Common antibiotics, azithromycin and amoxicillin, affect gut metagenomics within a household.

BACKGROUND: The microbiome of the human gut serves a role in a number of physiological processes, but can be altered through effects of age, diet, and disturbances such as antibiotics. Several studies have demonstrated that commonly used antibiotics can have sustained impacts on the diversity and the composition of the gut microbiome. The impact of the two most overused antibiotics, azithromycin, and amoxicillin, in the human microbiome has not been thoroughly described. In this study, we recruited a group of individuals and unrelated controls to decipher the effects of the commonly used antibiotics amoxicillin and azithromycin on their gut microbiomes. RESULTS: We characterized the gut microbiomes by metagenomic sequencing followed by characterization of the resulting microbial communities. We found that there were clear and sustained effects of the antibiotics on the gut microbial community with significant alterations in the representations of Bifidobacterium species in response to azithromycin (macrolide antibiotic). These results were supported by significant increases identified in putative antibiotic resistance genes associated with macrolide resistance. Importantly, we did not identify these trends in the unrelated control individuals. There were no significant changes observed in other members of the microbial community. CONCLUSIONS: As we continue to focus on the role that the gut microbiome plays and how disturbances induced by antibiotics might affect our overall health, elucidating members of the community most affected by their use is of critical importance to understanding the impacts of common antibiotics on those who take them. Clinical Trial Registration Number NCT05169255. This trial was retrospectively registered on 23-12-2021.

Soybean Hairy Root Transformation for the Analysis of Gene Function.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

RNA-Seq Dissects Incomplete Activation of Phytoalexin Biosynthesis by the Soybean Transcription Factors GmMYB29A2 and GmNAC42-1.

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (Glycine max). They play essential roles in providing resistance to the soil-borne pathogen Phytophthora sojae and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from P. sojae. GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene GmHSF6-1, the homolog of Arabidopsis HSFB2a that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway.

A panel of diverse Pseudomonas aeruginosa clinical isolates for research and development.

OBJECTIVES: Pseudomonas aeruginosa is a leading cause of community- and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, primarily through mutation. In response, WHO listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavour. METHODS: WGS was performed on 3785 P. aeruginosa isolates in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome MLST and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics. RESULTS: This 100-strain diversity panel contained representative strains from 91 different STs, including genetically distinct strains from major epidemic clones ST-111, ST-235, ST-244 and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-susceptible to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates. CONCLUSIONS: This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates and available metadata, including genome sequences, are available to industry, academia, federal and other laboratories at no additional cost.

Temporal variations in bacterial community diversity and composition throughout intensive care unit renovations.

BACKGROUND: Inanimate surfaces within a hospital serve as a reservoir of microbial life that may colonize patients and ultimately result in healthcare associated infections (HAIs). Critically ill patients in intensive care units (ICUs) are particularly vulnerable to HAIs. Little is known about how the microbiome of the ICU is established or what factors influence its evolution over time. A unique opportunity to bridge the knowledge gap into how the ICU microbiome evolves emerged in our health system, where we were able to characterize microbial communities in an established hospital ICU prior to closing for renovations, during renovations, and then after re-opening. RESULTS: We collected swab specimens from ICU bedrails, computer keyboards, and sinks longitudinally at each renovation stage, and analyzed the bacterial compositions on these surfaces by 16S rRNA gene sequencing. Specimens collected before ICU closure had the greatest alpha diversity, while specimens collected after the ICU had been closed for over 300 days had the least. We sampled the ICU during the 45 days after re-opening; however, within that time frame, the alpha diversity never reached pre-closure levels. There were clear and significant differences in microbiota compositions at each renovation stage, which was driven by environmental bacteria after closure and human-associated bacteria after re-opening and before closure. CONCLUSIONS: Overall, we identified significant differences in microbiota diversity and community composition at each renovation stage. These data help to decipher the evolution of the microbiome in the most critical part of the hospital and demonstrate the significant impacts that microbiota from patients and staff have on the evolution of ICU surfaces. Video Abstract.

Fecal Viral Community Responses to High-Fat Diet in Mice.

