RESUMEN

In forensic case investigations involving human traces, cell type identification has become increasingly important. No longer only the donor of a trace (sub-source level), but also the actions which could have led to the deposition of the trace ('beyond-the-source'/activity level) need to be evaluated by forensic experts. For this evaluation determining the cellular source of a DNA profile can be beneficial. In this report two criminal cases are described where both human STR profiling and microbial population profiling were applied to the same trace sample. Human STR profiling was used to evaluate the sub-source question and microbial population profiling was used to evaluate the source question. The Bayesian framework was used to evaluate the evidence.

Asunto(s)

Bacterias/genética , Dermatoglifia del ADN , Heces/microbiología , Repeticiones de Microsatélite , Crimen , Sondas de ADN , Bases de Datos de Ácidos Nucleicos , Femenino , Humanos , Funciones de Verosimilitud , Masculino , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa

RESUMEN

In forensic investigations involving human biological traces, cell type identification is often required. Identifying the cell type from which a human STR profile has originated can assist in verifying scenarios. Several techniques have been developed for this purpose, most of which focus on molecular characteristics of human cells. Here we present a microarray method focusing on the microbial populations that are associated with human cell material. A microarray with 863 probes targeting (sets of) species, specific genera, groups of genera or families was designed for this study and evaluated with samples from different body sites: hand, foot, groin, penis, vagina, mouth and faeces. In total 175 samples from healthy individuals were analysed. Next to human faeces, 15 feline and 15 canine faeces samples were also included. Both clustering and classification analysis were used for data analysis. Faecal and oral samples could clearly be distinguished from vaginal and skin samples, and also canine and feline faeces could be differentiated from human faeces. Some penis samples showed high similarity to vaginal samples, others to skin samples. Discriminating between skin samples from different skin sites proved to be challenging. As a proof of principle, twenty-one mock case samples were analysed with the microarray method. All mock case samples were clustered or classified within the correct main cluster/group. Only two of the mock case samples were assigned to the wrong sub-cluster/class; with classification one additional sample was classified within the wrong sub-class. Overall, the microarray method is a valuable addition to already existing cell typing techniques. Combining the results of microbial population analysis with for instance mRNA typing can increase the evidential value of a trace, since both techniques focus on independent targets within a sample.

Asunto(s)

Bacterias/aislamiento & purificación , Análisis por Micromatrices , Adolescente , Adulto , Anciano , Animales , Bacterias/genética , Biodiversidad , Gatos , Sondas de ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Perros , Heces/microbiología , Femenino , Pie/microbiología , Ingle/microbiología , Mano/microbiología , Humanos , Masculino , Persona de Mediana Edad , Boca/microbiología , Pene/microbiología , Reacción en Cadena de la Polimerasa , Análisis de Componente Principal , Piel/microbiología , Vagina/microbiología , Adulto Joven

RESUMEN

Although botanical trace evidence is often encountered in case investigations, the utilization of such traces in forensic investigations is still limited. Development of a forensic STR system for the two species of Betula (birch) indigenous to and abundant in North West Europe is a step in enhancing the applicability of traces from these species. We describe six microsatellite markers developed for birch species in detail, including repeat structure, and we propose a nomenclature for the encountered alleles. To assess the population characteristics, the genetic composition of wild, planted and intermediate populations of Betula pendula (a diploid species) and Betula pubescens (a tetraploid species) were investigated. The genetic differences between these two species were larger than the differences between populations of one species, even when both species co-occurred at one location. Therefore allele frequencies were estimated for both species separately. General, conservative random match probabilities were estimated for wild trees based on these allele frequencies (5∙10-6 for the diploid B. pendula and 1∙10-13 for the tetraploid B. pubescens), illustrating the potential relevance if trace evidence secured from a suspect is found to match a birch tree growing on or near a crime scene. Apart from wild trees, planted Betula trees also occur that may not originate from seeds, but may have been propagated through cloning. Based on the studied Betula trees, the random match probability of a potentially planted profile might be as high as 1.4∙10-2.

