Microbial analysis of bite marks by sequence comparison of streptococcal DNA

Human bite mark analysis can be a vital component in the investigation of violent offenses, providing crucial physical and biological evidence in criminal prosecutions. Variability in mechanical properties related to anatomical location, ageing and ethnicity of the skin undermine morphometric bite mark analysis. While the recovery of human DNA from bite marks may provide extremely compelling evidence, the presence of enzymes, especially deoxyribonuclease I, in saliva compromise the recovery of exposed DNA. The scientific rigor applied to the establishment of nuclear DNA analysis has highlighted the deficiencies in evidence underpinning other forensic disciplines, including bite mark analysis. Such inconsistencies now challenge the value and objectivity of morphometric bite mark evidence. Consequently, investigation in our laboratory persued an alternative method of analysis, based on bacterial genotyping. More than 700 bacterial taxa have been detected in the human oral cavity. The predominating species are of the genus Streptococcus and comprise the principal bacteria colonising the surface of the teeth. Streptococci exhibit extensive genetic diversity, which provides the premise for research aimed at exploring the forensic value of matching teeth to bite marks by bacterial genotyping. Streptococcal profiles may be distinctive among individuals to the degree that genotypic comparisons of isolates from bite marks and teeth can provide a correct match with a high level of confidence. The current research extends this approach by applying high-throughput sequencing to obtain streptococcal DNA sequences amplified directly from bite marks and teeth. Comparison of the sequences from the two sample types was used to establish the probability of matching a bite mark to the teeth responsible. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR using streptococcal-specific primers for three different regions of genomic DNA: 16S ribosomal RNA (16S rRNA), 16S-23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). The PCR products were elucidated using high throughput sequencing technology (GS FLX, Roche) and the sequence reads from each bite mark were compared to those generated from all teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of shared reads between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31 and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.9 for ITS, 1.0 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants in situations where the perpetrator’s DNA cannot be recovered