Forty-two pools of DNA extracts obtained from human scalp and pubic hairs were used to interrogate their microbial composition by next-generation sequencing. A total of 79,766 reads were generated, yielding 39,814 reads post control and abundance filtering. On average, the coverage per sample was 1,899 reads pre-filtered and 948 post-filtered. Whilst this depth of coverage is less than ideal given the advancement in NGS technology (i.e. Illumina and Ion Torrent platforms), these 454 data are still sufficient to explore the potential of hair microbial forensics for future development. Like all novel forensic techniques, metagenomic analyses of hairs will ultimately require robust evaluation and validation to ensure that these analyses are fit for purpose and able to withstand scientific scrutiny. Part of this validation should take into consideration: replication (spatial, temporal and PCR replicates); persistence of hair bacteria not only once they are transferred or deposited (during contact and stability during storage) and prevention of contamination during processing hairs in the laboratory. Budowle et al. [33] outline and discuss in detail the future validation criteria for metagenomic analyses in relation to microbial forensic applications, which they believe will require international participation. However, such an undertaking is beyond the scope of this initial evaluation into just one of many applications of forensic metagenomic investigations.

There are many ways to present metagenomic data such as generated here; the sections below explore the data using PCoA, taxonomy and OTU’s focusing on the value of the data in forensic applications. OTUs taxonomically assigned using RDP or UCLUST options revealed little to no difference in assignment to the rank of family. For this reason, all assignments refer to BLAST taxonomic assignments.

Principal coordinates plot

Of all the data generated in this study, the NGS data generated from pubic hair held the most potential for forensic applications. A general dichotomy was observed between taxa (OTUs) harboured on male and female pubic hair shafts (Figure 1).

Figure 1 Principal coordinate plots (PCoA). Clustering of microbial taxa from each individual at each collection time point. The lilac circle represents post-SI bacterial sequences, whilst the pale blue and yellow circles represent non-SI bacterial sequences—both circles relate solely to the co-habiting couple. Panel A represents pubic hair microbial taxa from male (orange) and female (red) participants. Panel B represents scalp hair microbial taxa from male (green) and female (blue) participants. Panel C represents microbial taxa present in male and female scalp and pubic hair samples. Full size image

In general, males were clustered close to the PC2 axis along the PC1 axis whilst females were more evenly spread along the PC1 axis and further from the PC2 axis than the males. Data relating to two individuals, who were a cohabiting couple, presented some interesting results. The red dots in the yellow ellipse at high PC1 represents the taxa present on the female partner of the couple at T0 and T2 whilst the two orange dots enclosed by small blue circles at low PC1 represent the taxa from the male partner at T0 and T2. The lilac circle encloses one red dot (taxa from the female at T5) and one orange dot (taxa from the male at T5). Microbial taxa extracted from the male and female at this time point were more similar to each other than to their other previous time points (T0, T2). Discreet enquiries revealed, unlike the preceding time points; the couple in question had engaged in sexual intercourse prior to the collection of T5 hair samples. It is noteworthy that intercourse had taken place 18 h prior to the collection of pubic hairs and both individuals had showered in the interim period. Cross-transference of bacteria during intercourse may account for the variation in taxa observed. Cross-transference, or shedding of skin micro flora, is not uncommon for individuals sharing living or communal spaces [34] or during contact sports in which Meadow et al. [35] observe ‘Our results are consistent with the hypothesis that the human skin microbiome shifts in composition during activities involving human to human contact’. The results we present here suggest that the pubic hair microbiome might be quite stable, even during cohabitation, but it might be shifted dramatically during sexual intercourse for some time. This present study is the first to suggest cross-transference of pubic/genital microbial taxa as a result of intercourse. Although further analyses need to be conducted, this initial finding bodes well for future forensic applications involving sexual crimes.

An additional advantage is that compared to other body areas such as the skin, gastrointestinal tract (GIT) and mouth, fewer bacterial species seem to comprise the vaginal microbiome [36]. The advantage of simpler communities and fewer taxa in the vaginal microbiome is one that may facilitate forensic investigations by providing results in a timely manner.

The clear microbial distinctions between pubic hairs from the sexes may largely be attributable to the prevalence of Lactobacillus spp. in the female pubic hair samples and the absence of these bacteria in the male samples (excepting the co-habiting male at T5) (Figure 1). Additionally, male pubic hair microbial taxa were clustered along axis PC2 suggesting that these taxa (OTUs) were common to the male microbiota.

In contrast, female pubic hair bacterial taxa showed elongation along axes PC1 and PC2. The elongation of data along PC1 may be attributable to females harbouring different lactobacilli species (Tables 2 and 3). However, the concomitant elongation of data along axis PC2 suggests the presence of secondary differences, differences that may be due to the presence of personalised taxa (Table 3).

