Necrophagous insect species succession on decomposed pig carcasses in North Dakota, USA.

Necrophagous insect species are widely used during death investigations primarily for the estimation of the minimum postmortem interval, since these species use decomposing organic matter for feeding, oviposition, and larval development. The development stages and successional colonization patterns provide important information for shorter or longer postmortem time intervals. Diptera species are the predominant taxa recovered from decomposed bodies. The temperature variance/seasonality is the main factor affecting the time presence and activity of these species. Other factors, such as geographical location, antemortem conditions, and cause of death, can influence the presence and succession of necrophagous entomofauna. Consequently, successional studies and information regarding species colonization patterns are needed for each geographical region to be used as reference data during death investigations. This study addressed the need to collect forensic entomological data for the State of North Dakota, within the first necrophagous entomofauna diversity report for the month of July 2022, using pig carcasses as human analogs. During the experimental period, 18 species of Diptera and Coleoptera were identified, with 10 new state records, while Calliphoridae was found to be the predominant family. The resulted data on necrophagous insect species diversity and dynamics from exposed pig carcasses will strengthen the current knowledge on forensic entomology in North Dakota and will serve as reference data to be used during death investigations in the Great Plains region.

Postmortem skin microbiome signatures associated with human cadavers within the first 12 h at the morgue.

Introduction: Forensic microbiome studies expanded during the last decade, aiming to identify putative bacterial biomarkers to be used for the postmortem interval (PMI) estimation. Bacterial diversity and dynamics during decomposition are influenced by each individual's micro and macroenvironment, ante and postmortem conditions, varying across body sites and time. The skin, the largest organ of the human body, hosts a diverse microbial diversity, representing the first line of defense of a living individual. Targeting the investigation of the postmortem skin microbiome could help understanding the role of microbes during decomposition, and association with the ante and postmortem conditions. Methods: The current study aimed to identify the postmortem skin microbiome signatures associated with eight human bodies, received at the Institute of Legal Medicine Iasi, Romania, during April and May 2021. A total of 162 samples (including triplicate) representing face and hands skin microbiome were investigated via Illumina MiSeq, upon arrival at the morgue (T0) and after 12 hours (T1). Results: The taxonomic characteristics of the skin microbiota varied across different body sites. However, there were no significant differences in taxonomic profiles between collection time, T0 and T1, except for some dynamic changes in the abundance of dominant bacteria. Moreover, different microbial signatures have been associated with a specific cause of death, such as cardiovascular disease, while an elevated blood alcohol level could be associated with a decrease in bacterial richness and diversity. Discussion: The places where the bodies were discovered seemed to play an important role in explaining the bacterial diversity composition. This study shows promising results towards finding common postmortem bacterial signatures associated with human cadavers within the first 12h at the morgue.

Bioprospecting for Novel Bacterial Sources of Hydrolytic Enzymes and Antimicrobials in the Romanian Littoral Zone of the Black Sea.

Marine microorganisms have evolved a large variety of metabolites and biochemical processes, providing great opportunities for biotechnologies. In the search for new hydrolytic enzymes and antimicrobial compounds with enhanced characteristics, the current study explored the diversity of cultured and uncultured marine bacteria in Black Sea water from two locations along the Romanian coastline. Microbial cell density in the investigated samples varied between 65 and 12.7 × 103 CFU·mL-1. The total bacterial community identified by Illumina sequencing of 16S rRNA gene comprised 185 genera belonging to 46 classes, mainly Gammaproteobacteria, Alphaproteobacteria, Flavobacteriia, and 24 phyla. The 66 bacterial strains isolated on seawater-based culture media belonged to 33 genera and showed variable growth temperatures, growth rates, and salt tolerance. A great fraction of these strains, including Pseudoalteromonas and Flavobacterium species, produced extracellular proteases, lipases, and carbohydrases, while two strains belonging to the genera Aquimarina and Streptomyces exhibited antimicrobial activity against human pathogenic bacteria. This study led to a broader view on the diversity of microbial communities in the Black Sea, and provided new marine strains with hydrolytic and antimicrobial capabilities that may be exploited in industrial and pharmaceutical applications.

Microbiome pattern of Lucilia sericata (Meigen) (Diptera: Calliphoridae) and feeding substrate in the presence of the foodborne pathogen Salmonella enterica.

