Introduction
Viruses are the most abundant biological entity on this planet. It is estimated that the ocean alone contains 100-billion times as many viruses than there are grains of sand on Earth [
1]. In addition to being the most abundant biological entity, viruses are arguably the most diverse biological entities on Earth due to their mutation rate and speed of replication. Despite their abundance and diversity, our understanding of viral ecology and function is in its infancy.
Viruses infect all forms of life and may influence host population dynamics, yet many viruses' functional influence has yet to be elucidated. Viruses may influence the host organism by increasing fitness through genetic transfer and expression of host-specific genes that confer fitness [
2‐
7]. Most microbiome studies have focused on the bacteriome [
8,
9]. However, the viral component of the microbiome is an essential factor contributing to community assembly and function. It has been demonstrated that bacteriophages can acquire genes that may provide the host with advantageous evolutionary adaptations, such as biofilm inducing genes, proviral genes that promote cell repair, toxin and virulence genes that promote host fitness [
4,
6,
10‐
12]. Additionally, specific bacteriophage genomes retain rate-limiting metabolic genes that increase host fitness [
4,
6,
13,
14]. Acquisition and expression of beneficial host metabolic genes were first described in marine bacteriophage populations, where marine bacteriophages were shown to carry auxiliary metabolic genes that contribute to photosynthetic processes [
15].
For animals and humans, the skin is the first barrier of defense against external pathogens. Shifts in the population dynamics of the skin microbiome have been shown to contribute to human diseases such as infections caused by
Staphylococcus aureus or
Streptococcus pyogenes [
8,
16]. Although studies have speculated about complex interactions of the human skin virome and its effects on bacterial pathogen colonization, the magnitude of host-viral interactions and the role of viruses in shaping the skin microbiome is poorly understood [
17‐
20]. Especially in regards to the viral contribution to human disease. Alternatively, bacteriophages have long been of interest in treating bacterial diseases and conditions by controlling bacterial population dysbiosis and antimicrobial resistance [
21‐
24], yet little is known about skin phage diversity and their role in shaping the skin microbiome.
Previous studies have demonstrated that bacteriophages on the skin carry auxiliary metabolic genes (AMGs), which have the potential to bolster host fitness and increase microbiome function [
17,
25]. We previously reported the persistence and abundance of viruses, including bacteriophages on the skin virome over time [
26]. In this present study, we investigated the diversity of the human skin virome and identified auxiliary metabolic genes present within the skin virome and their effect on microbiome function and host fitness. To this end, we evaluated the skin virome longitudinally across 60 human individuals across three different anatomical locations at five different time points by expanding the dataset developed previously by this team [
26]. Additionally, we identified and validated antimicrobial-resistant genes (AMR) using an amplicon sequencing approach in this study. We further evaluated the bacteriophage content of the human skin virome to identify factors that confer stability of the acquired gene content.
Discussion
Viruses play vital roles in shaping microbial communities by improving host fitness, controlling bacterial populations through predation, and helping increase metabolism by overcoming metabolic bottlenecks [
4,
6,
17,
55,
56]. For instance, over time, filamentous bacteriophages have acquired numerous host-specific genes that have allowed them to have symbiotic relationships with their bacterial hosts, thus promoting host survival and the production and spread of phage progeny [
6]. The retention of host genes and the metabolomic influence of phages on bacterial hosts have been described in aquatic communities [
13,
15], and human and animal gut microbiomes [
25,
31,
57].
Studies are limited that describe phage diversity and ecology in humans other than the gut [
17‐
19,
26,
58‐
66]. To date, only a few studies have addressed the human skin virome and its taxonomic composition [
17‐
19,
26,
59,
62]. Additionally, studies describing AMR genes in the skin virome and other auxiliary genes are limited [
17]. Here we describe bacteriophage diversity, AMGs, and AMR genes present in the human skin virome and possible roles of the human skin virome using temporal information from five time points spanning six-months across three anatomical skin locations (left hand, right hand, and scalp) from 60 human participants.
