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We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Numbers and types of bacterioplankton proliferating in blood samples mixed with water of various salinity levels were examined to determine the characteristics of species associated with salinity. Water samples (total n=88) were collected from the midstream of two rivers (freshwater; n=10; salinity <0.05%), from around their estuaries (areas of freshwater, n=20, salinity <0.05%; areas of brackish water, n=20, salinity <0.05-3.1%; areas of marine water beyond the mouths of the rivers, n=28, salinity 2.4-3.3%), and from the coast (areas of marine water; n=10; salinity 3.3-3.5%). Freshwater bacteria were identified in 41 of 42 blood samples mixed with water at ≤1.3% salinity, and the genus Aeromonas, which is universally distributed in freshwater environments, was predominant. Marine bacteria were identified in all of 46 blood samples mixed with water at ≥1.8% salinity, and most comprised the genera Vibrio and Photobacterium that are universally distributed in seawater environments. Aeromonas was undetectable in all blood samples mixed with brackish or sea water at ≥1.8% salinity although they are detectable even in seawater environments. Thus, the present results showed that bacterioplankton capable of proliferating in human blood reflects the salinity of water., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.