Unbalanced predatory communities and a lack of microbial degraders characterize the microbiota of a highly sewage-polluted Eastern-Mediterranean stream.

Wastewater pollution of water resources takes a heavy toll on humans and on the environment. In highly polluted water bodies, self-purification is impaired, as the capacity of the riverine microbes to regenerate the ecosystem is overwhelmed. To date, information on the composition, dynamics and functions of the microbial communities in highly sewage-impacted rivers is limited, in particular in arid and semi-arid environments. In this year-long study of the highly sewage-impacted Al-Nar/Kidron stream in the Barr al-Khalil/Judean Desert east of Jerusalem, we show, using 16S and 18S rRNA gene-based community analysis and targeted qPCR, that both the bacterial and micro-eukaryotic communities, while abundant, exhibited low stability and diversity. Hydrolyzers of organics compounds, as well as nitrogen and phosphorus recyclers were lacking, pointing at reduced potential for regeneration. Furthermore, facultative bacterial predators were almost absent, and the obligate predators Bdellovibrio and like organisms were found at very low abundance. Finally, the micro-eukaryotic predatory community differed from those of other freshwater environments. The lack of essential biochemical functions may explain the stream's inability to self-purify, while the very low levels of bacterial predators and the disturbed assemblages of micro-eukaryote predators present in Al-Nar/Kidron may contribute to community instability and disfunction.

Form follows function: Applying photographic content analysis to forensic firearm identification.

Drawing forensic conclusions from an image or a video is known as "photographic content analysis." It involves the analysis of an image, as well as objects, actions, and events depicted in images or video. In recent years, photographic depictions of objects suspected as illegal firearms have substantially increased, appearing on CCTV surveillance footage, captured by mobile phones and shared on social media. However, the law in Israel states that a person can be charged with illegally possessing a firearm only if it can be proven that the object is capable of shooting with lethal bullet energy. This becomes more challenging in cases where the firearm was not physically seized, and the evidence exclusively consists of images and video. In this study, photographic content analysis was applied to images and video where objects suspected as commercial or improvised firearms had been depicted. An image and event sequence reconstruction video databases of both firearms and replicas were created in order to better define firearm-specific functional morphological features. We demonstrate that it is possible to classify an object as a firearm by analyzing the functional, and not only the esthetic, morphology in images and video. It is also shown that event sequence reconstruction in video may be used to infer that an object suspected as a firearm has the capacity to shoot by confirming the occurrence of a shooting act or shooting process. Thus, photographic content analysis may be used to forensically establish that an object depicted in an image or a video is a firearm by ruling out other known scenarios, and without physically seizing it.

Preliminary investigation of the ability of fingerprint examiners in detection of sib-sib relationships based upon finger and palm prints similarities.

Variations in biometric parameters such as fingerprints between populations, genders and even twins are wide-ranging issues routinely examined by the forensic community. In this study we tested whether fingerprint examiners can detect if finger and palm prints originate from siblings. In total, 410 finger records and 300 palm records of siblings and non-siblings were examined by seven certified forensic fingerprint examiners with different levels of qualifications and experience. The examiners were asked to determine, based on friction ridge characteristics, if they could detect similarities to such a degree that it was possible to declare a familial relationship (FR) between two prints. The results indicated that among all seven fingerprint examiners, 'true negative' values were very high (95-98%), meaning that in cases where FRs were absent, the examiners' decision was usually correct. In cases where FR was declared by the expert, the probability of a 'true positive' was 25 and 10 times higher than the probability of a 'false positive' for finger prints and palm prints, respectively. We attempt to elucidate the specific fingerprint parameters which facilitate better sibling detection, and conclude that sibling detection by fingerprint similarity may have potential as a novel forensic tool that can be used for intelligence operations.

Technical note: The Next Step - a semi-automatic coding and comparison system for forensic footwear impressions.

Footwear impression evidence is a key tool in criminal investigations, connecting suspects to the crime scene; in addition, it may provide valuable forensic intelligence linking different crime scenes in the absence of a suspect. This paper presents the development of a computer database and semi-automatic system for shoeprint comparison. The database is based on structured manual coding of elements by CSI (Crime Scene Investigators). The computer algorithm then compares the pattern information of the query footwear impression to the entire database, returning a list of possible matches ranked by pattern similarity and crime scene geographic proximity. Initial results using a database of 284 footwear impressions from real crime scenes revealed that a "match" (i.e., the same shoe model) was found for 30% of the impressions; in most cases, the "match" ranked within the top five places of the "hit" list generated by the algorithm. Our results confirm that this semi-automatic footwear comparison system is simple, cost-effective and efficient, providing great potential for linking crime scenes.

