Time-dependent post mortem changes in the composition of intestinal bacteria using real-time quantitative PCR
© Tuomisto et al.; licensee BioMed Central Ltd. 2013
Received: 30 August 2013
Accepted: 20 November 2013
Published: 25 November 2013
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.
KeywordsForensic science Post mortem microbiology Fecal sample Real-time quantitative polymerase chain reaction Bacterial relative amount Time-dependent changes
Basic knowledge on the composition of intestinal bacterial populations and changes occurring after death is lacking. Even the normal composition of intestinal microbiota in life is not fully known . Only one study exists in which intestinal bacterial populations have been studied in three elderly women after death using PCR and sequencing .
Resident micro-organisms living in the intestinal tract influence host’s normal well-being and physiology including gut metabolism and the regulation of epithelial cell growth . Intestinal microbiota functions as a physical barrier against invading pathogens. It has been suggested that gut microbiota may have a role on the development of diseases, e.g. alcoholic liver cirrhosis  and atherosclerosis . Detailed bacterial population studies on the intestinal tract have mostly concentrated on fecal samples because they are easy to collect. Intestinal microbiota consists of a large and diverse community containing hundreds of commensal bacterial species . From sequencing libraries of 16S rRNA genes Durban et al. found that two dominant phyla, Firmicutes and Bacteroidetes accounted for nearly 85% of all sequences in stool samples . Compared to these two major phyla, Bifidobacterium genus is present in eight to ten-fold lower numbers . Although Bacteroides sp., Bifidobacterium sp. and bacteria belonging to the Clostridium coccoides–group (cluster XIVa) and Clostridium leptum–group (cluster IV) dominate in colon [9, 10] there is substantial inter- and intra-individual variation in species composition and distribution [7, 11].
This study aimed to investigate ratios of major intestinal bacterial populations in healthy volunteers and in rectum and cecum autopsy samples. Post mortem time-dependent changes were studied in order to see whether autopsy samples can be used for basic research concerning lifetime. Six species: Bacteroides sp. (phylum Bacteroidetes), Clostridium sp. (Firmicutes), Streptococcus sp. (Firmicutes), Lactobacillus sp. (Firmicutes), Bifidobacterium sp. (Actinobacteria) and Enterobactericaea (Proteobacteria) were chosen since they represent the major intestinal bacterial phyla .
Study design and results
This study comprises of 61 male cases collected in the Department of Forensic Medicine of the University of Tampere and 7 male volunteers. The selection criteria for the autopsies have been described elsewhere . None of the controls or cases was reported to has been used antibiotics. Deceased had been stored in +4°C within 24 hours after death. Written consent was obtained from the volunteers.
Demographic characteristics of the study subjects divided by post mortem time
Basic cause of death
Age mean (range)
BMI mean (range)
Heart diseases %
Other diseases %
Violent deaths (suicide, accident, poisoning) %
Used primers and probes
Primer and probe
Bifidobacterium sp .
Streptococcus sp. mainly Str. mitis- group*
The comparative Ct method (ΔΔCt, ΔCt sample – ΔCt reference sample), was used where mean values from healthy male volunteers were calculated and used as a reference to determine bacterial relative amount in rectum samples. The differences of the Ct values between the bacteria and the universal bacteria measurement (ΔCt) for each sample were calculated; the comparative Ct (ΔΔCt) for sample and reference samples was calculated. To determine relative amounts of bacteria in cecum samples the rectal sample was used as an inner reference.
Two standard curves were used to determine the total amount of bacteria. Tenfold dilution series of between 33 ng/ml and 0.00033 ng/ml from E. coli genomic DNA (ATCC 35401–5) as well as between 109 and 105 colony forming units (CFU) per milliliter from E .coli (ATCC 25922) were applied. The amount of CFU or bacterial DNA in the sample was calculated using values from universal measurement and the equation y = slope log (X) + intercept .
Statistical analyses were performed with Kruskal-Wallis median test with PASW Statistical Software, version 18 (SPSS Ltd, Quarry Bay, Hong Kong). If P-value was less than 0.05 (considered significant) pairwise Post Hoc comparisons using Mann–Whitney U-test were done.
The relative amounts (n-fold difference) of measured bacteria in cecum samples compared to rectum samples over post mortem time
The total amount of bacterial DNA in fecal samples
This study showed that relative amounts of major intestinal bacteria in rectum of autopsy cases were similar to stool donated by volunteers and remained quite stable over post mortem time up to 5 days, after which the total amount of bacteria started to increase. In contrast, in cecum significant post mortem time-dependent differences were observed as increase in ratio of strictly anaerobic Bacteroides sp. and decrease of facultative Lactobacillus sp. due to hypoxia after death. In cecum there is accumulation of undigested nutrients and metabolites produced by bacteria after death, which may be conducive to anaerobic bacterial growth. This study showed that autopsy rectum samples can be used to evaluate major intestinal bacterial populations concerning lifetime up to 5 days after death.
The excellent technical assistance from the personnel of the Department of Forensic Medicine, especially Mervi Seppänen, Kari Mänttäri and Olli Penttilä, University of Tampere, Finland is gratefully acknowledged. We also appreciate the participation of the volunteers.
This study was financially supported by the Tampere Graduate Program in Biomedicine and Biotechnology (TGPBB), the Finnish Foundation for Alcohol Studies, the Competitive Research Funding of the Pirkanmaa Hospital District, the European Union 7th Framework Program grant number 201668 for the AtheroRemo Project, the Pirkanmaa Regional Fund of the Finnish Cultural Foundation.
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