Linda D. Strausbaugh

The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

Routine use of the Ion Torrent AmpliSeq™ Cancer Hotspot Panel for identification of clinically actionable somatic mutations

Abstract Background: Somatic mutation analysis is standard of practice for solid tumors in order to identify therapeutic sensitizing and resistance mutations. Our laboratory routinely performed standalone PCR-based methods for mutations in several genes. Rapid discovery and introduction of new therapeutics has demanded additional genomic information for adequate management of the cancer patient. We evaluated a next generation sequencing assay, the Ion Torrent AmpliSeq Cancer Hotspot Panelv2 (CHPv2), capable of identifying multiple somatic mutations in 50 genes in a single assay. Methods: Accuracy, precision, limit of detection, and specificity were evaluated using DNA from well-characterized cell lines, genetically engineered cell lines fixed and embedded in paraffin, and previously tested mutation positive or negative, formalin-fixed, paraffin-embedded (FFPE) tissues. Normal kidney, tonsil and colon FFPE tissues were used as controls. Results: Accuracy studies showed 100% concordance in each patient sample between previous PCR results and the corresponding variants identified using the Ion Torrent panel. Precision studies gave consistent results when libraries were prepared from the same original DNA and were run on multiple 316 chips. The limit of detection was determined to be 5% for single nucleotide variants (SNVs) and 20% for insertions and deletions (indels). Specificity studies using normal FFPE tissue previously tested by PCR methods were also 100%. Conclusions: We have evaluated the performance of the AmpliSeq Cancer Panel Hotspotv2 and show that it is suitable for clinical testing. This next generation sequencing panel has allowed the laboratory to consolidate a broader range of molecular oncology testing to a single platform and single assay.

Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of Malassezia as a Prominent Commensal

Fungi are a large, complex group, increasingly recognized as emerging threats. Their roles as modifiers of health mandate accurate portrayals of fungal communities in humans. As an entry point into the airways and gastrointestinal tract, fungi in the mouth are relevant to several biocompartments. We have revised current practices in sequence-based taxonomy assignments and employed the improvements to address the question of the fungal genera present in the healthy human mouth. The human oral mycobiome was surveyed using massively parallel, high throughput sequencing of internal transcribed spacer 1 (ITS1) amplicons from saliva following robust extraction methods. Taxonomy was assigned by comparison to a curated reference dataset, followed by filtering with an empirically determined BLAST E-value match statistic (10−42). Nomenclature corrections further refined results by conjoining redundant names for a single fungal genus. Following these curation steps, about two-thirds of the initially identified genera were eliminated. In comparison with the one similar metagenomic study and several earlier culture-based ones, our findings change the current conception of the oral mycobiome, especially with the discovery of the high prevalence and abundance of the genus Malassezia. Previously identified as an important pathogen of the skin, and recently reported as the predominant fungal genus at the nostril and backs of the head and ear, this is the first account of Malassezia in the human mouth. Findings from this study were in good agreement with others on the existence of many consensus members of the core mycobiome, and on unique patterns for individual subjects. This research offered a cautionary note about unconditional acceptance of lengthy lists of community members produced by automated assignments, provided a roadmap for enhancing the likely biological relevance of sequence-based fungal surveys, and built the foundation for understanding the role of fungi in health and disease of the oral cavity.

The distribution of P element sequences in Drosophila: the willistoni and saltans species groups

SummaryThis report describes the distribution of P-element sequences among members of the closely relatedwillistoni andsaltans species groups of the subgenusSophophora. Gel-blotting analyses showed that many, but not all, species from each of these groups possess sequences with homology to the P transposable element ofDrosophila melanogaster, a sophophoran species belonging to themelanogaster species group. Furthermore, P-homologous fragments are present in lower numbers inwillistoni- andsaltans-group species than inD. melanogaster P strains, and, in some species of those two groups, exhibit species-characteristic hybridization patterns. On the basis of these results, it is proposed that P elements have had a long evolutionary history in thewillistoni andsaltans lineages.

Using high throughput sequencing to explore the biodiversity in oral bacterial communities

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.

On the origins of tandemly repeated genes: Does histone gene copy number in Drosophila reflect chromosomal location?

Widely regarded beliefs aboutDrosophila histone gene copy numbers and developmental requirements have been generalized from fairly limited data since studies on histone gene arrangements and copy numbers have been largely confined to a single species,D. melanogaster. Histone gene copy numbers and chromosomal locations were examined in three species:D. melanogaster, D. hydei andD. hawaiiensis. Quantitative whole genome blot analysis of DNA from diploid tissues revealed a tenfold variability in histone gene copy numbers for these three species. In situ hybridization to polytene chromosomes showed that the histone DNA (hDNA) chromosomal location is different in all three species. These observations lead us to propose a relationship between histone gene reiteration and chromosomal position.