Identifying protein function and functional links based on large-scale co-occurrence patterns.

OBJECTIVE: The vast majority of known proteins have not been experimentally tested even at the level of measuring their expression, and the function of many proteins remains unknown. In order to decipher protein function and examine functional associations, we developed "Cliquely", a software tool based on the exploration of co-occurrence patterns. COMPUTATIONAL MODEL: Using a set of more than 23 million proteins divided into 404,947 orthologous clusters, we explored the co-occurrence graph of 4,742 fully sequenced genomes from the three domains of life. Edge weights in this graph represent co-occurrence probabilities. We use the Bron-Kerbosch algorithm to detect maximal cliques in this graph, fully-connected subgraphs that represent meaningful biological networks from different functional categories. MAIN RESULTS: We demonstrate that Cliquely can successfully identify known networks from various pathways, including nitrogen fixation, glycolysis, methanogenesis, mevalonate and ribosome proteins. Identifying the virulence-associated type III secretion system (T3SS) network, Cliquely also added 13 previously uncharacterized novel proteins to the T3SS network, demonstrating the strength of this approach. Cliquely is freely available and open source. Users can employ the tool to explore co-occurrence networks using a protein of interest and a customizable level of stringency, either for the entire dataset or for a one of the three domains-Archaea, Bacteria, or Eukarya.

Community and single cell analyses reveal complex predatory interactions between bacteria in high diversity systems.

A fundamental question in community ecology is the role of predator-prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.

The risk of inter-stub contamination during SEM/EDS analysis of gunshot residue particles.

Detecting gunshot residue (GSR) particles on samples collected from individuals or their belongings can connect them to a shooting event. Scanning electron microscopy combined with energy-dispersive X-ray spectrometry (SEM/EDX) is currently the most common forensic method for detecting and characterizing GSR. At the forensic laboratory of the Israel Police, one inch (25 mm) diameter sticky stubs are used to collect samples from suspects' hands, hair, clothes and vehicles. To maximize testing capacity, stubs of samples collected from several different cases and persons may be analyzed side by side in a single run. This has raised concern in court that a clean sample taken from an innocent person may be contaminated during the analysis by GSR particles from an adjacent sample transferred inside the SEM chamber. several experiments were conducted where stubs that were known to contain GSR particles were run adjacent to stubs that were known to be clean. Not a single event of GSR particle transfer was detected, even when a clean stub was surrounded on all sides by stubs containing a total of over 100,000 particles. Thus, the probability of transfer of a single particle is at most 1:100,000. Since the total number of GSR particles found per run is usually three orders of magnitude lower than 100,000, we conclude that the risk of inter-stub contamination is highly negligible.

A walk on the dirt: soil microbial forensics from ecological theory to the crime lab.

Forensics aims at using physical evidence to solve investigations with science-based principles, thus operating within a theoretical framework. This however is often rather weak, the exception being DNA-based human forensics that is well anchored in theory. Soil is a most commonly encountered, easily and unknowingly transferred evidence but it is seldom employed as soil analyses require extensive expertise. In contrast, comparative analyses of soil bacterial communities using nucleic acid technologies can efficiently and precisely locate the origin of forensic soil traces. However, this application is still in its infancy, and is very rarely used. We posit that understanding the theoretical bases and limitations of their uses is essential for soil microbial forensics to be judiciously implemented. Accordingly, we review the ecological theory and experimental evidence explaining differences between soil microbial communities, i.e. the generation of beta diversity, and propose to integrate a bottom-up approach of interactions at the microscale, reflecting historical contingencies with top-down mechanisms driven by the geographic template, providing a potential explanation as to why bacterial communities map according to soil types. Finally, we delimit the use of soil microbial forensics based on the present technologies and ecological knowledge, and propose possible venues to remove existing bottlenecks.

Population-dependent migration shift caused by sequence variation at the alpha fibrinogen (FGA) short tandem repeat (STR) locus.

