Analysis of bacterial dynamics and biochemical markers in postmortem interval estimation

Abstract

The determination of the postmortem interval (PMI) is fundamental to forensic investigations but remains challenging due to numerous influencing factors, including cause of death and environmental conditions. Traditional indicators such as Algor Mortis, Rigor Mortis, and Livor Mortis are useful early on but often unreliable beyond initial stages. Complementary approaches from entomology, botany, and microbiology show promise but vary in precision. Recent advances in high-throughput sequencing have underscored the potential of postmortem microbiology and biochemistry. However, while sequencing dominates the field recently, culture-based approaches, critical for accurate species-level resolution, remain underexplored in PMI research. This study aimed to perform a combined microbial and biochemical analysis of different organs (lungs, heart, kidneys, liver, and brain) at six PMI (0, 12, 24, 48, 72, and 96 hours) in male C57BL/6J SPF mice (n=3 per timepoint). For microbial analysis, total bacterial load, and the distribution of Escherichia coli and Enterococcus faecalis were quantified. Feces and organs were homogenized in buffered peptone water and cultured on non-selective (Blood agar) and selective (MacConkey for E. coli, and Slanetz-Bartley for E. faecalis) media. Colony-forming units (CFU) per gram of tissue (total and species-specific bacterial loads) were determined after aerobic incubation. For biochemical analysis, tissue homogenates in phosphate buffer (pH 7.4) were processed using the Accent MC240 autoanalyzer (Cormay®). Selected biochemical markers included urea, lactate, uric acid, glucose, total proteins, magnesium, ethanol, and iron. To develop a mathematical model for PMI estimation, markers were normalized to magnesium, and those showing a clear and consistent increasing or decreasing trend, with a Pearson correlation coefficient (r) greater than 0.900, were selected. Postmortem microbiome dynamics were strongly tissue- and time-dependent, with total aerobic bacterial loads peaking at 72 hours in most organs, except the brain, which peaked at 96 hours, likely due to its anatomical protection. E. coli was absent in the liver, heart, and brain. E. faecalis exhibited consistent colonization patterns, particularly in kidneys, lungs, and brain, with initial invasion observed at 24 hours and peaking between 72 and 96 hours. These findings underscore the value of culture-based methods in detecting taxa relevant to PMI estimation. Biochemical markers showed distinct tissue- and time-dependent trends. Lactate increased early in most organs, peaking at 24-48 hours PMI, with a sustained increase in the brain, likely consequent to anaerobic metabolism. Uric acid levels also increased progressively, especially in the kidney, heart, and brain, likely reflecting purine degradation and autolysis. Urea levels exhibited a marked increase at later PMI. Iron increased steadily in the kidney and heart, peaking earlier in the liver. Among the different biochemical markers, lactate was most informative at early PMI stages, while uric acid and urea correlated strongly with later intervals. These findings suggest that culture-based microbiological data, combined with biochemical markers, provide a robust multiparametric support for PMI estimation. Particularly, E. faecalis colonization in the brain and the dynamics of lactate, uric acid, urea, and iron in different organs emerged as reliable indicators of postmortem progression. Nevertheless, further validation in complex forensic conditions, including in humans, is necessary.