Fratini Riccardo

Background: Listeria monocytogenes is an ubiquitous foodborne pathogen capable of persisting in food-processing environments, domestic settings, and water systems; consequently, the mere co-detection of the microorganism in a patient and in a food product is insufficient to support criminal causation. In judicial proceedings, attribution of invasive listeriosis to a specific food and food business operator requires a pathogen-specific evidentiary chain that exceeds the standards used for public health surveillance. Methods: We performed a retrospective medico-legal and methodological analysis of a fatal listeriosis case that triggered criminal prosecution for foodborne homicide, systematically assessing the investigative chain according to temporal plausibility, food traceability, compliance with European and ISO microbiological standards, interpretation of whole-genome sequencing (WGS) data for an ubiquitous pathogen, and clinical causation of death. Results: The analysis identified critical weaknesses in the accusatory reconstruction, including incompatibility with established incubation periods, lack of proof that the sampled food lot corresponded to the product actually consumed, deviations from standard microbiological procedures, reliance on a poorly documented “clinical” isolate for WGS, non-specific genomic findings within a widely disseminated clonal complex, and the presence of plausible alternative environmental reservoirs. Clinically, the immediate cause of death was more consistent with hypovolemic shock due to uncontrolled gastrointestinal bleeding than with ongoing listerial sepsis. Conclusions: This article demonstrates how, in cases involving ubiquitous pathogens, failure to adopt a pathogen-specific investigative and interpretative framework may lead to methodologically fragile criminal allegations, potentially increasing litigation burden and costs for the justice system. A methodologically robust approach integrating microbiology, genomics, epidemiology and medico-legal causation analysis is essential for fair and scientifically sound criminal proceedings.

This study builds on a prior proof-of-concept metabolomic analysis of post-mortem pericardial fluid to assess its reproducibility and validate its utility for estimating the post-mortem interval. Sixty-five pericardial fluid samples were collected during medico-legal autopsies in two different Forensic Medicine Institutes with post-mortem intervals spanning 16 to 199 h. Samples underwent liquid-liquid extraction and ¹ H NMR analysis, quantifying 50 metabolites. Multivariate statistical analyses were employed to develop post-mortem interval estimation models, controlling for age to minimize its confounding effects. Reproducibility was confirmed, with 92% of metabolites showing high similarity (cosine similarity ≥ 0.90) in 23 re-analyzed samples, demonstrating robust intra-laboratory consistency. For post-mortem intervals of 16 to 100 h, the regression model achieved presented a prediction error of 16.7 h, identifying nine key predictors, including choline, glycine, citrate, betaine, ethanolamine, glutamate, ornithine, uracil, and β-alanine. For intervals of 16 to 130 h, the prediction error was 23.2 h, and for 16 to 199 h, it was 42.1 h. A classification model distinguishing intervals below 48 h from those above 48 h showed high accuracy for detecting longer intervals, with key predictors including aspartate, histidine, and proline. These findings underscore the stability and reproducibility of pericardial fluid metabolomics, establishing its potential as a reliable forensic tool for post-mortem interval estimation, particularly beyond 48 h, with significant implications for forensic investigations.