage therapy has been attracting renewed attention as an alternative to antibiotics. In this study, we aimed to isolate and maintain R. equi bacteriophages and characterize them as a potential agent for phage therapy to treat R. equi infection. Phages with lytic activity, ReT1, were induced with mitomycin C from R. equi Kuma83-10 strain that genomic analysis revealed to possess phage-like sequences. ReT1 showed two plaque types immediately after induction, and the cloned ones were designated ReT1v-1 and ReT1v-2. These phages could be maintained in the laboratory without titer reduction, were morphologically Siphoviridae-like phages, and were stable under the environmental conditions expected for use as phage therapy. ReT1v-2 was altered into a lytic phage lacking the region around the integrase gene from the ReT1v-1 genome. Both phages did not differ in their host range (narrow range), that is, formed spots only in R. equi JID03-27, of the 18 strains examined. However, ReT1v-2 showed the most pronounced bactericidal effect against JID03-27:pVAPA strains, which infected cultured macrophages. This result was consistent in vivo, with the ReT1v-2-treated group showing growth inhibition in mice. The collection of more phages with lysogenic activity against R. equi and with different characteristics will lead to more options for phage cocktail formulations in anticipation of resistance. The results of this study support the possibility of developing formulations based on these phages.