High Genetic Diversity Among Isolates Contaminating Donated Milk at a Canadian Human Milk Bank.

poses a persistent challenge for human milk banks (HMBs) due to its ability to survive Holder pasteurization (HoP; 62.5 °C for 30 min). To ensure neonatal safety, any milk found to be contaminated post-HoP must be discarded, which impacts milk supply and adds to the operational demands of HMBs. In this study, we analyzed 688 isolates from human milk (pre- and post-HoP), as well as from patient and environmental sources, to investigate human milk contamination by at a Canadian HMB. Despite the limited temporal and geographic scope of the collection, the isolates exhibited remarkable genomic diversity, comparable to global collections. Phylogenetic analysis at the core genome level revealed no clear clustering by isolate source, suggesting multifactorial pathways of contamination. Isolates surviving HoP displayed gene variants linked to sporulation and cell wall integrity, suggesting a potential basis for HoP tolerance. Our findings emphasize that while genomic analyses offer major valuable insights, they alone are insufficient to address the complexities of contamination in HMBs. Addressing this challenge will require combining genomic tools with robust monitoring systems, improved human milk-handling protocols, and pasteurization strategies better-suited to countering resilience.