Asunto(s)

Betula/genética , Dermatoglifia del ADN , Repeticiones de Microsatélite , Europa (Continente) , Genética Forense , Frecuencia de los Genes , Reacción en Cadena de la Polimerasa Multiplex

RESUMEN

Typing of different portions of the feline mitochondrial control region has illustrated pronounced differences in haplotype distributions between cats from the Netherlands and other parts of the world. To gain a better understanding of the haplotype distribution of North West Continental Europe, 605bp of mitochondrial DNA was typed from randomly selected cats from the Netherlands (N=146), Belgium (N=64) and South West Germany (N=128). The genetic differences between these randomly sampled European populations correlate to the geographical distances, with the Dutch and the South West German populations furthest apart and the Belgian population as an intermediate (Fst values 0.01-0.03). Comparison of North West European mainland distributions to published feline mitochondrial haplotype distributions illustrated moderate to large genetic differentiation (Fst values 0.01-0.32). In this comparison, the correlation between geographical and genetic distance was absent, leading to founder effects and human impact on cat population structure and dispersion being considered as important parameters. When an accurate estimation of feline haplotype distribution is required in forensics, care should be taken when deciding whether extrapolating the frequency data from a certain source to a larger area (country/continent) is justified or whether additional typing of local populations is necessary. This may differ from case to case as local frequencies can be relevant, but can also be deceitful. To improve the applicability of forensic feline mitochondrial DNA studies, documentation and publishing of sampling strategies is advised, as is the implementation of measures to help eliminate potentially erroneous haplotypes.

Asunto(s)

Gatos/genética , ADN Mitocondrial/genética , Haplotipos , Animales , Europa (Continente) , Efecto Fundador , Variación Genética

RESUMEN

The forensic science community has a growing interest in microbial population analysis, especially the microbial populations found inside and on the human body. Both their high abundance, microbes outnumber human cells by a factor 10, and their diversity, different sites of the human body harbour different microbial communities, make them an interesting tool for forensics. Faecal material is a type of trace evidence which can be found in a variety of criminal cases, but is often being ignored in forensic investigations. Deriving a human short tandem repeat (STR) profile from a faecal sample can be challenging. However, the microbial communities within faecal material can be of additional criminalistic value in linking a faecal trace to the possible donor. We present a microarray technique in which the faecal microbial community is used to differentiate between faecal samples and developed a decision model to predict the possible common origin of questioned samples. The results show that this technique may be a useful additional tool when no or only partial human STR profiles can be generated.

Asunto(s)

Bacterias/genética , Chaperonina 60/genética , Heces/microbiología , ARN Ribosómico 16S , ARN Ribosómico 18S , Técnicas de Apoyo para la Decisión , Genética Forense/métodos , Humanos , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Sondas ARN , Reproducibilidad de los Resultados

RESUMEN

Soil can play an important role in forensic cases in linking suspects or objects to a crime scene by comparing samples from the crime scene with samples derived from items. This study uses an adapted ED-XRF analysis (sieving instead of grinding to prevent destruction of microfossils) to produce elemental composition data of 20 elements. Different data processing techniques and statistical distances were evaluated using data from 50 samples and the log-LR cost (Cllr ). The best performing combination, Canberra distance, relative data, and square root values, is used to construct a discriminative model. Examples of the spatial resolution of the method in crime scenes are shown for three locations, and sampling strategy is discussed. Twelve test cases were analyzed, and results showed that the method is applicable. The study shows how the combination of an analysis technique, a database, and a discriminative model can be used to compare multiple soil samples quickly.

Asunto(s)

Bacterias/genética , Crimen , Genética Forense/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas Microbiológicas/métodos , Humanos , Microbiota/genética

RESUMEN

Different portions of the feline mitochondrial DNA control region (CR) were evaluated for their informative value in forensic investigations. The 402bp region located between RS2 and RS3 described most extensively in the past is not efficient for distinguishing between the majority of Dutch cats, illustrated by a random match probability (RMP) of 41%. Typing of the whole region between RS2 and RS3, and additional typing of the 5'portion of the feline CR decreases the RMP to 29%, increasing the applicability of such analyses for forensic investigations. The haplotype distribution in Dutch random bred cats (N=113) differs greatly from the distributions reported for other countries, with a single haplotype NL-A1 present in 54% of the population. The three investigated breeds showed haplotype distributions differing from each other and the random bred cats with haplotype NL-A1 accounting for 4%, 29% and 32% of Maine Coon, Norwegian forest cats and Siamese & Oriental cats. These results indicate the necessity of validating haplotype frequencies within continents and regions prior to reporting the value a mtDNA match. In cases where known purebred cats are involved, further investigation of the breed may be valuable.