Alterations in diet can have significant impact on the host, with high-fat diet (HFD) leading to obesity, diabetes, and inflammation of the gut. Although membership and abundances in gut bacterial communities are strongly influenced by diet, substantially less is known about how viral communities respond to dietary changes. Examining fecal contents of mice as the mice were transitioned from normal chow to HFD, we found significant changes in the relative abundances and the diversity in the gut of bacteria and their viruses. Alpha diversity of the bacterial community was significantly diminished in response to the diet change but did not change significantly in the viral community. However, the diet shift significantly impacted the beta diversity in both the bacterial and viral communities. There was a significant shift away from the relatively abundant Siphoviridae accompanied by increases in bacteriophages from the Microviridae family. The proportion of identified bacteriophage structural genes significantly decreased after the transition to HFD, with a conserved loss of integrase genes in all four experimental groups. In total, this study provides evidence for substantial changes in the intestinal virome disproportionate to bacterial changes, and with alterations in putative viral lifestyles related to chromosomal integration as a result of shift to HFD.IMPORTANCE Prior studies have shown that high-fat diet (HFD) can have profound effects on the gastrointestinal (GI) tract microbiome and also demonstrate that bacteria in the GI tract can affect metabolism and lean/obese phenotypes. We investigated whether the composition of viral communities that also inhabit the GI tract are affected by shifts from normal to HFD. We found significant and reproducible shifts in the content of GI tract viromes after the transition to HFD. The differences observed in virome community membership and their associated gene content suggest that these altered viral communities are populated by viruses that are more virulent toward their host bacteria. Because HFD also are associated with significant shifts in GI tract bacterial communities, we believe that the shifts in the viral community may serve to drive the changes that occur in associated bacterial communities.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

The Virome of Cerebrospinal Fluid: Viruses Where We Once Thought There Were None.

Traditionally, medicine has held that some human body sites are sterile and that the introduction of microbes to these sites results in infections. This paradigm shifted significantly with the discovery of the human microbiome and acceptance of these commensal microbes living across the body. However, the central nervous system (CNS) is still believed by many to be sterile in healthy people. Using culture-independent methods, we examined the virome of cerebrospinal fluid (CSF) from a cohort of mostly healthy human subjects. We identified a community of DNA viruses, most of which were identified as bacteriophages. Compared to other human specimen types, CSF viromes were not ecologically distinct. There was a high alpha diversity cluster that included feces, saliva, and urine, and a low alpha diversity cluster that included CSF, body fluids, plasma, and breast milk. The high diversity cluster included specimens known to have many bacteria, while other specimens traditionally assumed to be sterile formed the low diversity cluster. There was an abundance of viruses shared among CSF, breast milk, plasma, and body fluids, while each generally shared less with urine, feces, and saliva. These shared viruses ranged across different virus families, indicating that similarities between these viromes represent more than just a single shared virus family. By identifying a virome in the CSF of mostly healthy individuals, it is now less likely that any human body site is devoid of microbes, which further highlights the need to decipher the role that viral communities may play in human health.

VIPdb, a genetic Variant Impact Predictor Database.

Genome sequencing identifies vast number of genetic variants. Predicting these variants' molecular and clinical effects is one of the preeminent challenges in human genetics. Accurate prediction of the impact of genetic variants improves our understanding of how genetic information is conveyed to molecular and cellular functions, and is an essential step towards precision medicine. Over one hundred tools/resources have been developed specifically for this purpose. We summarize these tools as well as their characteristics, in the genetic Variant Impact Predictor Database (VIPdb). This database will help researchers and clinicians explore appropriate tools, and inform the development of improved methods. VIPdb can be browsed and downloaded at https://genomeinterpretation.org/vipdb.

Shared and Distinct Features of Human Milk and Infant Stool Viromes.