Fungal-bacterial interactions and their relevance to oral health: linking the clinic and the bench

High throughput sequencing has accelerated knowledge on the oral microbiome. While the bacterial component of oral communities has been extensively characterized, the role of the fungal microbiota in the oral cavity is largely unknown. Interactions among fungi and bacteria are likely to influence oral health as exemplified by the synergistic relationship between Candida albicans and oral streptococci. In this perspective, we discuss the current state of the field of fungal-bacterial interactions in the context of the oral cavity. We highlight the need to conduct longitudinal clinical studies to simultaneously characterize the bacterial and fungal components of the human oral microbiome in health and during disease progression. Such studies need to be coupled with investigations using disease-relevant models to mechanistically test the associations observed in humans and eventually identify fungal-bacterial interactions that could serve as preventive or therapeutic targets for oral diseases.

Transplantation-Associated Long-Term Immunosuppression Promotes Oral Colonization by Potentially Opportunistic Pathogens without Impacting Other Members of the Salivary Bacteriome

ABSTRACT Solid-organ transplant recipients rely on pharmacological immunosuppression to prevent allograft rejection. The effect of such chronic immunosuppression on the microflora at mucosal surfaces is not known. We evaluated the salivary bacterial microbiome of 20 transplant recipients and 19 nonimmunosuppressed controls via 454 pyrosequencing of 16S rRNA gene amplicons. Alpha-diversity and global community structure did not differ between transplant and control subjects. However, principal coordinate analysis showed differences in community membership. Taxa more prevalent in transplant subjects included operational taxonomic units (OTUs) of potentially opportunistic Gammaproteobacteria such as Klebsiella pneumoniae, Pseudomonas fluorescens, Acinetobacter species, Vibrio species, Enterobacteriaceae species, and the genera Acinetobacter and Klebsiella. Transplant subjects also had increased proportions of Pseudomonas aeruginosa, Acinetobacter species, Enterobacteriaceae species, and Enterococcus faecalis, among other OTUs, while genera with increased proportions included Klebsiella, Acinetobacter, Staphylococcus, and Enterococcus. Furthermore, in transplant subjects, the dose of the immunosuppressant prednisone positively correlated with bacterial richness, while prednisone and mycophenolate mofetil doses positively correlated with the prevalence and proportions of transplant-associated taxa. Correlation network analysis of OTU relative abundance revealed a cluster containing potentially opportunistic pathogens as transplant associated. This cluster positively correlated with serum levels of C-reactive protein, suggesting a link between the resident flora at mucosal compartments and systemic inflammation. Network connectivity analysis revealed opportunistic pathogens as highly connected to each other and to common oral commensals, pointing to bacterial interactions that may influence colonization. This work demonstrates that immunosuppression aimed at limiting T-cell-mediated responses creates a more permissive oral environment for potentially opportunistic pathogens without affecting other members of the salivary bacteriome.

Microbiome Profiles in Periodontitis in Relation to Host and Disease Characteristics

Periodontitis is an inflammatory condition that affects the supporting tissues surrounding teeth. The occurrence of periodontitis is associated with shifts in the structure of the communities that inhabit the gingival sulcus. Although great inter-subject variability in the subgingival microbiome has been observed in subjects with periodontitis, it is unclear whether distinct community types exist and if differences in microbial signatures correlate with host characteristics or with the variable clinical presentations of periodontitis. Therefore, in this study we explored the existence of different community types in periodontitis and their relationship with host demographic, medical and disease-related clinical characteristics. Clustering analyses of microbial abundance profiles suggested two types of communities (A and B) existed in the 34 subjects with periodontitis evaluated. Type B communities harbored greater proportions of certain periodontitis-associated taxa, including species historically associated with the disease, such as Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, and taxa recently linked to periodontitis. In contrast, subjects with type A communities had increased proportions of different periodontitis-associated species, and were also enriched for health-associated species and core taxa (those equally prevalent in health and periodontitis). Periodontitis subgingival clusters were not associated with demographic, medical or disease-specific clinical parameters other than periodontitis extent (proportion of sites affected), which positively correlated with the total proportion of cluster B signature taxa. In conclusion, two types of microbial communities were detected in subjects with periodontitis. Host demographics and underlying medical conditions did not correlate with these profiles, which instead appeared to be related to periodontitis extent, with type B communities present in more widespread disease cases. The two identified periodontitis profiles may represent distinct dysbiotic processes potentially requiring community-tailored therapeutic interventions.

Molecular analysis of P element behavior in Drosophila simulans transformants.

SummaryIn this report we describe the successful transformation of Drosophila simulans with an autonomous P element from Drosophila melanogaster without the use of a selectable marker. This result demonstrates that there is no species barrier for P element transposition. Utilizing gel blotting and in situ hybridization techniques, we have monitored the behavior of newly-introduced P elements in several D. simulans transformed lines over twelve generations. In most instances, an overall increase in the number of P elements was observed. An examination of the frequency of P-element-bearing individuals in one line revealed the rapid spread of P elements through the population. Analysis of well-characterized sublines confirmed that P elements increase in number by transposition to new genomic sites. The formation of degenerate elements occurred in at least one case. These observations suggest that P elements may behave similarly in D. melanogaster and D. simulans.