Asunto(s)

Gatos/genética , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , Genética Forense/métodos , Análisis de Secuencia de ADN/veterinaria , Animales , Cabello/química , Haplotipos , Países Bajos , Análisis de Secuencia de ADN/métodos

Asunto(s)

Vestuario , Diatomeas , Manejo de Especímenes/métodos , Humanos

RESUMEN

Forensic analysis of biological traces generally encompasses the investigation of both the person who contributed to the trace and the body site(s) from which the trace originates. For instance, for sexual assault cases, it can be beneficial to distinguish vaginal samples from skin or saliva samples. In this study, we explored the use of microbial flora to indicate vaginal origin. First, we explored the vaginal microbiome for a large set of clinical vaginal samples (n = 240) by next generation sequencing (n = 338,184 sequence reads) and found 1,619 different sequences. Next, we selected 389 candidate probes targeting genera or species and designed a microarray, with which we analysed a diverse set of samples; 43 DNA extracts from vaginal samples and 25 DNA extracts from samples from other body sites, including sites in close proximity of or in contact with the vagina. Finally, we used the microarray results and next generation sequencing dataset to assess the potential for a future approach that uses microbial markers to indicate vaginal origin. Since no candidate genera/species were found to positively identify all vaginal DNA extracts on their own, while excluding all non-vaginal DNA extracts, we deduce that a reliable statement about the cellular origin of a biological trace should be based on the detection of multiple species within various genera. Microarray analysis of a sample will then render a microbial flora pattern that is probably best analysed in a probabilistic approach.

Asunto(s)

Bacterias/genética , Bacterias/aislamiento & purificación , Metagenoma/genética , Vagina/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sangre/microbiología , Dermatoglifia del ADN , Heces/microbiología , Femenino , Ciencias Forenses , Variación Genética/genética , Mano/microbiología , Humanos , Masculino , Persona de Mediana Edad , Análisis por Matrices de Proteínas , Saliva/microbiología , Semen/microbiología , Piel/microbiología , Orina/microbiología , Adulto Joven

RESUMEN

The possibilities and strategies for using DNA characteristics to link a botanical sample to a specific source plant or location vary with its breeding system. For inbreeding species, which often form small patches of identical genotypes, knotgrass (Polygonum aviculare L.) is a suitable model species because of its (1) occurrence in a wide range of natural environments, (2) abundant presence in pieces of evidence, and (3) ease in molecular processing. The value of knotgrass for forensic casework was demonstrated using data from a homicide case. Using the DNA fingerprinting technique AFLP(®) we were able to identify the knotgrass population at the crime site as the most likely origin of the botanical evidence. We expect that the development of tailored marker systems for knotgrass and other frequently occurring (model) species will considerably accelerate the use of botanical DNA evidence in criminal cases.

Asunto(s)

Dermatoglifia del ADN/métodos , ADN de Plantas/genética , Ciencias Forenses , Polygonum/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Análisis por Conglomerados , Código de Barras del ADN Taxonómico , Variación Genética , Homicidio , Humanos , Semillas/genética

RESUMEN

Soil can play an important role in forensic investigations in linking suspects or objects to a crime scene. Bacterial populations are one of the biotic parameters in soil which can be used for comparisons. Terminal restriction fragment length polymorphism (t-RFLP) is used to visualize these populations. Here we present a method to compare soil t-RFLP profiles based on Bray-Curtis distances. We developed a decision model to predict the possible common source of unknown samples. Test cases in cooperation with the Police Academy of the Netherlands were used to validate the decision model. The results of these test cases are very promising, indicating that bacterial profiling is a useful additional tool in forensic soil comparisons.

