Sari Tuomisto

The microbiome serves important functions in human health, and postmortem, the microbial signatures of colonized organ tissue could be useful in helping to predict the manner of death in cases where this information is not known. We surveyed the microbiota (16S rRNA V4 amplicon sequencing) of 265 organ tissue samples including liver, blood, brain, heart, prostate, spleen and uterus from cadavers in Italy, Finland and the United States with confirmed manners of death comprising either accidental death, natural death, homicide, and suicide. Geographic locality (i.e. nationality) had a strong effect on observed microbial composition. Differing PERMANOVA results between unweighted and weighted UniFrac (nearly inverse results) suggest that specific bacteria may be associated with ethnicity and age, but that these differences are negligible when taking into account the relative abundance of bacterial taxa; weighted UniFrac measures suggest that although taxonomic composition may not vary significantly between different manners of death, PMI, or BMI categories, the relative abundance of specific taxa vary significantly. Various tissues exhibit differential associations with bacteria, and prostate and uterus were substantially different compared to other organs. For example, in Italian cadavers, the bacteria MLE1-12 permeated nearly all tissues, except the prostate and uterus. We identified specific bacterial ASVs as biomarkers of either natural or accidental death and suicide, but not for homicide. While the manner of death may have an impact on microbial associations, further investigation under more controlled conditions will be needed to validate whether these associations are predictive in forensic determinations. Importance: The utilization of microbial data in the context of forensic investigations holds great promise for the field of forensic science. Identification of taxa that are associated with postmortem interval (PMI), specific manners of death (MOD), or other traits such as age, sex, ethnicity, and nationality may allow investigators to refine the circumstantial details surrounding the death of an individual. In this study we find nationality (geographic location of cadaver) to be a dominant predictor of cadaver microbiome composition. We also identify a number of cadaver-specific traits to be associated with microbial alpha- and beta diversity, as well as bacterial taxa that are differentially associated with these traits.

The liver is the first line of defence against continuously occurring influx of microbial-derived products and bacteria from the gut. Intestinal bacteria have been implicated in the pathogenesis of alcoholic liver cirrhosis. Escape of intestinal bacteria into the ascites is involved in the pathogenesis of spontaneous bacterial peritonitis, which is a common complication of liver cirrhosis. The association between faecal bacterial populations and alcoholic liver cirrhosis has not been resolved. Relative ratios of major commensal bacterial communities (Bacteroides spp., Bifidobacterium spp., Clostridium leptum group, Enterobactericaea and Lactobacillus spp.) were determined in faecal samples from post mortem examinations performed on 42 males, including cirrhotic alcoholics (n = 13), non-cirrhotic alcoholics (n = 15), non-alcoholic controls (n = 14) and in 7 healthy male volunteers using real-time quantitative PCR (RT-qPCR). Translocation of bacteria into liver in the autopsy cases and into the ascites of 12 volunteers with liver cirrhosis was also studied with RT-qPCR. CD14 immunostaining was performed for the autopsy liver samples. Relative ratios of faecal bacteria in autopsy controls were comparable to those of healthy volunteers. Cirrhotics had in median 27 times more bacterial DNA of Enterobactericaea in faeces compared to the healthy volunteers (p = 0.011). Enterobactericaea were also the most common bacteria translocated into cirrhotic liver, although there were no statistically significant differences between the study groups. Of the ascites samples from the volunteers with liver cirrhosis, 50% contained bacterial DNA from Enterobactericaea, Clostridium leptum group or Lactobacillus spp.. The total bacterial DNA in autopsy liver was associated with the percentage of CD14 expression (p = 0.045). CD14 expression percentage in cirrhotics was significantly higher than in the autopsy controls (p = 0.004). Our results suggest that translocation of intestinal bacteria into liver may be involved as a one factor in the pathogenesis of alcoholic liver cirrhosis.

Postmortem bacteriology can be a valuable tool for evaluating deaths due to bacterial infection or for researching the involvement of bacteria in various diseases. In this study, time‐dependent postmortem bacterial migration into liver, mesenteric lymph node, pericardial fluid, portal, and peripheral vein was analyzed in 33 autopsy cases by bacterial culturing and real‐time quantitative polymerase chain reaction (RT‐qPCR). None suffered or died from bacterial infection. According to culturing, pericardial fluid and liver were the most sterile samples up to 5 days postmortem. In these samples, multigrowth and staphylococci were not or rarely detected. RT‐qPCR was more sensitive and showed higher bacterial positivity in all samples. Relative amounts of intestinal bacterial DNA (bifidobacteria, bacteroides, enterobacter, clostridia) increased with time. Sterility of blood samples was low during the studied time periods (1–7 days). The best postmortem microbiological sampling sites were pericardial fluid and liver up to 5 days after death.

Post mortem or even normal changes during life occurring in major gut bacterial populations are not known. We investigated Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea ratios in 7 fecal samples from healthy volunteers and in 61 autopsies rectum and cecum samples and studied the effect of post mortem time using quantitative real-time PCR. Bacterial ratios in stool samples from volunteers and rectum samples from autopsy cases were similar and did not change significantly up to 5 days post mortem. In cecum, significant post mortem time-dependent differences were observed in ratios of Bacteroides sp. (p = 0.014) and Lactobacillus sp. (p = 0.024). Our results showed that ratios of Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea can be investigated in autopsy rectum samples up to 5 days after death.

Milk is an important source of nutrients. The consumption of milk, however, may cause abdominal complaints in lactose intolerant individuals. The frequency of -13910C/C genotype is known to be high among Northern Russians, exceeding the prevalence in northern Europe. In our study we tested two hypotheses: 1) subjects with lactase non-persistent genotype (-13910C/C) have more gastrointestinal (GI) symptoms associated with milk 2) subjects with lactase non-persistence avoid using milk.