Table 2 Shared taxa from pairwise comparisons of all data located in scalp (Sc) and/or pubic (Pu) hairs Full size table

Table 3 Personalised (unique) bacterial taxa colonising male and female scalp and pubic hair Full size table

The PCoA plot of male and female scalp hair microbiota over the 5-month time period did not demonstrate any significant clustering (Figure 1). This is most likely attributable to male and female scalp hairs harbouring similar bacterial taxa. However, some of the female taxa are slightly spread out along axis PC1 suggesting that there may be some variation in microbial taxa in the hairs of these individuals. The distribution and composition of the microbial communities colonising scalp and pubic hair is discussed in further detail below.

Hair microbiota

Bacteria colonising male and female scalp and pubic hair samples are classed as either ‘core’ or transient (Tr) bacteria (Figure 2, see the ‘Methods’ section). In relation to the number of OTUs extracted from scalp and pubic hair microbiomes, far less bacterial sequences were lost post control filtering for pubic microbiomes in comparison to scalp hair. Pubic hairs in general contained more OTUs than scalp hair (approximately 50 male OTUs/55 female for scalp hairs c.f. approximately 73/76 for pubic hairs). Therefore, in general, pubic hair microbiomes appear to be less influenced by environmental bacteria than scalp hairs and possible harbour more niche specific bacteria. Zhou et al. [37] support this premise by demonstrating that (in comparison to other areas of the body) vaginal microbiota consisted of less stable bacteria (i.e. more transient bacteria) and showed lower alpha diversity (i.e. low species richness), supporting the premise of pubic hair harbouring niche specific bacteria.

Figure 2 Microbial data extracted from scalp and pubic hairs. Diagrams illustrating core and transient (Tr) bacterial taxa on male and female scalp and pubic hair samples. Full size image

Pubic hair microbiota

Male pubic hairs could be readily distinguished from female pubic hairs on the basis of their respective microbiota. Lactobacillus was the most prevalent taxon that clearly differentiated male and female pubic hair microbiota (Figure 2). Whilst the prevalence of Lactobacillus spp. in the vagina and vaginal secretions is well established [38]-[40], this present study is the first to discuss these bacteria colonising pubic hairs, and general pubic area, in the context of probative value in forensic investigations. Fleming and Harbison [41] suggested the presence of two Lactobacillus spp. (Lactobacillus crispatus and Lactobacillus gasseri) as suitable forensic markers to identify vaginal secretions. However, microbial data garnered in this present study suggest that a NGS metagenomic approach may be preferable to those that target specific species. The variety of Lactobacillus spp. detected in pubic hairs from the female cohort consisted of 11 OTUs (taxa) in total; three Lactobacillus spp. were unique to Female 5, one Lactobacillus spp. occurred in Female 1, and four Lactobacillus spp. were uniquely between the cohabiting couple. In addition, two Lactobacillus spp. were uniquely shared between F4 and F5, and one OTU was uniquely shared between F4 and F1 (Tables 2 and 3).

Compared to male pubic hairs, female pubic hairs harboured fewer transient bacteria (Figure 2); the number of bacterial sequences comprising transient bacteria of female pubic hairs was approximately half the number of those found in male pubic hair (Table 4). This disparity may be attributable to lactobacilli conferring ‘antimicrobial protection’ to the vagina by preventing colonisation by other microorganisms [38]. Li et al. [42] also found that in comparison to other body areas, the vaginal microbiome is less transient (i.e. more stable). This stability was apparent in the differences between the number of OTUs detected in the scalp and pubic hair controls; there were significantly less OTUs present in controls from the pubic hairs in comparison to the scalp hairs. Post control filtering for FSH and MSH samples there were 33% and 43% (respectively) of sequences left. In comparison, for FPH and MPH there were 70% and 72% (respectively) sequences left, post-filtering. The disparity between the two somatic origins suggests that the bacterial taxa in scalp hair extracts had a high proportion of environmental bacteria that readily appear in controls.

Table 4 Number of bacterial 16S sequences found in core microbiomes compared with transient number of sequences for each sex/somatic origin sampled Full size table

Scalp hair microbiota

In contrast to the pubic hairs, scalp hair microbiota showed no correlation with the sex of the donor (Figure 2). Male and female scalp hair bacterial taxa consisted of normal human skin commensals, e.g. Anaerococcus spp., and environmentally derived taxa, e.g. Knoellia subterranea, many of which occurred in both male and female samples (Table 5). In the present study, the most significant difference observed in male and female scalp hairs was the disparate proportions of the transient bacterial taxa (Figure 2). Almost twice as many transient bacterial taxa were present in female scalp hair compared to males (Table 4). This may be due to the greater frequency of females grooming and/or washing and/or dyeing or bleaching their hair in comparison to males. Such grooming practices may prevent establishment of more stable bacterial colonies in favour of less stable (transient) bacterial colonies. Irrespective of the cause of this disparity, this observation cannot be regarded significant in relation to forensic investigations.

