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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). 100 90 80 70 60 50 40 30 20 10 0 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 100 100 80 80 60 60 40 40 20 20 0 0 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.