Infants acquire many of their microbes from their mothers during the birth process. The acquisition of these microbes is believed to be critical in the development of the infant immune system. Bacteria also are transmitted to the infant through breastfeeding, and help to form the microbiome of the infant gastrointestinal (GI) tract; it is unknown whether viruses in human milk serve to establish an infant GI virome. We examined the virome contents of milk and infant stool in a cohort of mother-infant pairs to discern whether milk viruses colonize the infant GI tract. We observed greater viral alpha diversity in milk than in infant stool, similar to the trend we found for bacterial communities from both sites. When comparing beta diversity, viral communities were mostly distinguishable between milk and infant stool, but each was quite distinct from adult stool, urine, and salivary viromes. There were significant differences in viral families in the infant stool (abundant bacteriophages from the family Siphoviridae) compared to milk (abundant bacteriophages from the family Myoviridae), which may reflect significant differences in the bacterial families identified from both sites. Despite the differences in viral taxonomy, we identified a significant number of shared viruses in the milk and stool from all mother-infant pairs. Because of the significant proportion of bacteriophages transmitted in these mother-infant pairs, we believe the transmission of milk phages to the infant GI tract may help to shape the infant GI microbiome.

Draft Genome Sequence of an Enterococcus faecalis ATCC 19433 Siphovirus Isolated from Raw Domestic Sewage.

We previously isolated and characterized an Enterococcus faecalis ATCC 19433 siphovirus from raw domestic sewage as a viral indicator of human fecal pollution. Here, we report the draft genome sequence of this bacteriophage.

Transmission of viruses via our microbiomes.

BACKGROUND: Bacteria inhabiting the human body have important roles in a number of physiological processes and are known to be shared amongst genetically-related individuals. Far less is known about viruses inhabiting the human body, but their ecology suggests they may be shared between close contacts. RESULTS: Here, we report the ecology of viruses in the guts and mouths of a cohort and demonstrate that substantial numbers of gut and oral viruses were shared amongst genetically unrelated, cohabitating individuals. Most of these viruses were bacteriophages, and each individual had distinct oral and gut viral ecology from their housemates despite the fact that some of their bacteriophages were shared. The distribution of bacteriophages over time within households indicated that they were frequently transmitted between the microbiomes of household contacts. CONCLUSIONS: Because bacteriophages may shape human oral and gut bacterial ecology, their transmission to household contacts suggests they could have substantial roles in shaping the microbiota within a household.

Microbial diversity in individuals and their household contacts following typical antibiotic courses.

BACKGROUND: Antibiotics are a mainstay of treatment for bacterial infections worldwide, yet the effects of typical antibiotic prescriptions on human indigenous microbiota have not been thoroughly evaluated. We examined the effects of the two most commonly prescribed antibiotics (amoxicillin and azithromycin) in the USA to discern whether short-term antibiotic courses may have prolonged effects on human microbiota. RESULTS: We sampled the feces, saliva, and skin specimens from a cohort of unrelated, cohabitating individuals over 6 months. An individual in each household was given an antibiotic, and the other a placebo to discern antibiotic impacts on microbiota, as well as determine whether antibiotic use might reshape the microbiota of each household. We observed household-specific patterns of microbiota on each body surface, which persevered despite antibiotic perturbations. While the gut microbiota within an individual became more dissimilar over time, there was no evidence that the use of antibiotics accelerated this process when compared to household members. There was a significant change in microbiota diversity in the gut and mouth in response to antibiotics, but analogous patterns were not observed on the skin. Those who received 7 days of amoxicillin generally had greater reductions in diversity compared to those who received 3 days, in contrast to those who received azithromycin. CONCLUSIONS: As few as 3 days of treatment with the most commonly prescribed antibiotics can result in sustained reductions in microbiota diversity, which could have implications for the maintenance of human health and resilience to disease.

Chemostat culture systems support diverse bacteriophage communities from human feces.

BACKGROUND: Most human microbiota studies focus on bacteria inhabiting body surfaces, but these surfaces also are home to large populations of viruses. Many are bacteriophages, and their role in driving bacterial diversity is difficult to decipher without the use of in vitro ecosystems that can reproduce human microbial communities. RESULTS: We used chemostat culture systems known to harbor diverse fecal bacteria to decipher whether these cultures also are home to phage communities. We found that there are vast viral communities inhabiting these ecosystems, with estimated concentrations similar to those found in human feces. The viral communities are composed entirely of bacteriophages and likely contain both temperate and lytic phages based on their similarities to other known phages. We examined the cultured phage communities at five separate time points over 24 days and found that they were highly individual-specific, suggesting that much of the subject-specificity found in human viromes also is captured by this culture-based system. A high proportion of the community membership is conserved over time, but the cultured communities maintain more similarity with other intra-subject cultures than they do to human feces. In four of the five subjects, estimated viral diversity between fecal and cultured communities was highly similar. CONCLUSIONS: Because the diversity of phages in these cultured fecal communities have similarities to those found in humans, we believe these communities can serve as valuable ecosystems to help uncover the role of phages in human microbial communities.

