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Bacterial Succession of the Aquatic Necrobiome as
an Indicator of Post Mortem Submersion Interval
Samantha A. Lehan1, Professor John P. Cassella1, Dr. Claire Gwinnett1, Dawn Williams2
1. Department of Forensic and Crime Science, Faculty of Computing, Science and Engineering, Staffordshire University, U.K
2. Department of Clinical Microbiology, University Hospitals of Leicester, Infirmary Square, Leicester, LE1 5WW, U.K
Introduction
Table 1: Potentially useful bacteria for PMSI estimation
Water Quality Data
The soft tissue decomposition of human remains has been extensively studied,
resulting in identification of sequential morphological change and patterns of
insect colonisation. These studies have led to formulae being derived for post
mortem interval estimation. Despite 140,000 annual police investigations
around the world involving water, much of the present knowledge is only
applicable to terrestrial decomposition [1].
ORGANISM
All bodies of water may be separated into layers according to how much light
penetrates the water and the oxygen concentration. Hence, a difference in
water quality data will be observed for water taken from the surface and the
bed [5] (Figures 3, 4, 5). This is particularly interesting in this case as the depth
of the brook was only approximately 15cm deep due to dry, warm weather.
The difference in water quality data shows that the population of
microorganisms within a body of water may change with depth due to their
environmental requirements.
It is well documented that micro-organisms are involved in decomposition
processes, and are ubiquitous in water sources and human bodies [2].
Investigating the sequential colonisation of a body by microorganisms in water
as well as the bacterial migration from the gut may provide a useful method
for estimating post mortem submersion interval (PMSI).
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Matrix assisted laser desorption/ionisation time-of-flight mass spectrometry
(MALDI-TOF MS) has been described as a revolutionary new technique within
the field of microbiology [3] and is expected to become the method of choice
over other biochemical microbial identification techniques [4]. This study
utilised MALDI-TOF MS as a rapid, inexpensive and sensitive method for
identifying bacteria from a freshwater system.
Temperature (ᵒC) Dissolved Oxygen Dissolved Oxygen
(%)
(mg/L)
Surface
Surface Water
The agar used were chocolate agar, Columbia blood agar (CBA), CystineLactose-Electrolyte-Deficient (CLED) agar and Fastidious Anaerobe Agar (FAA).
This agar was chosen as these are typical, non-selective culture media used in
NHS laboratories. The agar plates were incubated at 37ᵒC. After 24 hours
incubation, purity plates were prepared by ‘picking’ colonies from the mixed
cultures and inoculated onto fresh agar and incubated at 37ᵒC (Figure 2). After
24 hours incubation, the purity plates were removed from incubation. Each
colony was Gram stained and a Vitek MS-DS target slide was prepared for
identification using MALDI-TOF MS.
Figure 1. Primary CBA plate
showing the full range of
bacterial colonies cultured
after 24 hours incubation
Figure 2. Secondary CBA
plate showing pure culture
for identification by MALDITOF MS
Bed Water
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pH
Day of Collection
Temperature (ᵒC)
Conclusions
Further Research
Bed
Figure 3. Average water quality data taken on the day of
collection for both surface and bed water samples
Water samples were collected from Barkby Brook, Barkby, U.K and processed
in the Clinical Microbiology Department at the Leicester Royal Infirmary. Water
from the brook’s surface and bed were collected into sterile universal
containers. Water quality data was recorded before testing. At the laboratory,
samples were centrifuged 3500 rpm for 10 minutes to concentrate the
bacterial cells. The supernatant was removed and the pellet was re-suspended
in 1ml of phosphate buffered saline. 10µl of the suspension was spread onto
each type of agar (Figure 1).
Clostridium histolyticum
Enterococcus hirae
Paenibacillus pabuli
Rahnella aquatilis
Vibrio furnissi
Vibrio metschnikovii
The results show that MALDI-TOF MS provides rapid identification of
bacteria in freshwater to species level and may provide a useful tool in
the investigation of the bacterial succession of the necrobiome. The study
also shows that the depth of submersion may affect the bacteria
identified from remains.
pH
Materials and Methods
Aeromonas hydrophila/caviae
Aeromonas sobria
Bacillus licheniformis
Bacillus simplex
Buttiauxella agrestis
Citrobacter freundii
Dissolved Oxygen
(%)
2 days post collection
Dissolved Oxygen
(mg/L)
7 days post collection
Figure 4. Water quality data changes over
time for surface water samples stored at 4°C
pH
Day of Collection
Temperature (ᵒC) Dissolved Oxygen (%) Dissolved Oxygen
(mg/L)
2 days post collection
7 days post collection
 Continue sampling Barkby Brook at a consistent point and depth to
maintain database of organisms over seasons.
 Water sampling at another freshwater location to determine if there
are common bacteria.
 Perform animal tissue submersion experiments to identify successive
bacteria colonisation of the remains and the bacterial changes to the
water.
 Investigate the role of bacterial biofilms on the rate of decomposition
and necrobiome community structure.
 Compare laboratory results with veterinary case studies.
Figure 5. Water quality data changes over
time for bed water samples stored at 4°C
References
Bacterial Identification
To date, MALDI-TOF MS identification has yielded 116 bacterial identifications
from water samples collected from Barkby Brook. Of these identifications, a
number of bacterial species may prove useful for determining PMSI as they are
not considered normal human microbiota (Table 1). This suggests that these
bacteria may form part of the decomposition microbiology, or necrobiome.
However, the necrobiome consists of both bacteria from the water source as
well as bacteria from within the living body that is able to thrive in the
decomposition environment. MALDI-TOF MS will be used in further
investigations throughout this project to assess the composition of the aquatic
necrobiome over time to identify succession patterns of potential use for PMSI
estimation.
1. Dickson, G.C, R.T.M. Poulter, E.W. Maas, K. Probert and J.A. Kieser. 2011. Marine bacterial
succession as a potential indicator of post-mortem submersion interval. Forensic Science
International. 209: 1 – 10
2. Kakizaki, E, O. Yoshitoshi, S. Kozawa, S. Nishida, T. Uchiyama, T. Hayashi and N. Yukawa. 2012.
Detection of diverse aquatic microbes in blood and organs of drowning victims: First metagenomic
approach using high-throughput 454-pyrosequencing. Forensic Science International. 220: 135 –
146
3. Yaman, G, I. Akyar and S. Can. 2012. Evaluation of the MALDI-TOF MS method for identification
of Candida strains isolated from blood cultures. Diagnostic Microbiology and Infectious Disease.
73: 65 -67
4. Patel, R. 2013.Matrix-assisted laser desorption ionisation time of flight mass spectrometry in
clinical microbiology. Clinical Infectious Diseases. 57(4): 564 – 72
5. Hogg, S. 2013. Essential Microbiology. Wiley-Blackwell, 2nd Ed
Acknowledgements
Thank you to the staff in Clinical Microbiology at University Hospitals of Leicester
for technical advice and the use of the laboratory.