Co-occurrence of Yersinia pestis and a lytic bacteriophage in brain tissue of Eothenomys miletus

ABSTRACT We surveyed plague in the Apodemus chevrieri–Eothenomys miletus natural plague focus on the Yunnan Plateau, China. From 2023 to 2025, plague-positive E. miletus were detected in Gaomei, Xiaomachang, and Tangshang. In the brain tissue of an E. miletus carcass, both Yersinia pestis and a Yersinia pestis -specific bacteriophage (YN2024) were simultaneously isolated, and their simultaneous presence was observed via transmission electron microscopy (TEM). This observation was supported by previous analysis of a similar co-occurrence of Y. pestis and bacteriophage from the Marmota himalayana plague focus. After 72 h of in vitro liquid culture, TEM imaging revealed multiple morphological stages that were indicative of interaction, including intact bacteria, bacteria enveloped internally and externally with phages, lysed bacteria, and numerous released phages. These findings suggest that such co-occurrence may be a recurring phenomenon across different plague foci, as it has now been observed in two distinct ecosystems. Genomic analysis demonstrated that the phage YN2024 exhibited a high degree of homology with the phage AKS2021-55F0913 from the M. himalayana plague focus (average nucleotide identity, 96.94%). A comparison of growth curves indicated that the lytic capacity of phage AKS2021-55F0913 was stronger than that of phage YN2024. IMPORTANCE Overall, our findings support the recognition of an ecologically connected Apodemus chevrieri–Eothenomys miletus natural plague focus and also provide a basis for understanding the co-occurrence of Yersinia pestis and its phage in natural hosts across multiple foci. The observed ecological linkage among plague foci previously attributed to different administrative regions highlights the importance of considering ecological connectivity in plague surveillance and control. In addition, these findings may contribute to a better understanding of host-phage interactions involving Y. pestis under natural conditions and may inform future phage-related studies in plague research.