Defining new Buruli ulcer endemic areas in urban southeastern Australia using bacterial genomics-informed possum excreta surveys.

Buruli ulcer in southeastern Australia is a zoonosis caused by infection with Mycobacterium ulcerans. Australian native possums are a major wildlife reservoir, and infected possums shed M. ulcerans in their excreta, with mosquitoes being the major transmitting vector in this region. Buruli ulcer is geographically restricted, and this feature, combined with an average 4.8-month incubation period, makes tracking M. ulcerans environmental spread and timely identification of new Buruli ulcer endemic areas challenging. While human mobility complicates transmission tracing, we used the highly territorial behavior of native possums and high-resolution pathogen genomics to confidently identify new Buruli ulcer endemic areas in Melbourne's inner northwest and southern suburbs of Geelong. Using pathogen genomic phylodynamic modeling, we estimated that M. ulcerans was introduced to these areas 2-6 years before the emergence of human Buruli ulcer cases. This study shows how possum excreta surveys combined with pathogen genome data can pinpoint new Buruli ulcer endemic areas, thus providing critical local knowledge for targeted public health interventions to reduce exposure risk and ensure early diagnosis.IMPORTANCEBuruli ulcer is a debilitating skin disease caused by Mycobacterium ulcerans, with transmission in southeastern Australia involving native possums as wildlife reservoirs and mosquitoes as vectors. Early detection of new endemic areas is critical to preventing human disease, yet is hindered by the pathogen's long incubation period, highly focal transmission, and the mobility of human hosts. This study demonstrates how structured possum excreta surveys, combined with high-resolution pathogen genomics, can confidently identify new Buruli ulcer endemic areas in southeastern Australia. By confirming local transmission in Melbourne's inner northwest and Geelong through genome matching between possum- and human-derived M. ulcerans, we provide a framework for targeted, timely public health interventions. This integrated wildlife-environment-human approach exemplifies the power of One Health surveillance to detect and manage emerging zoonotic threats, offering a scalable model for other regions where Buruli ulcer is endemic.