We identified 3230 bacteriophage contigs from human skin viral metagenomes. Bacteriophage contigs identified in this study predominantly originated from the viral Families
Myoviridae and
Siphoviridae, which both belong to Order
Caudovirales. Previous studies have reported that the skin virome consists primarily of
Caudovirales bacteriophages [
17,
18,
26,
62]. While our study is consistent with previous reports, current viral reference databases consist of a disproportionate amount of
Caudovirales viral genomes, and this fact could contribute to an annotation bias in virome studies, ours included. This is further reflected by the fact that only 16% of the putative phage contigs we assembled could be taxonomically classified using current reference databases. This fact underscores the lack of knowledge regarding phage diversity in the human skin virome. Therefore, we speculate that most of the phages that we have identified in this study are novel phages. Additional work assessing bacteriophage identification, isolation, annotation, culture, and comparative genomics is needed to fully understand bacteriophage taxonomic composition and diversity in the human skin virome. However, our filtration using 0.22 μm filters may have resulted in removal of large viruses from the skin virome and may be lacking large viral particles. Additionally, the whole genome amplification used in this study may have led to amplification bias.
Though many studies have shown the presence of host-specific genes such as AMR genes in
S. aureus and that transduction via
S. aureus phage is a common form of horizontal gene transfer of these AMR genes, very few culture-independent studies have been done that look at the human skin virome and its composition and abundance of host-related genes in phages [
17]. Studies have investigated the skin virome composition and the temporal stability and diversity of the virome; however, many of these works did not address the presence of AMG or AMR genes or were smaller-scale studies with lower sample numbers with short sampling periods of only a couple of weeks without repeated sampling periods [
17‐
19,
26,
62].
The acquisition of bacteria-specific genes, especially genes associated with host immune defense evasion and host genes associated with increased replication or cell proliferation to aid in viral replication and spread, is not a new concept in virology [
4,
17,
31,
67,
68]. However, it is crucial to understand what genes are enriched in viromes and how expression of such genes could affect host function and persistence. To this end, our phage contigs contained 648 AMGs within the human skin virome. This study identified the human skin virome to carry genes associated with carbohydrate metabolism and amino acid synthesis. These genes are critical when the virus overtakes the cell for replication as carbohydrate metabolism could lead to increased energy for replication, and amino acid synthesis genes could help synthesize viral capsid during replication. Recently, it was shown that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) could use host folate and one-carbon metabolism to bolster replication [
69]. Similarly, it may be possible that bacteriophages carry folate metabolism genes to increase de novo purine synthesis to increase viral replication. The presence of auxiliary metabolic genes associated with folate biosynthesis has been reported in rumen viruses and has been suggested in one-carbon metabolism [
57]. As such, the increased folate metabolism genes may help utilize one-carbon substrates to provide energy during replication.
We also identified AMR genes associated with three main antimicrobial drug resistance mechanisms among the bacteriophages identified. This included efflux transport pumps found in gram-positive and gram-negative bacteria that help transport toxic compounds and antibiotic drugs out of the cell [
70]. Of the five main families of efflux pumps associated with antibiotic resistance, genes encoding efflux pumps belonging to ATP-binding cassette (ABC), the major facilitator superfamily (MFS), and the small multidrug resistance family (SMR) were all identified within the phage population. We identified the MFS efflux pump protein
mef(A) and the ABC efflux pump protein
msr(D), present on the same contigs. In
Streptococcus pneumoniae and
Staphylococcus epidermidis, when dually expressed, these two proteins act as a dual efflux pump that gives moderate resistance against 14- and 15- membered macrolides such as erythromycin, clarithromycin, and azithromycin [
71]. Having the
mef(A) gene alone results in high resistance levels to varying macrolide drugs and is considered a common gene associated with multidrug-resistant pathogens such as MRSA. The presence of the dual efflux pump of
mef(A)-
msr(D) (also commonly referred to as
mef(A)-
mel) confers a higher level of resistance to macrolides than that of just
mef(A) alone [
72]. Both
mef(A) alone and dual
mef(A)-msr(D) genes have been identified in prophage regions of bacteria and in phage genomes such as that of the
Streptococcus infecting phages Tn1207.3 and Tn1207.1 [
73‐
75]. The detection of dual efflux pumps within the phage population suggests that AMR genes can be moved across bacteria via phage-mediated transfection.
