Impact of microbiological testing protocols on discard rates in donor human milk with a focus on Bacillus cereus

Microbiological testing protocols are key components of human milk bank operations, yet the lack of consensus on screening schedules and microbiological criteria leads to variability in discard rates and uncertainty about safety standards. This study provides a large-scale characterization of pre-pasteurization donor human milk microbiota and evaluates the impact of testing protocols on operational outcomes. Over 59 months, 7,921 donor human milk batches were analysed. Pre-pasteurization analyses were dominated by skin staphylococci, with environmental Gram-negative bacilli and enteric genera detected at lower prevalence. Among pathogens of concern, only Staphylococcus aureus, Escherichia coli and Bacillus cereus sensu lato were identified. A selective 24-h workflow, incorporating BACARA® plates with a detection limit of 1 CFU/mL, was validated and implemented to target these organisms within the pre-pasteurization 24-h storage interval. This protocol significantly increased B. cereus s.l. detection (2.2% vs. 8.8%), without affecting S. aureus or Enterobacteriaceae detection, indicating improved analytical sensitivity. B. cereus s.l. exhibited a reproducible warm-season excess (5.5% vs. 2.4%) and accounted for 72% of post-pasteurization positives. In a sub-study of 491 batches, agreement between pre- and post-pasteurization B. cereus s.l. results was good (κ ≈ 0.61) with a high negative predictive value (95%), supporting the predictive value of early screening. Protocol design was a major determinant of discard rates, and using higher microbiological thresholds (2 × 10⁵ CFU/mL) reduced avoidable losses without compromising safety. These findings hightlight the importance of integrating rapid, selective methods and advancing towards harmonized, risk-based microbiological criteria in human milk banking to improve both operational efficiency and microbiological safety.