Asunto(s)

Bacterias/genética , ADN Bacteriano/genética , Polimorfismo de Longitud del Fragmento de Restricción , Microbiología del Suelo , ADN Ribosómico/genética , Técnicas de Apoyo para la Decisión , Electroforesis Capilar , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados

RESUMEN

Diatoms in clothing can be used to determine contact with surface water and contact with a specific water source, which can help link suspects to crime scenes. However, for the study of diatoms it is imperative that they are first extracted from the clothing under investigation. In this study we tested three methods for extracting diatoms from cotton clothing: rinsing with water (RW), rinsing with ethanol (RE) and the dissolution of cotton with nitric and sulphuric acid (DI). The DI method produced the highest average yield and can be used to determine contact with water. The RE method extracted reproducible numbers of diatoms from two different T-shirts and the resulting species compositions were similar to their relevant reference water samples. Therefore, we present rinsing with ethanol as an effective extraction method for the qualitative and quantitative analysis of diatoms in (cotton) clothing.

Asunto(s)

Vestuario , Fibra de Algodón , Diatomeas , Ahogamiento/diagnóstico , Etanol , Medicina Legal , Humanos , Microscopía , Ácido Nítrico , Solventes , Manejo de Especímenes/métodos , Ácidos Sulfúricos

RESUMEN

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Asunto(s)

Factores Quimiotácticos/fisiología , Quimiotaxis , Ácido Fusárico , Fusarium , Hifa , Pseudomonas fluorescens/fisiología , Solanum lycopersicum/microbiología , Microscopía Confocal , Enfermedades de las Plantas , Raíces de Plantas/microbiología

RESUMEN

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A. G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.

Asunto(s)

Meristema/microbiología , Control Biológico de Vectores , Pseudomonas/fisiología , ADN Glicosilasas/genética , ADN Glicosilasas/fisiología , Farmacorresistencia Bacteriana/genética , Mutación , Pseudomonas/genética , Rifampin/farmacología

RESUMEN

Worldwide, contamination of soil and ground water is a severe problem. The negative effects of pollutants on the environment and on human health are diverse and depend on the nature of the pollution. The search for alternative methods for excavation and incineration to clean polluted sites resulted in the application of bioremediation techniques. In this review, we describe some generally accepted bioremediation tools and subsequently focus on the combination of two approaches, phytoremediation and bioaugmentation, resulting in rhizoremediation. During rhizoremediation, exudates derived from the plant can help to stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. The root system of plants can help to spread bacteria through soil and help to penetrate otherwise impermeable soil layers. The inoculation of pollutant-degrading bacteria on plant seed can be an important additive to improve the efficiency of phytoremediation or bioaugmentation.

Asunto(s)

Raíces de Plantas/microbiología , Plantas/microbiología , Contaminantes del Suelo/metabolismo , Contaminantes Químicos del Agua/metabolismo , Biodegradación Ambiental , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo

RESUMEN

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Asunto(s)

Proteínas Bacterianas/química , Biopelículas/efectos de los fármacos , Lipoproteínas/química , Péptidos Cíclicos/química , Pseudomonas putida/fisiología , Tensoactivos/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Cromatografía Líquida de Alta Presión , Cinética , Lipoproteínas/aislamiento & purificación , Lipoproteínas/farmacología , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Péptidos Cíclicos/farmacología , Pseudomonas putida/clasificación , Pseudomonas putida/crecimiento & desarrollo , Tensoactivos/farmacología

RESUMEN

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

Asunto(s)

Quimiotaxis/fisiología , Flagelos/fisiología , Raíces de Plantas/metabolismo , Pseudomonas fluorescens/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Aminoácidos/metabolismo , Metabolismo de los Hidratos de Carbono , Carbohidratos/análisis , Quimiotaxis/genética , Ácido Cítrico/metabolismo , Ácido Láctico/metabolismo , Solanum lycopersicum/microbiología , Malatos/metabolismo , Mutación , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Pseudomonas fluorescens/genética

RESUMEN

Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies. After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv. Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots. Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components. High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids. Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P. stutzeri. Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase. Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid. Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase. When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid. These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P. putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components. Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.

Asunto(s)

Naftalenos/metabolismo , Raíces de Plantas/microbiología , Pseudomonas putida/fisiología , Carbono/metabolismo , Cromatografía Líquida de Alta Presión , Genes Bacterianos , Pseudomonas putida/genética