The efflux pump transcribing gene,
qacF, was also observed in multiple viral contigs. These efflux pumps confer quaternary ammonium compound resistance and have been hypothesized to increase bacterial tolerance to antibiotics, especially antibiotics that inhibit cell wall synthesis [
76,
77]. Bacteria containing
qacF and
qac-related genes have been reported in soil and agriculture related environments, including livestock related industries. The presence of this AMR gene in bacteriophages is not surprising since multiple study participants reported being in recent contact and working with livestock. This demonstrates that the phage diversity in the skin virome constantly changes and is, at least in part, acquired from the environment. Additionally, this suggests that mobile genetic elements may be a route in which AMR genes are horizontally transferred.
One of the most common AMR genes associated with antibiotic resistance is genes encoding proteins that directly interact with or modify bacterial ribosomes. This mode of action confers resistance to antibiotic agents that target transcription and translation. The 23S rRNA interacting methyltransferases
RlmH,
erm(C), and phosphotransferase
MphE were all identified in the phage population. These genes confer strong antimicrobial resistance to many drugs, such as aminoglycosides, macrolides, oxazolidinones, and streptogramins [
70]. The presence of
erm(C) in multiple contigs is of clinical importance since
erm(C) is the best-studied resistance mechanism to MLSB (macrolide, lincosamide, and streptogramin B) in bacteria and is one of the leading AMRs associated with MRSA [
71,
78]. Due to its high abundance within the population and its clinical relevance to human health, we investigated the abundance and distribution of the
erm(C) gene within the study population using targeted amplicon-based sequencing to identify the SNP variation within the
erm(C) gene to evaluate functional capabilities and phylogeny of this gene within our study population.
The
erm(C) gene was highly abundant and temporally stable in some individuals. The stability of the
erm(C) gene across the skin virome of study participants over a six-month period suggests that the gene plays an important role in phage stability and, in turn, bacterial host fitness. One can hypothesize that temporal stability and evolutionary retention of functional versions of AMR genes give associated phages an evolutionary advantage over viral strains that do not carry AMR genes or do not have functional versions of AMR genes. Thus, to establish the importance of AMR genes for persistence and to identify the evolutionary advantage of phages having AMR genes, we investigated the abundance and the presence of functional sequence variants of the
erm(C) gene using the amplicon variants obtained through targeted sequencing. This analysis revealed that only functional variants of the
erm(C) gene persist within the viral population at high abundance (Fig.
5). This suggests that
erm(C) provides an advantage for the phages to persist in the human virome and demonstrates that phages can acquire multiple host genes that can impact microbiome community diversity and evolutionary selection, including genes that transcribe antimicrobial activity resistance.
Conclusions
Viruses play an important role in modulating bacterial population and diversity. Here we investigated the human skin virome and skin associated bacteriophage population diversity, dynamics, and the auxiliary metabolic genes associated with these phages. Human skin viral metagenome samples revealed that the bacteriophage population on the skin is mainly composed of tailed bacteriophages in the viral order Caudovirales. Nevertheless, many phage contigs could not be classified due to the poor representation of human skin viruses in viral reference databases. We identified 648 different bacterial host-derived AMGs related to varying types of bacterial cell processes and functions. Additionally, we identified the antimicrobial-resistant genes erm(C), par(E), par(EF), par(C), fus(B), msr(D), msr(E), mef(A), mph(E), and rlm(H). These genes were, in some cases, subject-specific, whereas genes such as erm(C) were abundant across multiple individuals and were stable over time. This study demonstrates that the phages in the human skin virome carry auxiliary metabolic genes that increase host fitness and help with the persistence of the bacterial host and contribute greatly to bacterial-viral (phage) interactions. Findings from this study suggest that viral-host relationships are more complex than previously thought and highlight the importance of utilizing system-based approaches to study ecosystem interactions in order to fully understand microbiome diversity and function.
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