The microbial diversity and quantitative dynamics during the insect's development stages constitute recently developed putative tools in forensic and medical studies. Meanwhile, little is known on the role of insects in spreading foodborne pathogenic bacteria and on the impact of these pathogens on the overall insects and feeding substrate microbiome composition. Here, we provide the first characterization of the bacterial communities harbored in adult and immature stages of Lucilia sericata, one of the first colonizers of decomposed human remains, in the presence of the foodborne pathogen Salmonella enterica using 16S rRNA Illumina sequencing and qPCR. The pathogen transmission from the wild adults to the second generation was observed, with a 101.25× quantitative increase. The microbial patterns from both insect and liver samples were not influenced by the artificial introduction of this pathogenic foodborne bacteria, being dominated by Firmicutes and Proteobacteria. Overall, our results provided a first detailed overview of the insect and decomposed substrate microbiome in the presence of a human pathogen, advancing the knowledge on the role of microbes as postmortem interval estimators and the transmission of pathogenic bacteria.

Draft Genome Sequence of Flavobacterium sp. Strain PL002, Isolated from Antarctic Porphyra Algae.

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.

Potential bacterial biomarkers for insect colonization in forensic cases: preliminary quantitative data on Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica dynamics.

For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 (Diptera: Calliphoridae), and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng-1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.

Characterizing forensically important insect and microbial community colonization patterns in buried remains.

During violent criminal actions in which the perpetrator disposes of the victim's remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters' influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Bacterial and archaeal community structures in perennial cave ice.

Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 104 up to 7.5 105 cells mL-1, with 59-78% viability. Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.

Temperature Influence on Prevailing Necrophagous Diptera and Bacterial Taxa With Forensic Implications for Postmortem Interval Estimation: A Review.

The decomposition process of human (and other mammalian) remains is influenced by numerous factors such as the environmental temperature, relative humidity, precipitation, geographical location of the remains, as well as the medical conditions of and any injuries sustained by the deceased person. The decomposition process generally follows a consistent pattern (fresh, bloat, active decomposition, advanced decomposition, and dry stage). Understanding the time-span of each stage of decomposition and the influence of the biotic and abiotic factors involved is imperative when trying to estimate the time elapsed since death (postmortem interval [PMI]). Over the course of decomposition, the tissues are gradually consumed by necrophagous insects and bacteria. The environmental temperature and its variations influence how insects colonize the remains, having a significant impact on their presence and developmental cycle. Additionally, the bacterial community colonizing decomposing tissues is also greatly affected by variations in environmental temperature. Because both the rate of decomposition of human remains and the relative abundance of certain insects and bacterial species are all temperature-dependent, insect and bacterial colonization data are useful as key points for the PMI estimation. This article reviews the current literature documenting the data collected on the occurrence and development cycles of predominant necrophagous Diptera (Calliphoridae, Muscidae, Sarcophagidae) and bacterial taxa involved in the decomposition of various carcasses at different temperatures. This review will impact the forensic community by providing an overview on the temperature, insect and bacterial records for the PMI estimation, seeking to aid forensic entomologists, microbiologists, pathologists, and the legal community.

Fungi in perennial ice from Scarișoara Ice Cave (Romania).

Screening of 1,000-years old ice layers from the perennial ice block of Scarișoara Ice Cave (NW Romania) revealed the presence of fungal communities. Using culture-dependent methods and molecular techniques based on DGGE fingerprinting of 18S rRNA gene fragments and sequencing, we identified 50 cultured and 14 uncultured fungi in presently-forming, 400 and 900 years old ice layers, corresponding to 28 distinct operational taxonomic units (OTUs). The dominant ice-contained fungal OTUs were related to Ascomycota, Basidiomycota and Cryptomycota phyla. Representatives of Mucoromycota and Chytridiomycota were also isolated from recent and 400 years old ice samples. The cryophilic Mrakia stokesii was the most abundant fungal species found in the cave ice samples of all prospected ages, alongside other cryophilic fungi also identified in various glacial environments. Ice deposits formed during the Little Ice Age (dated between AD 1,250 and 1,850) appeared to have a higher fungal diversity than the ice layer formed during the Medieval Warm Period (prior to AD 1,250). A more complex fungal community adapted to low temperatures was obtained from all analyzed ice layers when cultivated at 4 °C as compared to 15 °C, suggesting the dominance of cold-adapted fungi in this glacial habitat. The fungal distribution in the analyzed cave ice layers revealed the presence of unique OTUs in different aged-formed ice deposits, as a first hint for putative further identification of fungal biomarkers for climate variations in this icy habitat. This is the first report on fungi from a rock-hosted cave ice block.

Characterization and microbial analysis of first recorded observation of Conicera similis Haliday (Diptera: Phoridae) in forensic decomposition study in Romania.