Effects of Long Term Antibiotic Therapy on Human Oral and Fecal Viromes.

Viruses are integral members of the human microbiome. Many of the viruses comprising the human virome have been identified as bacteriophage, and little is known about how they respond to perturbations within the human ecosystem. The intimate association of phage with their cellular hosts suggests their communities may change in response to shifts in bacterial community membership. Alterations to human bacterial biota can result in human disease including a reduction in the host's resilience to pathogens. Here we report the ecology of oral and fecal viral communities and their responses to long-term antibiotic therapy in a cohort of human subjects. We found significant differences between the viral communities of each body site with a more heterogeneous fecal virus community compared with viruses in saliva. We measured the relative diversity of viruses, and found that the oral viromes were significantly more diverse than fecal viromes. There were characteristic changes in the membership of oral and fecal bacterial communities in response to antibiotics, but changes in fecal viral communities were less distinguishing. In the oral cavity, an abundance of papillomaviruses found in subjects on antibiotics suggests an association between antibiotics and papillomavirus production. Despite the abundance of papillomaviruses identified, in neither the oral nor the fecal viromes did antibiotic therapy have any significant impact upon overall viral diversity. There was, however, an apparent expansion of the reservoir of genes putatively involved in resistance to numerous classes of antibiotics in fecal viromes that was not paralleled in oral viromes. The emergence of antibiotic resistance in fecal viromes in response to long-term antibiotic therapy in humans suggests that viruses play an important role in the resilience of human microbial communities to antibiotic disturbances.

Transcriptome analysis of bacteriophage communities in periodontal health and disease.

BACKGROUND: The role of viruses as members of the human microbiome has gained broader attention with the discovery that human body surfaces are inhabited by sizeable viral communities. The majority of the viruses identified in these communities have been bacteriophages that predate upon cellular microbiota rather than the human host. Phages have the capacity to lyse their hosts or provide them with selective advantages through lysogenic conversion, which could help determine the structure of co-existing bacterial communities. Because conditions such as periodontitis are associated with altered bacterial biota, phage mediated perturbations of bacterial communities have been hypothesized to play a role in promoting periodontal disease. Oral phage communities also differ significantly between periodontal health and disease, but the gene expression of oral phage communities has not been previously examined. RESULTS: Here, we provide the first report of gene expression profiles from the oral bacteriophage community using RNA sequencing, and find that oral phages are more highly expressed in subjects with relative periodontal health. While lysins were highly expressed, the high proportion of integrases expressed suggests that prophages may account for a considerable proportion of oral phage gene expression. Many of the transcriptome reads matched phages found in the oral cavities of the subjects studied, indicating that phages may account for a substantial proportion of oral gene expression. Reads homologous to siphoviruses that infect Firmicutes were amongst the most prevalent transcriptome reads identified in both periodontal health and disease. Some genes from the phage lytic module were significantly more highly expressed in subjects with periodontal disease, suggesting that periodontitis may favor the expression of some lytic phages. CONCLUSIONS: As we explore the contributions of viruses to the human microbiome, the data presented here suggest varying expression of bacteriophage communities in oral health and disease.

Identification of staphylococcal phage with reduced transcription in human blood through transcriptome sequencing.

Many pathogenic bacteria have bacteriophage and other mobile genetic elements whose activity during human infections has not been evaluated. We investigated the gene expression patterns in human subjects with invasive Methicillin Resistant Staphylococcus aureus (MRSA) infections to determine the gene expression of bacteriophage and other mobile genetic elements. We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation. We compared ex vivo to in vitro profiles from 10 human subjects to determine MRSA gene expression in blood. Using RNA sequencing, we found that there were distinct and significant differences between ex vivo and in vitro MRSA gene expression profiles. Among the major differences between ex vivo and in vitro gene expression were virulence/disease/defense and mobile elements. While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression. Five subjects had MRSA with bacteriophage that were inhibited by the presence of blood in the media, supporting that the lysogeny state was preferred in human blood. Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood. By comparing the gene expression cultured in media with and without the blood of patients, we gain insights into the specific adaptations made by MRSA and its bacteriophage to life in the human bloodstream.

