Elsevier

Legal Medicine

Volume 25, March 2017, Pages 75-82
Legal Medicine

Terminal restriction fragment length polymorphism profiling of bacterial flora derived from single human hair shafts can discriminate individuals

https://doi.org/10.1016/j.legalmed.2017.01.002Get rights and content

Highlights

  • Sufficient amount of bacterial DNA could be recovered from all single hair shaft.

  • T-RFLP profiles of same individual are reproducible and higher similarity.

  • Similar T-RFLP profiles were obtained in most volunteers collected after 6 months.

  • Most of unidentified hairs were assigned to a few clusters including correct person.

Abstract

Human hairs are the trace evidence most commonly encountered at many crime scenes. However, they have not been effectively utilized for actual criminal investigations because of the low accuracy of their morphological inspection, low detection rate of short tandem repeat (STR) typing, and the problem of heteroplasmy in mitochondrial DNA analysis. Here, we examined the possibility of individual discrimination by comparing profiles of bacterial flora on hair. We carried out the profiling of terminal restriction fragment length polymorphisms (T-RFLP) of the amplified bacterial 16S ribosomal RNA (rRNA) gene from hair samples. Compared with existing STR typing methods that use hair roots, this method using hair shafts allowed the detection of stable bacterial DNA. We successfully obtained the T-RFLP profile from single hair shafts of all volunteers tested. The profiles were specific to each individual, and multiple profiles obtained from the individual him/herself showed higher similarity than those from different individuals. These individual-specific profiles were stably obtained from samples from most volunteers, when collected again after 6 months. Storage of the collected hair samples at −30 °C was effective for obtaining reproducible T-RF profiles. When unidentified hair samples collected in the laboratory were compared with a pre-constructed database, 17 of 22 hairs were assigned to a small group of people, including the corresponding individuals. These results show that T-RFLP analysis of bacterial flora on a hair shaft found at a crime scene could provide useful information for narrowing down a suspect.

Introduction

Among the evidence that an offender leaves at a crime scene, human hairs are often found in criminal investigation [1], [2], [3]. Their judgment is mainly carried out by morphological observation using microscopy [1], [4] and human short tandem repeat (STR) typing [3], [5]. It is estimated that 50–150 strands of human hair are shed naturally every day [2], [6]. Therefore, there is some possibility of collecting an offender’s hair as forensic evidence, without their knowledge. If personal discrimination of the shed hairs were possible, it would be very helpful for identifying an offender, or for establishing an offender's action [1].

The human scalp contains 100,000–150,000 hairs on average [2], [7]. Of these, 90–95% are in anagen stage and the others (5–10%) are in telogen stage [8]. The telogen hairs have stopped growing, and apoptosis and keratinization of the hair root are progressing [2], [3], [6], [9]. As a result, it is easy for telogen hairs to fall out without external force due to their weak adhesion to the scalp [2], [6]. In general, the hair root is used for nuclear DNA extraction for human DNA typing [1], [2], [3], [5]. However, it is considered to be difficult to extract nuclear DNA from hairs recovered at a crime scene, because 90–95% of those hairs are at telogen stage [2], [7]. In fact, Bourguignon et al. found that enough DNA could be extracted from only 3.3% of 3242 telogen stage hairs from 27 volunteers for STR typing [3].

Because of these limitations, the mitochondrial DNA test is sometimes performed, since it is a more sensitive appraisal technique than STR typing [7], [10]. However, mitochondrial DNA testing has not been adopted in routine forensic examination in Japan because 1) there is no database like that for STR typing for comparison, 2) mitochondrial DNA shows maternal heredity, and 3) there is a possibility of the occurrence of an irregular heteroplasmy [6], [9]. Therefore, we focused on DNA derived from bacteria attached to the hair instead of human DNA from the hairs themselves.

It has been already reported that bacteria attached to human hair forms a complex flora [11], [12], [13]. For example, Tridico et al. performed metagenomic evaluations of bacteria on human hairs using next-generation sequencing data, and revealed that Corynebacteriaceae and Tissierellacea families were major bacterial taxa on human hairs [13]. In Japan, the terminal restriction fragment length polymorphism (T-RFLP) technique is currently the most applicable among all the general forensic laboratories [14]. We have already reported the possibility of discriminating an individual from the bacterial flora existing in their handprints using T-RFLP [15]. To date, there are no reports on discriminating an individual based on the bacterial flora on human hairs using T-RFLP. In preliminary experiments, we found that sufficient bacterial DNA could be recovered from a single shed hair. In this study, we aimed to analyze bacterial flora on single strands of human hair by T-RFLP to discriminate individuals as an alternative approach to STR typing or mitochondrial DNA judgment, without impairing the conventional inspection technique.

Section snippets

Samples and collection

Hair samples were collected from 24 Japanese adults (18 males and 6 females) who consented to take part in this study. None of the volunteers was taking medication during the experimental period. Samples of naturally shed or plucked hairs were collected while wearing nitrile gloves.

To examine the variation of T-RFLP patterns within an individual over time, a follow-up sampling from 16 volunteers was conducted after 6 months. The hair sample collections were carried out during 2 periods, April

Bacterial DNA yields from hair for T-RFLP analysis

We first investigated the yield of bacterial DNA extracted from each piece of hair. Hair roots and shafts from 50 hairs derived from 18 volunteers were analyzed separately. The average length of hairs used was 6.98 ± 4.99 cm, and the amount of bacterial DNA obtained from a hair root and a hair shaft were 40.06 ± 89.93 pg and 17.99 ± 18.30 pg, with the minimum amount of 0 pg and 1.32 pg, respectively. The average amount of bacterial DNA obtained from hair roots was twofold higher than that from hair

Discussion

Our previous study on T-RFLP bacterial profiling of handprints suggested that bacterial flora analysis is effective for discrimination among individuals [15]. Further to this, we aimed to establish a novel method of hair bacterial profiling, which could be routinely applied in crime scene analysis without any additional instrumentation. In this study, we investigated the possibility of individual discrimination using T-RFLP analysis of the hair bacterial floras by targeting 16S rRNA genes.

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