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.

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.

Characterization of bacteriophage communities and CRISPR profiles from dental plaque.

BACKGROUND: Dental plaque is home to a diverse and complex community of bacteria, but has generally been believed to be inhabited by relatively few viruses. We sampled the saliva and dental plaque from 4 healthy human subjects to determine whether plaque was populated by viral communities, and whether there were differences in viral communities specific to subject or sample type. RESULTS: We found that the plaque was inhabited by a community of bacteriophage whose membership was mostly subject-specific. There was a significant proportion of viral homologues shared between plaque and salivary viromes within each subject, suggesting that some oral viruses were present in both sites. We also characterized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in oral streptococci, as their profiles provide clues to the viruses that oral bacteria may be able to counteract. While there were some CRISPR spacers specific to each sample type, many more were shared across sites and were highly subject specific. Many CRISPR spacers matched viruses present in plaque, suggesting that the evolution of CRISPR loci may have been specific to plaque-derived viruses. CONCLUSIONS: Our findings of subject specificity to both plaque-derived viruses and CRISPR profiles suggest that human viral ecology may be highly personalized.

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.

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.

Altered oral viral ecology in association with periodontal disease.

UNLABELLED: The human oral cavity is home to a large and diverse community of viruses that have yet to be characterized in patients with periodontal disease. We recruited and sampled saliva and oral biofilm from a cohort of humans either periodontally healthy or with mild or significant periodontal disease to discern whether there are differences in viral communities that reflect their oral health status. We found communities of viruses inhabiting saliva and the subgingival and supragingival biofilms of each subject that were composed largely of bacteriophage. While there were homologous viruses common to different subjects and biogeographic sites, for most of the subjects, virome compositions were significantly associated with the oral sites from which they were derived. The largest distinctions between virome compositions were found when comparing the subgingival and supragingival biofilms to those of planktonic saliva. Differences in virome composition were significantly associated with oral health status for both subgingival and supragingival biofilm viruses but not for salivary viruses. Among the differences identified in virome compositions was a significant expansion of myoviruses in subgingival biofilm, suggesting that periodontal disease favors lytic phage. We also characterized the bacterial communities in each subject at each biogeographic site by using the V3 hypervariable segment of the 16S rRNA and did not identify distinctions between oral health and disease similar to those found in viral communities. The significantly altered ecology of viruses of oral biofilm in subjects with periodontal disease compared to that of relatively periodontally healthy ones suggests that viruses may serve as useful indicators of oral health status. IMPORTANCE: Little is known about the role or the constituents of viruses as members of the human microbiome. We investigated the composition of human oral viral communities in a group of relatively periodontally healthy subjects or significant periodontitis to determine whether health status may be associated with differences in viruses. We found that most of the viruses present were predators of bacteria. The viruses inhabiting dental plaque were significantly different on the basis of oral health status, while those present in saliva were not. Dental plaque viruses in periodontitis were predicted to be significantly more likely to kill their bacterial hosts than those found in healthy mouths. Because oral diseases such as periodontitis have been shown to have altered bacterial communities, we believe that viruses and their role as drivers of ecosystem diversity are important contributors to the human oral microbiome in health and disease states.

Association between living environment and human oral viral ecology.

The human oral cavity has an indigenous microbiota known to include a robust community of viruses. Very little is known about how oral viruses are spread throughout the environment or to which viruses individuals are exposed. We sought to determine whether shared living environment is associated with the composition of human oral viral communities by examining the saliva of 21 human subjects; 11 subjects from different households and 10 unrelated subjects comprising 4 separate households. Although there were many viral homologues shared among all subjects studied, there were significant patterns of shared homologues in three of the four households that suggest shared living environment affects viral community composition. We also examined CRISPR (clustered regularly interspaced short palindromic repeat) loci, which are involved in acquired bacterial and archaeal resistance against invading viruses by acquiring short viral sequences. We analyzed 2 065 246 CRISPR spacers from 5 separate repeat motifs found in oral bacterial species of Gemella, Veillonella, Leptotrichia and Streptococcus to determine whether individuals from shared living environments may have been exposed to similar viruses. A significant proportion of CRISPR spacers were shared within subjects from the same households, suggesting either shared ancestry of their oral microbiota or similar viral exposures. Many CRISPR spacers matched virome sequences from different subjects, but no pattern specific to any household was found. Our data on viromes and CRISPR content indicate that shared living environment may have a significant role in determining the ecology of human oral viruses.