The alpha fibrinogen (FGA) is a core short tandem repeat (STR) locus commonly used in forensic laboratories and is part of most of the commercial multiplex forensic STR kits. There are two distinct groups of FGA alleles based on their size: alleles 16-34.2 and 42.2-51.2. A thorough survey revealed that the long (>33) FGA alleles appear exclusively in populations of sub-Saharan Africans, Caribbean of African descent, non-African Arabs and peoples of non-African Arab descent. Our survey revealed that the long FGA alleles are rare and appear in only 0.01-1% of these populations, with the rarest allele being 47.2. The Israel Police DNA database includes about 470,000 DNA profiles, of which 193 bear an FGA allele longer than 33.2 and only 64 bearing the 47.2 allele. 30 samples with DNA profiles known to contain long FGA alleles were re-analyzed using three different STR multiplex kits. The regions of each long allele were sequenced in addition to STR analysis. The allele sequences revealed a striking difference in the pattern of repeats between the population groups of African and Arab descent. Eight of our samples contained the 47.2 allele, with a clear distinction between 47.2 sequences derived from African vs. Arab populations. In STR analysis, all 47.2 alleles displayed a shift from the allelic ladder bin center in all three kits. In all kits, the shift was significantly larger in the Arab population than in the African population. Hence, there is a population-dependent migration shift which may help differentiate profiles derived from different populations.

Weight-of-evidence for DNA identification of missing persons and human remains using CODIS.

DNA Identification of unidentified human remains (UHR) is performed in Israel by comparing the UHR's short tandem repeat (STR) profiles to a national database of STR profiles taken from relatives of missing persons. Kinship analysis is performed using the CODIS 7.0 software and results are stated as a Joint Pedigree Likelihood Ratio (JPLR). The weight-of-evidence for JPLR has never been studied, making it difficult to interpret the meaning of specific values in terms of whether UHR are related to specific pedigrees. Therefore, the aim of this study was to statistically determine the practical meaning and context of the JPLR. We used 440 million pairs of simulated DNA profiles and 294 pairs of real ones from known siblings, parent/offspring and unrelated persons. A Score-Based Likelihood Ratio (SBLR) was empirically constructed, validated and compared to both JPLR and the LR produced by CODIS. Our results show that CODIS's JPLR and LR values for single-person pedigrees overestimate the level of support for both "parent/child" and "siblings" propositions relative to the "unrelated" proposition, by up to two orders of magnitude. A practical table is given for correcting this phenomenon, with statistical interpretation (i.e. SBLR) for each JPLR score, including verbal levels of propositional support ranging from "no support" (SBLR<2) to "extremely strong" (SBLR>1 Million).

Forensic Applications of Microbiomics: A Review.

The rise of microbiomics and metagenomics has been driven by advances in genomic sequencing technology, improved microbial sampling methods, and fast-evolving approaches in bioinformatics. Humans are a host to diverse microbial communities in and on their bodies, which continuously interact with and alter the surrounding environments. Since information relating to these interactions can be extracted by analyzing human and environmental microbial profiles, they have the potential to be relevant to forensics. In this review, we analyzed over 100 papers describing forensic microbiome applications with emphasis on geolocation, personal identification, trace evidence, manner and cause of death, and inference of the postmortem interval (PMI). We found that although the field is in its infancy, utilizing microbiome and metagenome signatures has the potential to enhance the forensic toolkit. However, many of the studies suffer from limited sample sizes and model accuracies, and unrealistic environmental settings, leaving the full potential of microbiomics to forensics unexplored. It is unlikely that the information that can currently be elucidated from microbiomics can be used by law enforcement. Nonetheless, the research to overcome these challenges is ongoing, and it is foreseeable that microbiome-based evidence could contribute to forensic investigations in the future.

Spatial heterogeneity stabilizes predator-prey interactions at the microscale while patch connectivity controls their outcome.