Table 5 Natural habitats of shared bacterial taxa identified by pairwise comparisons Full size table

Costello et al. [3] identified two dominant 16S sequences from scalp swabs: Propionibacterinae in which members are predominant bacteria in hair follicles and other sebaceous sites [39] and Streptophyta (a plant phylum). In contrast, the predominant bacterial taxa from hair shafts in this study were Corynebacteriaceae and Tissierellacea fam.nov (‘new family’) (Figure 2). The difference may be attributable to either environmental differences (i.e. different study sites) or the collection technique employed by Costello et al. [3] where swabbing the top of the head might have favoured the removal of scalp/follicular bacteria (i.e. propionibacteria rather than hair shaft bacteria).

Personalised and shared bacterial taxa

Forensic investigations seek to establish ‘common origin’ or ‘source attribution’ of evidence, that is, to establish with reasonable scientific certainty that a particular individual is the source of an evidentiary sample. In relation to biological evidence, this question may be addressed through the detection of individualising biological characteristics, for example, a human DNA profile, characteristics which excludes other individuals as being the source. Ideally, these characteristics should not commonly occur within the general population or one that is solely found in males or females.

Inside the confines of the 16S V4 region, with the exception of one male (co-habiting male at T5), all individuals harboured unique taxa on their pubic hairs (Figure 3). In addition to personalised bacteria that were part of the normal skin flora, e.g. Corynebacteriaceae, pubic hairs were also colonised by environmentally derived bacterial taxa, e.g. Methylobacteriaceae (Table 3).

Figure 3 Personalised microbial data. Diagrammatic summary of unique bacterial taxa found in male and female scalp and pubic hair samples. Male individuals left to right: Individuals M2 (cohabiting male), M3 and M7. Female individuals left to right: Individuals F1, F4, F5 (cohabiting female) and F6. Full size image

Hairs from scalp and pubic regions, for both sexes, included shared taxa that are common inhabitants of human skin or scalp, e.g. Corynebacteria, or were environmental in origin, e.g. Rhodobacteriaceae (Tables 2 and 5). At first glance, the commonality of these bacteria may appear to be of minimal probative value; as discussed in a preceding section, personalised features should be uncommon traits or features. However, common bacteria may harbour single nucleotide polymorphisms (SNPs) within their genome, which may further discriminate between individuals.

Among all mammals, the microbiota composition is extensively conserved at the high taxonomic levels such as phylum or class. At these taxonomic levels, humans are very similar to each other (and other mammals) but variation increases progressively at the lower taxonomic levels. Personalised taxa, which allow discrimination between individuals (the goal of forensic applications), are likely to be detected at these lower taxonomic rankings. Personalised taxa may be present in high or low abundance; detection of low abundance taxa may only be detected by ultra-deep sequencing of the extracted bacterial DNA. In this regard, higher depth of coverage afforded by NGS platforms such as Ion Torrent or Illumina may be more informative than the 454 data presented here. As Ursell et al. [43] noted ‘it is important to realise that sampling depth may be critical for distinguishing taxa that are absent from those that are merely rare’. Under these circumstances, it is critical to discount bacterial taxa present in all control samples in order for the results to be not only robust but also scientifically accurate and capable of withstanding scientific and legal scrutiny.

Temporal stability data garnered in this study broadly suggest that bacteria on scalp hairs may be more prone to fluctuations in comparison to pubic hairs (in addition to being more prone to environmental contaminants). The data shows that, on average and post-filtering, approximately 17% (range 6%–25%) of pubic hair bacterial OTUs were temporally stable across all time points; whilst, on average, scalp hair harbour approximately 5% (range 0%–13%) of bacterial OTUs (Table 6). These preliminary findings suggest that pubic hair bacteria may be more temporally stable than scalp hair bacteria and therefore potentially of more probative value than scalp hair bacteria.

Table 6 Temporal stability of bacterial taxa (OTUs) Full size table

Although temporal stability of an individual’s bacterial taxa may appear to be an important prerequisite for metagenomics to have forensic value, the most relevant attributes will mostly likely be transference of bacteria (during contact), persistence of bacteria post transfer and storage conditions. Consider a case of unlawful sexual intercourse (of an adult female), the most relevant microbial data will be the taxa available for transfer at the time of the assault (rather than what it was weeks, months or days before or after) and the persistence of the victim’s bacteria on the offender’s genitals/pubic area (and vice versa). This, of course, is reliant upon collection of evidence from the victim and suspect(s) within several hours of the time of the assault rather than several days. Microbial data from the cohabiting couple, albeit preliminary, are encouraging, in supporting the suggestion of bacterial transfer and persistence following sexual intercourse.