Global transcription of CRISPR loci in the human oral cavity.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are active in acquired resistance against bacteriophage and plasmids in a number of environments. In the human mouth, CRISPR loci evolve to counteract oral phage, but the expression of these CRISPR loci has not previously been investigated. We sequenced cDNA from CRISPR loci found in numerous different oral bacteria and compared with oral phage communities to determine whether the transcription of CRISPR loci is specifically targeted towards highly abundant phage present in the oral environment. RESULTS: We found that of the 529,027 CRISPR spacer groups studied, 88 % could be identified in transcripts, indicating that the vast majority of CRISPR loci in the oral cavity were transcribed. There were no strong associations between CRISPR spacer repertoires and oral health status or nucleic acid type. We also compared CRISPR repertoires with oral bacteriophage communities, and found that there was no significant association between CRISPR transcripts and oral phage, regardless of the CRISPR type being evaluated. We characterized highly expressed CRISPR spacers and found that they were no more likely than other spacers to match oral phage. By reassembling the CRISPR-bearing reads into longer CRISPR loci, we found that the majority of the loci did not have spacers matching viruses found in the oral cavities of the subjects studied. For some CRISPR types, loci containing spacers matching oral phage were significantly more likely to have multiple spacers rather than a single spacer matching oral phage. CONCLUSIONS: These data suggest that the transcription of oral CRISPR loci is relatively ubiquitous and that highly expressed CRISPR spacers do not necessarily target the most abundant oral phage.

The human urine virome in association with urinary tract infections.

While once believed to represent a sterile environment, the human urinary tract harbors a unique cellular microbiota. We sought to determine whether the human urinary tract also is home to viral communities whose membership might reflect urinary tract health status. We recruited and sampled urine from 20 subjects, 10 subjects with urinary tract infections (UTIs) and 10 without UTIs, and found viral communities in the urine of each subject group. Most of the identifiable viruses were bacteriophage, but eukaryotic viruses also were identified in all subjects. We found reads from human papillomaviruses (HPVs) in 95% of the subjects studied, but none were found to be high-risk genotypes that are associated with cervical and rectal cancers. We verified the presence of some HPV genotypes by quantitative PCR. Some of the HPV genotypes identified were homologous to relatively novel and uncharacterized viruses that previously have been detected on skin in association with cancerous lesions, while others may be associated with anal and genital warts. On a community level, there was no association between the membership or diversity of viral communities based on urinary tract health status. While more data are still needed, detection of HPVs as members of the human urinary virome using viral metagenomics represents a non-invasive technique that could augment current screening techniques to detect low-risk HPVs in the genitourinary tracts of humans.

Conservation of streptococcal CRISPRs on human skin and saliva.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are utilized by bacteria to resist encounters with their viruses. Human body surfaces have numerous bacteria that harbor CRISPRs, and their content can provide clues as to the types and features of viruses they may have encountered. RESULTS: We investigated the conservation of CRISPR content from streptococci on skin and saliva of human subjects over 8-weeks to determine whether similarities existed in the CRISPR spacer profiles and whether CRISPR spacers were a stable component of each biogeographic site. Most of the CRISPR sequences identified were unique, but a small proportion of spacers from the skin and saliva of each subject matched spacers derived from previously sequenced loci of S. thermophilus and other streptococci. There were significant proportions of CRISPR spacers conserved over the entire 8-week study period for all subjects, and salivary CRISPR spacers sampled in the mornings showed significantly higher levels of conservation than any other time of day. We also found substantial similarities in the spacer repertoires of the skin and saliva of each subject. Many skin-derived spacers matched salivary viruses, supporting that bacteria of the skin may encounter viruses with similar sequences to those found in the mouth. Despite the similarities between skin and salivary spacer repertoires, the variation present was distinct based on each subject and body site. CONCLUSIONS: The conservation of CRISPR spacers in the saliva and the skin of human subjects over the time period studied suggests a relative conservation of the bacteria harboring them.