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

<i>Bacillus cereus</i> 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 <i>B. cereus</i> isolates from human milk (pre- and post-HoP), as well as from patient and environmental sources, to investigate human milk contamination by <i>B. cereus</i> at a Canadian HMB. Despite the limited temporal and geographic scope of the collection, the isolates exhibited remarkable genomic diversity, comparable to global <i>B. cereus</i> collections. Phylogenetic analysis at the core genome level revealed no clear clustering by isolate source, suggesting multifactorial pathways of <i>B. cereus</i> 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 <i>B. cereus</i> 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 <i>B. cereus</i> resilience.