Multi-omics Analysis of Periodontal Pocket Microbial Communities Pre- and Posttreatment.

Periodontitis is a polymicrobial infectious disease that causes breakdown of the periodontal ligament and alveolar bone. We employed a meta-omics approach that included microbial 16S rRNA amplicon sequencing, shotgun metagenomics, and tandem mass spectrometry to analyze sub- and supragingival biofilms in adults with chronic periodontitis pre- and posttreatment with 0.25% sodium hypochlorite. Microbial samples were collected with periodontal curettes from 3- to 12-mm-deep periodontal pockets at the baseline and at 2 weeks and 3 months. All data types showed high interpersonal variability, and there was a significant correlation between phylogenetic diversity and pocket depth at the baseline and a strong correlation (rho = 0.21; P = 0.008) between metabolite diversity and maximum pocket depth (MPD). Analysis of subgingival baseline samples (16S rRNA and shotgun metagenomics) found positive correlations between abundances of particular bacterial genera and MPD, including Porphyromonas, Treponema, Tannerella, and Desulfovibrio species and unknown taxon SHD-231. At 2 weeks posttreatment, we observed an almost complete turnover in the bacterial genera (16S rRNA) and species (shotgun metagenomics) correlated with MPD. Among the metabolites detected, the medians of the 20 most abundant metabolites were significantly correlated with MPD pre- and posttreatment. Finally, tests of periodontal biofilm community instability found markedly higher taxonomic instability in patients who did not improve posttreatment than in patients who did improve (UniFrac distances; t = -3.59; P = 0.002). Interestingly, the opposite pattern occurred in the metabolic profiles (Bray-Curtis; t = 2.42; P = 0.02). Our results suggested that multi-omics approaches, and metabolomics analysis in particular, could enhance treatment prediction and reveal patients most likely to improve posttreatment. IMPORTANCE Periodontal disease affects the majority of adults worldwide and has been linked to numerous systemic diseases. Despite decades of research, the reasons for the substantial differences among periodontitis patients in disease incidence, progressivity, and response to treatment remain poorly understood. While deep sequencing of oral bacterial communities has greatly expanded our comprehension of the microbial diversity of periodontal disease and identified associations with healthy and disease states, predicting treatment outcomes remains elusive. Our results suggest that combining multiple omics approaches enhances the ability to differentiate among disease states and determine differential effects of treatment, particularly with the addition of metabolomic information. Furthermore, multi-omics analysis of biofilm community instability indicated that these approaches provide new tools for investigating the ecological dynamics underlying the progressive periodontal disease process.

Significant Impacts of Increasing Aridity on the Arid Soil Microbiome.

Global deserts occupy one-third of the Earth's surface and contribute significantly to organic carbon storage, a process at risk in dryland ecosystems that are highly vulnerable to climate-driven ecosystem degradation. The forces controlling desert ecosystem degradation rates are poorly understood, particularly with respect to the relevance of the arid-soil microbiome. Here we document correlations between increasing aridity and soil bacterial and archaeal microbiome composition along arid to hyperarid transects traversing the Atacama Desert, Chile. A meta-analysis reveals that Atacama soil microbiomes exhibit a gradient in composition, are distinct from a broad cross-section of nondesert soils, and yet are similar to three deserts from different continents. Community richness and diversity were significantly positively correlated with soil relative humidity (SoilRH). Phylogenetic composition was strongly correlated with SoilRH, temperature, and electrical conductivity. The strongest and most significant correlations between SoilRH and phylum relative abundance were observed for Acidobacteria, Proteobacteria, Planctomycetes, Verrucomicrobia, and Euryarchaeota (Spearman's rank correlation [rs] = >0.81; false-discovery rate [q] = ≤0.005), characterized by 10- to 300-fold decreases in the relative abundance of each taxon. In addition, network analysis revealed a deterioration in the density of significant associations between taxa along the arid to hyperarid gradient, a pattern that may compromise the resilience of hyperarid communities because they lack properties associated with communities that are more integrated. In summary, results suggest that arid-soil microbiome stability is sensitive to aridity as demonstrated by decreased community connectivity associated with the transition from the arid class to the hyperarid class and the significant correlations observed between soilRH and both diversity and the relative abundances of key microbial phyla typically dominant in global soils. IMPORTANCE We identify key environmental and geochemical factors that shape the arid soil microbiome along aridity and vegetation gradients spanning over 300 km of the Atacama Desert, Chile. Decreasing average soil relative humidity and increasing temperature explain significant reductions in the diversity and connectivity of these desert soil microbial communities and lead to significant reductions in the abundance of key taxa typically associated with fertile soils. This finding is important because it suggests that predicted climate change-driven increases in aridity may compromise the capacity of the arid-soil microbiome to sustain necessary nutrient cycling and carbon sequestration functions as well as vegetative cover in desert ecosystems, which comprise one-third of the terrestrial biomes on Earth.