Natural landscapes are both fragmented and heterogeneous, affecting the distribution of organisms, and their interactions. While predation in homogeneous environments increases the probability of population extinction, fragmentation/heterogeneity promotes coexistence and enhances community stability as shown by experimentation with animals and microorganisms, and supported by theory. Patch connectivity can modulate such effects but how microbial predatory interactions are affected by water-driven connectivity is unknown. In soil, patch habitability by microorganisms, and their connectivity depend upon the water saturation degree (SD). Here, using the obligate bacterial predator Bdellovibrio bacteriovorus, and a Burkholderia prey, we show that soil spatial heterogeneity profoundly affects predatory dynamics, enhancing long-term co-existence of predator and prey in a SD-threshold dependent-manner. However, as patches and connectors cannot be distinguished in these soil matrices, metapopulations cannot be invoked to explain the dynamics of increased persistence. Using a set of experiments combined with statistical and physical models we demonstrate and quantify how under full connectivity, predation is independent of water content but depends on soil microstructure characteristics. In contrast, the SD below which predation is largely impaired corresponds to a threshold below which the water network collapses and water connectivity breaks down, preventing the bacteria to move within the soil matrix.

Bacterial predation under changing viscosities.

Bdellovibrio and like organisms (BALOs) are largely distributed in soils and in water bodies obligate predators of gram-negative bacteria that can affect bacterial communities. Potential applications of BALOs include biomass reduction, their use against pathogenic bacteria in agriculture, and in medicine as an alternative against antibiotic-resistant pathogens. Such different environments and uses mean that BALOs should be active under a range of viscosities. In this study, the predatory behaviour of two strains of the periplasmic predator B. bacteriovorus and of the epibiotic predator Micavibrio aeruginosavorus was examined in viscous polyvinylpyrrolidone (PVP) solutions at 28 and at 37°C, using fluorescent markers and plate counts to track predator growth and prey decay. We found that at high viscosities, although swimming speed was largely decreased, the three predators reduced prey to levels similar to those of non-viscous suspensions, albeit with short delays. Prey motility and clumping did not affect the outcome. Strikingly, under low initial predator concentrations, predation dynamics were faster with increasing viscosity, an effect that dissipated with increasing predator concentrations. Changes in swimming patterns and in futile predator-predator encounters with viscosity, as revealed by path analysis under changing viscosities, along with possible PVP-mediated crowding effects, may explain the observed phenomena.

Bacteria and microeukaryotes are differentially segregated in sympatric wastewater microhabitats.

Wastewater purification is mostly performed in activated sludge reactors by bacterial and microeukaryotic communities, populating organic flocs and a watery liquor. While there are numerous molecular community studies of the bacterial fraction, those on microeukaryotes are rare. We performed a year-long parallel 16S rRNA gene and 18S rRNA-gene based analysis of the bacterial and of the microeukaryote communities, respectively, of physically separated flocs and particle-free liquor samples from three WWTPs. This uncovered a hitherto unknown large diversity of microeukaryotes largely composed of potential phagotrophs preferentially feeding on either bacteria or other microeukaryotes. We further explored whether colonization of the microhabitats was selective, showing that for both microbial communities, different but often closely taxonomically and functionally related populations exhibiting different dynamic patterns populated the microhabitats. An analysis of their between plants-shared core populations showed the microeukaryotes to be dispersal limited in comparison to bacteria. Finally, a detailed analysis of a weather-caused operational disruption in one of the plants suggested that the absence of populations common to the floc and liquor habitat may negatively affect resilience and stability.

Applying microbial biogeography in soil forensics.

The ubiquity, heterogeneity and transferability of soil makes it useful as evidence in criminal investigations, especially using new methods that survey the microbial DNA it contains. However, to be used effectively and reliably, more needs to be learned about the natural distribution patterns of microbial communities in soil. In this study we examine these patterns in detail, at local to regional scales (2 m-260 km), across an environmental gradient in three different soil types. Geographic location was found to be more important than soil type in determining the microbial community composition: communities from the same site but different soil types, although significantly different from each other, were still much more similar to each other than were communities from the same soil type but from different sites. At a local scale (25-1000 m), distance-decay relationships were observed in all soil types: the farther apart two soil communities were located, even in the same soil type, the more they differed. At regional-scale distances (1-260 km), differences between communities did not increase with increased geographic distance between them, and the dominant factor determining the community profile was the physico-chemical environment, most notably annual precipitation (R2 = 0.69), soil sodium (R2 = 0.49) and soil ammonium (R2 = 0.47) levels. We introduce a likelihood-ratio framework for quantitative evaluation of soil microbial DNA profile evidence in casework. In conclusion, these profiles, along with detailed knowledge of natural soil microbial biogeography, provide valuable forensic information on soil sample comparison and allow the determination of approximate source location on large (hundreds of km) spatial scales. Moreover, at small spatial scales it may enable pinpointing the source location of a sample to within at least 25 m, regardless of soil type and environmental conditions.

Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5-10 days post-partum.

Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n = 35) were sampled immediately following clinical classification as healthy (n = 21), suffering from metritis (n = 13) or septic metritis (n = 1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8 ± 9.3%), Firmicutes (27.9 ± 8.4%) and Bacteroidetes (19.7 ± 7.2%), while Bacteroidetes (60.3 ± 10.3%), Fusobacteria (13.4 ± 5.9%) and Firmicutes (10.5 ± 3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7 ± 7.2% vs. 60.3 ± 10.3%), Fusobacteria (7.5 ± 5.2% vs. 13.4 ± 5.9%) and Proteobacteria (31.8 ± 9.3% vs. 7.3 ± 5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.

Automatic identification of optimal marker genes for phenotypic and taxonomic groups of microorganisms.

Finding optimal markers for microorganisms important in the medical, agricultural, environmental or ecological fields is of great importance. Thousands of complete microbial genomes now available allow us, for the first time, to exhaustively identify marker proteins for groups of microbial organisms. In this work, we model the biological task as the well-known mathematical "hitting set" problem, solving it based on both greedy and randomized approximation algorithms. We identify unique markers for 17 phenotypic and taxonomic microbial groups, including proteins related to the nitrite reductase enzyme as markers for the non-anammox nitrifying bacteria group, and two transcription regulation proteins, nusG and yhiF, as markers for the Archaea and Escherichia/Shigella taxonomic groups, respectively. Additionally, we identify marker proteins for three subtypes of pathogenic E. coli, which previously had no known optimal markers. Practically, depending on the completeness of the database this algorithm can be used for identification of marker genes for any microbial group, these marker genes may be prime candidates for the understanding of the genetic basis of the group's phenotype or to help discover novel functions which are uniquely shared among a group of microbes. We show that our method is both theoretically and practically efficient, while establishing an upper bound on its time complexity and approximation ratio; thus, it promises to remain efficient and permit the identification of marker proteins that are specific to phenotypic or taxonomic groups, even as more and more bacterial genomes are being sequenced.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Soil characterisation by bacterial community analysis for forensic applications: A quantitative comparison of environmental technologies.

The ubiquity and transferability of soil makes it a resource for the forensic investigator, as it can provide a link between agents and scenes. However, the information contained in soils, such as chemical compounds, physical particles or biological entities, is seldom used in forensic investigations; due mainly to the associated costs, lack of available expertise, and the lack of soil databases. The microbial DNA in soil is relatively easy to access and analyse, having thus the potential to provide a powerful means for discriminating soil samples or linking them to a common origin. We compared the effectiveness and reliability of multiple methods and genes for bacterial characterisation in the differentiation of soil samples: ribosomal intergenic spacer analysis (RISA), terminal restriction fragment length polymorphism (TRFLP) of the rpoB gene, and five methods using the 16S rRNA gene: phylogenetic microarrays, TRFLP, and high throughput sequencing with Roche 454, Illumina MiSeq and IonTorrent PGM platforms. All these methods were also compared to long-chain hydrocarbons (n-alkanes) and fatty alcohol profiling of the same soil samples. RISA, 16S TRFLP and MiSeq performed best, reliably and significantly discriminating between adjacent, similar soil types. As TRFLP employs the same capillary electrophoresis equipment and procedures used to analyse human DNA, it is readily available for use in most forensic laboratories. TRFLP was optimized for forensic usage in five parameters: choice of primer pair, fluorescent tagging, concentrating DNA after digestion, number of PCR amplifications per sample and number of capillary electrophoresis runs per PCR amplification. This study shows that molecular microbial ecology methodologies are robust in discriminating between soil samples, illustrating their potential usage as an evaluative forensic tool.

Draft Genome Sequence of the Bactrocera oleae Symbiont "Candidatus Erwinia dacicola".

"Candidatus Erwinia dacicola" is a Gammaproteobacterium that forms a symbiotic association with the agricultural pest Bactrocera oleae Here, we present a 2.1-Mb draft hybrid genome assembly for "Ca. Erwinia dacicola" generated from single-cell and metagenomic data.