The identification of necrophagous insect diversity and dynamics has forensic significance for postmortem interval estimation specific to burial. Few studies regarding the necrophagous entomofauna from buried remains have been performed to date. In contrast to the exposed carcasses, the accessibility of soil to insects is limited due to burial depth and is dependent on soil type. This study highlights the colonization behavior of Conicera similis (Haliday 1833) (Diptera: Phoridae) during carcass decomposition, a previously unobserved taxon in Romania. Adult and larvae specimens were collected from rat (Rattus norvegicus) carcasses buried at 40 cm depth in an urban park environment in June 2016 during active decomposition and their presence and activity period was correlated with the environmental parameters variation. Bacterial diversity from C. similis female adult and larvae specimens was determined via 16S rRNA gene Illumina sequencing to further characterize these commonly encountered and forensically important necrophagous insects. This report signals the easternmost geographical location in Europe (Bucharest, Romania) of C. similis to date.

Dynamics of Necrophagous Insect and Tissue Bacteria for Postmortem Interval Estimation During the Warm Season in Romania.

The estimation of postmortem interval (PMI) is affected by several factors including the cause of death, the place where the body lay after death, and the weather conditions during decomposition. Given the climatic differences among biogeographic locations, the understanding of necrophagous insect species biology and ecology is required when estimating PMI. The current experimental model was developed in Romania during the warm season in an outdoor location. The aim of the study was to identify the necrophagous insect species diversity and dynamics, and to detect the bacterial species present during decomposition in order to determine if their presence or incidence timing could be useful to estimate PMI. The decomposition process of domestic swine carcasses was monitored throughout a 14-wk period (10 July-10 October 2013), along with a daily record of meteorological parameters. The chronological succession of necrophagous entomofauna comprised nine Diptera species, with the dominant presence of Chrysomya albiceps (Wiedemann 1819) (Calliphoridae), while only two Coleoptera species were identified, Dermestes undulatus (L. 1758) and Creophilus maxillosus Brahm 1970. The bacterial diversity and dynamics from the mouth and rectum tissues, and third-instar dipteran larvae were identified using denaturing gradient gel electrophoresis analysis and sequencing of bacterial 16S rRNA gene fragments. Throughout the decomposition process, two main bacterial chronological groups were differentiated, represented by Firmicutes and Gammaproteobacteria. Twenty-six taxa from the rectal cavity and 22 from the mouth cavity were identified, with the dominant phylum in both these cavities corresponding to Firmicutes. The present data strengthen the postmortem entomological and microbial information for the warm season in this temperate-continental area, as well as the role of microbes in carcass decomposition.

Using bacterial and necrophagous insect dynamics for post-mortem interval estimation during cold season: Novel case study in Romania.

Considering the biogeographical characteristics of forensic entomology, and the recent development of forensic microbiology as a complementary approach for post-mortem interval estimation, the current study focused on characterizing the succession of necrophagous insect species and bacterial communities inhabiting the rectum and mouth cavities of swine (Sus scrofa domesticus) carcasses during a cold season outdoor experiment in an urban natural environment of Bucharest, Romania. We monitored the decomposition process of three swine carcasses during a 7 month period (November 2012-May 2013) corresponding to winter and spring periods of a temperate climate region. The carcasses, protected by wire cages, were placed on the ground in a park type environment, while the meteorological parameters were constantly recorded. The succession of necrophagous Diptera and Coleoptera taxa was monitored weekly, both the adult and larval stages, and the species were identified both by morphological and genetic characterization. The structure of bacterial communities from swine rectum and mouth tissues was characterized during the same time intervals by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. We observed a shift in the structure of both insect and bacterial communities, primarily due to seasonal effects and the depletion of the carcass. A total of 14 Diptera and 6 Coleoptera species were recorded on the swine carcasses, from which Calliphora vomitoria and C. vicina (Diptera: Calliphoridae), Necrobia violacea (Coleoptera: Cleridae) and Thanatophilus rugosus (Coleoptera: Silphidae) were observed as predominant species. The first colonizing wave, primarily Calliphoridae, was observed after 15 weeks when the temperature increased to 13°C. This was followed by Muscidae, Fanniidae, Anthomyiidae, Sepsidae and Piophilidae. Families belonging to Coleoptera Order were observed at week 18 when temperatures raised above 18°C, starting with Cleridae, Silphidae, and followed by Histeridae, Staphylinidae and Dermestidae. For bacteria, 53 taxa belonging to phyla Proteobacteria (Gammaproteobacteria, Betaproteobacteria), Firmicutes and Bacteroidetes were identified from the mouth cavity (36 OTUs) and rectum cavity (17 OTUs). These shifts in insect and bacterial communities indicated their complementary role in the carcass decomposition process. These results represent the first forensic entomological and microbiological survey in Romania. This study shows the value of forensic entomology as a tool for forensic investigations in Romania and neighboring areas with similar biogeographical characteristics. Moreover, this study represents a novel approach for understanding taphonomy and estimating post-mortem interval during cold season by combining entomological and microbiological methods.