Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning
Introduction
The diagnosis of death by drowning remains a problematic issue in forensic medicine. Drowning is a process resulting in primary respiratory impairment from submersion/immersion in a liquid medium [1]. This definition is the consensus of a group of international investigators during a conference convened in Amsterdam in June 2002. In 1918, Roll already gave a similar definition: “death by drowning is the result of a hampering of the respiration by obstruction of mouth and nose by a fluid medium (usually water)” [2]. These definitions do not necessarily mean aspiration of water (true drowning). Drowning without aspiration of water (immersion death, dry-drowning, hydrocution, “Badetod”) as a result of cardiac arrest or respiratory arrest due to autonomous neural reflex or laryngospasm, is also mentioned by many authors [3], [4]. Although it remains a controversial issue, this kind of drowning, estimated at about 10–15% of death occurring in water, is mostly seen in situations with ethanol intoxication and cold water. According to Modell et al. it is risky to ascribe drowning as a cause of death to a body found in water without some evidence of the effect of having aspirated water [5]. Therefore, there is a need for good diagnostic markers for drowning. The study of fresh water ecology to aid this type of medico-legal investigations belongs to the field of forensic limnology and has generated a number of potential diagnostic markers. During immersion, water dissolved compounds and suspended particles with small diameter from the drowning medium such as electrolytes, diatoms [6], [7], [8], [9], [10], [11], [12] and bacterioplankton [13], [14], [15], [16], [17] are able to reach the pulmonary circulation through diffusion, osmosis or small lesions in the alveolar membranes and are further distributed to other tissues including liver, kidney and bone marrow. Although controversial because of problems regarding risk of contamination, lack of sensitivity and the false-positive recovery of diatoms from tissue samples of non-drowned persons, digestion of tissue samples for analyses of unicellular diatoms currently remains the gold standard to assist the medico-legal diagnosis of death by drowning [18], [19].
In an attempt to find a more reliable and/or sensitive alternative to the diatom test, several studies have explored the possibility to use bacterial cultures from blood samples of water cadavers [13], [15], [16], [17], [20], [21], [22], [23], [24], [25], [26]. In recent literature, members of the bacterial genus Aeromonas such as the species Aeromonas sobria, Aeromonas hydrophila and Aeromonas caviae have been proposed as potential bacterial markers for drowning [24], [26]. Aeromonads comprise facultatively aerobic gram-negative bacteria that are ubiquitous in freshwater and brackish water environments, and although to a lesser extent, they are also found in marine ecosystems [27]. Their widespread occurrence in virtual any type of natural aquatic environment and the fact that they are not considered natural inhabitants of the human body makes these bacteria highly suitable as potential diagnostic markers in the medico-legal investigations of drowning cases. On the other hand, however, Aeromonas species are well recognized as opportunistic human pathogens associated with gastroenteritis and a range of extraintestinal infections including pulmonary infections. In fact, Aeromonas hydrophila is the most frequently reported Aeromonas species causing near-drowning-associated pneumonia [28], [29], [30], [31], [32], [33], [34]. Furthermore, the presence of Aeromonas in lungs from drowned victims that are considered for transplantation may hold a potential risk for subsequent development of Aeromonas infection in the implanted organ [28].
The detection of Aeromonas in organs other than the lung is crucial to prove that the victim was still alive when the drowning medium was aspirated and bacteria were transported to the organs via the systematic circulation.
So far, detection of aeromonads in drowning cases has been based on culture-independent detection of DNA fragments specific for one or more Aeromonas spp. from blood samples [24], [26]. In the present study, we explored an alternative approach that combines culture-based selective enrichment of aeromonads with a genus-specific PCR. In addition, the practical benefits of bone marrow collection by aseptic sternal puncture as an alternative post-mortem sample technique in drowning cases were evaluated.
Section snippets
Bacterial strains and culture conditions
The following Aeromonas reference strains were obtained from the BCCM/LMG Bacteria Collection, Belgium (http://bccm.belspo.be/about/lmg.php): Aeromonas veronii biovar veronii LMG 16332, Aeromonas bestiarum LMG 13446, Aeromonas media LMG 14688 and Aeromonas hydrophila subsp. hydrophila LMG 13439. As negative controls for the PCR assay, reference strains of Acinetobacter baumannii, Bacillus cereus, Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus
Sampling strategy
Due to the likely presence of various microorganisms on outer skin surfaces originating from the autochthonous skin microbiota or resulting from contact with the drowning medium, aseptic sampling remains the most critical step in any procedure that is based on detection of bacterial infiltration in blood and tissue samples of drowning victims. In the current study, the possible presence of skin-associated aeromonads was investigated by including swab samples of the skin of the disinfected
Conclusions
The study confirms previous suggestions that the presence of aeromonads in tissue samples of water bodies can be used as a marker to aid the diagnosis of death by drowning. The use of ADA medium formed the basis of a sensitive, inexpensive and quick screening procedure for the detection of aeromonads in human tissue samples. To our knowledge, this study is the first to report the use of bone marrow of the sternum as an alternative matrix and aseptic sternal puncture as an alternative sampling
Acknowledgement
The authors wish to thank Margo Cnockaert for excellent technical assistance in the PCR work.
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2022, Forensic Science InternationalCitation Excerpt :Moreover, if the number of diatoms detected in the lungs is low, it is difficult to assume inhalation of water because the water may have invaded the lungs after death, contaminated the surface of the lungs during sampling (via gloves touching the surface of the body), or contaminated flasks or conical tubes during diatom testing. In such cases, however, additional testing that targets other aquatic microorganisms, such as aquatic bacteria (bacterioplankton) [38–46], could be effective when used in conjunction with conventional diatom testing. Indeed, the cause of death of cadaver No. 67 was non-drowning, but 4 diatoms were found in the right lung, 0 in the left lung, 5 in the kidney (17 in re-testing), and 2 in the liver (0 in re-testing), and the cadaver was immersed in an irrigation ditch for 20 days.
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2022, Forensic Science InternationalCitation Excerpt :The presence of these bacteria in the blood, closed organs and bone marrow has been linked to ante-mortem water aspiration. Microbiomes, such as bacteria and cyanobacteria [66], as well as phytoplankton including microalgae [67], have been studied to find out the cause of death using a variety of techniques, including traditional culture-dependent methods [68], PCR-based techniques [69], or light electron microscopy [70]. These microbiomes are substantially more abundant and have smaller sizes (ranging from 0.2 to 2 µm) than diatoms and can be of substantive use if sequenced and compared.
Conventional diatom testing using strong acid: Notable false-positive results caused by an underestimated contamination source (blind spot)
2022, Forensic Science InternationalDevelopment of 18S rRNA gene arrays for forensic detection of diatoms
2020, Forensic Science InternationalCitation Excerpt :In some cases, molecular techniques appeared to be more sensitive and specific than diatom test [52–57]. Molecular technique has also been attempted to tackle the problem of drowning site [56–63], a challenging but important aspect of diagnosis of drowning. Other recently developed high-throughput detection techniques such as next generation sequencing [64] and gene microarrays [65–68] have the potential to make an inroad in diatom detection.