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history.

Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague-related die-offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67-0.87) in all colonies. Two other DRB1 alleles appear to be trans-species polymorphisms shared with the black-tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F ST = 0.033) than at microsatellite markers (F ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.

Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence.

Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.

Redefining the differences in gene content between Yersinia pestis and Yersinia pseudotuberculosis using large-scale comparative genomics.

Yersinia pestis, the causative agent of plague, is best known for historical pandemics, but still actively causes disease in many parts of the world. Y. pestis is a recently derived clone of the pathogenic species Yersinia pseudotuberculosis, but is more associated with human infection. Numerous studies have documented genomic changes since the two species differentiated, although all of these studies used a relatively small sample set for defining these differences. In this study, we compared the complete genomic content between a diverse set of Y. pestis and Y. pseudotuberculosis genomes, and identified unique loci that could serve as diagnostic markers or for better understanding the evolution and pathogenesis of each group. Comparative genomics analyses also identified subtle variations in gene content between individual monophyletic clades within these species, based on a core genome single nucleotide polymorphism phylogeny that would have been undetected in a less comprehensive genome dataset. We also screened loci that were identified in other published studies as unique to either species and generally found a non-uniform distribution, suggesting that the assignment of these unique genes to either species should be re-evaluated in the context of current sequencing efforts. Overall, this study provides a high-resolution view into the genomic differences between Y. pestis and Y. pseudotuberculosis, demonstrating fine-scale differentiation and unique gene composition in both species.

Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere.

Hematodinium is a genus of parasitic dinoflagellates that infects crustaceans worldwide including Tanner crabs Chionoecetes bairdi and snow crabs C. opilio in the Northeast Pacific Ocean. The present study describes the optimization of a PCR-based assay for the detection and monitoring of Hematodinium sp. in snow and Tanner crabs. Two fragments, 1682 and 187 bp, were amplified from the 18S ribosomal DNA region of the parasite. The assay performed well in 6 additional decapod species (1 lobster and 5 crabs) infected with Hematodinium spp., suggesting that it could be used to detect Hematodinium spp. in other decapods. We also report Hematodinium spp. infections in the majid crab, Hyas coarctatus, and the lithodid crab, Lithodes couesi. Sequencing of 18S rDNA and the adjacent internal transcribed spacer 1 (ITS1) region of Hematodinium spp. isolated from 7 host species in the present study revealed the presence of 2 Hematodinium clades, one in the blue crab Callinectes sapidus and a second in all other host species. The ITS1 sequences of the 2 clades could not be aligned, but showed a conserved secondary structure that may be related to a functional diversification during a host switch. Comparison of our data with 18S and ITS1 sequence data available in GenBank placed the north Pacific Hematodinium sp. in a clade separate from the Hematodinium sp. infecting the portunoids, C. sapidus, Liocarcinus depurator and Scylla serrata, and within a second clade that infected all other decapod hosts located in the North Pacific and North Atlantic Oceans.