Microbial DNA fingerprinting of human fingerprints: dynamic colonization of fingertip microflora challenges human host inferences for forensic purposes
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Human fingertip microflora is transferred to touched objects and may provide forensically relevant information on individual hosts, such as on geographic origins, if endogenous microbial skin species/strains would be retrievable from physical fingerprints and would carry geographically restricted DNA diversity. We tested the suitability of physical fingerprints for revealing human host information, with geographic inference as example, via microbial DNA fingerprinting. We showed that the transient exogenous fingertip microflora is frequently different from the resident endogenous bacteria of the same individuals. In only 54% of the experiments, the DNA analysis of the transient fingertip microflora allowed the detection of defined, but often not the major, elements of the resident microflora. Although we found microbial persistency in certain individuals, time-wise variation of transient and resident microflora within individuals was also observed when resampling fingerprints after 3 weeks. While microbial species differed considerably in their frequency spectrum between fingerprint samples from volunteers in Europe and southern Asia, there was no clear geographic distinction between Staphylococcus strains in a cluster analysis, although bacterial genotypes did not overlap between both continental regions. Our results, though limited in quantity, clearly demonstrate that the dynamic fingerprint microflora challenges human host inferences for forensic purposes including geographic ones. Overall, our results suggest that human fingerprint microflora is too dynamic to allow for forensic marker developments for retrieving human information.
KeywordsMicrobial forensics Fingertip microflora Microbial DNA analysis Microbial fingerprint Forensic PFGE Staphylococcus
Microbes can negatively interfere with the postmortem assessment of alcohol abuse and in this way pose problems for forensic investigators . However, microbial forensics is often chiefly associated with the detection of highly pathogenic microbes to which humans are deliberately exposed in cases of biological terrorism [2, 3]. However, human fingertip microflora left behind on touched objects at crime scenes may potentially contain forensically relevant information that may be useful for human host inferences accessible via microbial DNA fingerprinting of physical fingerprints. For example, if endogenous microbial skin species/strains with a geographically restricted distribution could be retrieved from touched objects via microbial DNA analysis, the geographic origin of the human host individual could be determined indirectly. Information about the geographic region of origin can be relevant in suspect-less forensic cases where the evidence DNA sample does not match either a suspect’s DNA profile or any in a criminal DNA database. In such cases, geographic information derived from crime scene samples is expected to reduce the potential pool of suspects by allowing police investigations to concentrate on specific groups of people, i.e., those from a restricted geographic region. Numerous human genetic markers have been suggested for inferring human genetic ancestry mostly to the continental level [4, 5, 6, 7], and a recent study indicated that inferring the subregion of origin of an unknown European may be feasible from autosomal genetic data . However, direct ancestry inference based on human genetic markers is currently far from perfect, initiating the question whether microbial DNA may be used to supplement human DNA markers in reliable ancestry reconstruction of unknown persons. Recently, it has been shown that the gastric pathogenic bacteria Helicobacter pylori has intimately coevolved with its human host [9, 10]. Although this example may be of limited direct relevance for forensics, because samples containing H. pylori are usually not found at crime scenes (with the exception of bodies in cases of missing persons), it shows that in principle human geographic signatures are inferable from microbial genomes. The human skin is a complex microbial ecosystem consisting of multiple niches, which can differ drastically from each other . Interactions between skin microbes and the human host, as well as between the microbial occupants, are still poorly understood. The current knowledge on skin microbiota primarily derives from cultivation-based studies [12, 13], although molecular fingerprinting techniques have been employed more recently [14, 15]. If a comparable relationship exists between humans and their skin microbiota, as has been observed for H. pylori, new methods for human geographic origin determination could be developed based on DNA analysis of fingertip microflora, with interesting new applications to molecular analyses of physical fingerprints left at crime scenes.
Material and methods
Volunteers and microbiological procedures
Five Dutch European subjects from Rotterdam and three volunteers from Dhaka, Bangladesh, placed their right index finger on a Colombia III blood agar plate (bioMerieux, Marcy-l’Etoile, France). Fingerprint sampling for Dutch volunteers was performed in Rotterdam whereas that of Bangladeshi volunteers was performed in Dhaka. After washing this finger with hospital soap and air drying, another fingerprint, from the same finger, was placed next to the first. Whereas the first fingerprint was expected to provide a mixture of transient and resident microbiota, the second one was assumed to provide resident microbiota only as soap removes the upper dead-skin layers including their transient microflora. After overnight incubation at 37°C, colonies with different morphologies were isolated, with 20 per fingerprint (two each) from the five Dutch individuals (n = 200 strains). Three weeks later, this procedure was repeated for assessment of bacterial persistence in the Dutch volunteers. Agar plates from Bangladeshi volunteers were shipped by courier to Rotterdam after overnight incubation at 37°C for further analyses. Given the diversity in genotypes encountered in the time-wise resampling experiments using Dutch volunteers (see below), this procedure was not repeated for the volunteers from Bangladesh. Gram staining and the determination of catalase activity were applied for the identification of staphylococci using commercially available test kits (bioMérieux, Marcy-l’Etoile, France). Staphylococcus epidermidis sensu stricto was identified among the isolates by a species-specific polymerase chain reaction assay as described elsewhere . A coagulase test with Slidex™ Staph Plus (bioMérieux, Marcy-l’Etoile, France) was performed to detect Staphylococcus aureus isolates. Staphylococcus spp. were classified with the ID 32 Staph API system (bioMérieux, Marcy-l’Etoile, France). Strains were suspended in 15% glycerol in water and stored at −80°C.
Genotyping bacterial isolates
In order to generate DNA fingerprints for the most relevant bacterial isolates, all staphylococcal isolates were subjected to pulsed-field gel electrophoresis (PFGE). PFGE can be used to electrophoretically separate DNA restriction fragments ranging in length between 50 and 1,000 kb. Bacteria were embedded in low melting agarose. After lysostaphin and proteinase treatment and SmaI macrorestriction , PFGE was performed in a 1% InCert agarose (FMC) gel in 0.5× TBE at 14°C, using a constant electric field of 6 V cm−1 with pulse ramping from 0.1 to 30 s at a 60°/−60° angle for 18 h. PFGE patterns were analyzed for levels of similarity with BioNumerics 4.5 (Applied-Maths, Sint Martens Latem, Belgium).
Results and discussion
Staphylococcus species and PFGE types from Dutch fingerprints before washing with soap representing elements of the transient (TM) microflora and after washing representing resident fingertip microflora (RM)
Staphylococcus species and PFGE types from Bangladeshi fingerprints before washing with soap representing elements of the transient (TM) microflora and after washing representing resident fingertip microflora (RM)
To conclude, we see only limited geographic differentiation between microbial DNA fingerprints from Dutch Europeans and Bangladeshi south Asians, indicating that geographic inferences of human hosts from fingertip microbial DNA analysis is not feasible. Furthermore, our results from DNA profiling of transient and resident fingertip microflora show that human fingertip microflora is too dynamic and thus does not fulfill the criteria required for forensic marker developments to infer any human host information from physical fingerprints.
We are grateful to all volunteers for participation and thank Kaye Ballantyne for useful comments on the manuscript. The study was supported by the Erasmus University Medical Center Rotterdam and additionally by funds from the Netherlands Forensic Institute.
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