Biogeography of the Oral Microbiome: The Site-Specialist Hypothesis.

Microbial communities are complex and dynamic, composed of hundreds of taxa interacting across multiple spatial scales. Advances in sequencing and imaging technology have led to great strides in understanding both the composition and the spatial organization of these complex communities. In the human mouth, sequencing results indicate that distinct sites host microbial communities that not only are distinguishable but to a meaningful degree are composed of entirely different microbes. Imaging suggests that the spatial organization of these communities is also distinct. Together, the literature supports the idea that most oral microbes are site specialists. A clear understanding of microbiota structure at different sites in the mouth enables mechanistic studies, informs the generation of hypotheses, and strengthens the position of oral microbiology as a model system for microbial ecology in general.

A patient journey audit tool (PJAT) to assess quality indicators in a nuclear medicine service.

PURPOSE: To develop a nuclear medicine specific patient journey audit tool (PJAT) to survey and audit patient journeys in a nuclear medicine department such as staff interaction with patients, equipment, quality of imaging and laboratory procedures, patient protection, infection control and radiation safety, with a view to optimising patient care and providing a high-quality nuclear medicine service. METHODS: The PJAT was developed specifically for use in nuclear medicine practices. Thirty-two questions were formulated in the PJAT to test the department's compliance to the Australian National Safety and Quality Health Service Standards, namely clinical governance, partnering with consumers, preventing and controlling health care infection, medication safety, comprehensive care, communicating for safety, blood management and recognising and responding to acute deterioration. The PJAT was also designed to test our department's adherence to diagnostic reference levels (DRL). A total of 60 patient journey audits were completed for patients presenting for nuclear medicine, positron emission tomography and bone mineral density procedures during a consecutive 4-week period to audit the range of procedures performed. A further 120 audits were captured for common procedures in nuclear medicine and positron emission tomography during the same period. Thus, a total of 180 audits were completed. A subset of 12 patients who presented for blood labelling procedures were audited to solely assess the blood management standard. RESULTS: The audits demonstrated over 85% compliance for the Australian national health standards. One hundred percent compliance was noted for critical aspects such as correct patient identification for the correct procedure prior to radiopharmaceutical administration, adherence to prescribed dose limits and distribution of the report within 24 h of completion of the imaging procedure. CONCLUSION: This PJAT can be applied in nuclear medicine departments to enhance quality programmes and patient care. Austin Health has collaborated with the IAEA to formulate the IAEA PJAT, which is now available globally for nuclear medicine departments to survey patient journeys.

Outcomes and Complications of Revision Reverse Shoulder Arthroplasty Following Failed Primary Anatomical Shoulder Arthroplasty or Hemiarthroplasty: A Systematic Review.

BACKGROUND: The purpose of this study is to systematically review the evidence in the literature to ascertain the functional outcomes, range of motion (ROM) and complication and reoperation rates following revision reverse shoulder arthroplasty (RSA) for a failed primary total shoulder arthroplasty (TSA) or hemiarthroplasty (HA). METHODS: Two independent reviewers performed the literature search based on PRISMA guidelines, utilizing the EMBASE, MEDLINE, and The Cochrane Library Databases. Studies were included if they reported clinical outcomes for revision RSA for a failed primary TSA or HA. RESULTS: Our review found 23 studies including 1,041 shoulders (627 TSA, 414 HA) meeting our inclusion criteria. The majority of patients were female (66.1%), with an average age of 69.0 years (range 39-93) with a mean follow-up of 46.3 months. ASES and VAS pain scores improved from 32.6 to 61.9 and 6.7 to 2.7, respectively. ROM results including forward flexion, abduction and external rotation that improved from 59.4° to 107.7°, 50.7° to 104.4°, and 19.8° to 26.3° respectively. Only one out of the ten studies reporting internal rotation found a statistically significant difference with mean internal rotation improving from S1-S3 preoperatively to L4-L5 postoperatively for patients undergoing HA. The overall complication rate and reoperation rate were 23.4% and 12.5% respectively. The most common complications were glenoid component loosening (6.0%), fracture (periprosthetic, intraoperative, or other scapula fractures) (n=4.7%), and infection (n=3.3%). CONCLUSION: Revision RSA for a failed primary TSA and HA has been shown to result in excellent functional outcomes and improved ROM suggesting patients who have failed TSA or HA may benefit from a revision RSA.

Prodromal dementia with Lewy bodies in REM sleep behavior disorder: A multicenter study.

INTRODUCTION: Isolated/idiopathic rapid eye movement sleep behavior disorder (iRBD) is a powerful early predictor of dementia with Lewy bodies (DLB) and Parkinson's disease (PD). This provides an opportunity to directly observe the evolution of prodromal DLB and to identify which cognitive variables are the strongest predictors of evolving dementia. METHODS: IRBD participants (n = 754) from 10 centers of the International RBD Study Group underwent annual neuropsychological assessment. Competing risk regression analysis determined optimal predictors of dementia. Linear mixed-effect models determined the annual progression of neuropsychological testing. RESULTS: Reduced attention and executive function, particularly performance on the Trail Making Test Part B, were the strongest identifiers of early DLB. In phenoconverters, the onset of cognitive decline began up to 10 years prior to phenoconversion. Changes in verbal memory best differentiated between DLB and PD subtypes. DISCUSSION: In iRBD, attention and executive dysfunction strongly predict dementia and begin declining several years prior to phenoconversion. HIGHLIGHTS: Cognitive decline in iRBD begins up to 10 years prior to phenoconversion. Attention and executive dysfunction are the strongest predictors of dementia in iRBD. Decline in episodic memory best distinguished dementia-first from parkinsonism-first phenoconversion.

The Use and Downstream Associations of Magnetic Resonance Imaging for Lateral Epicondylitis.

PURPOSE: Low-value imaging is associated with wasteful health care spending and patient harm. The routine use of magnetic resonance imaging (MRI) for the work-up of lateral epicondylitis is an example of low-value imaging. As such, our aim was to investigate the use of MRIs ordered for lateral epicondylitis, the characteristics of those undergoing an MRI, and the downstream associations of MRI with other care. METHODS: We identified patients aged ≥18 years with a diagnosis of lateral epicondylitis between 2010 and 2019 using a Humana claims database. We identified patients with a Current Procedural Terminology code corresponding to an elbow MRI. We analyzed the use and downstream treatment cascades in those undergoing MRI. Multivariable logistic regression models were used to assess the odds of undergoing an MRI, adjusting for age, sex, insurance type, and comorbidity index. Separate multivariable logistic regression analyses were used to determine the association between undergoing an MRI and the incidence of secondary outcomes (eg, receiving surgery). RESULTS: A total of 624,102 patients met the inclusion criteria. Of 8,209 (1.3%) patients undergoing MRI, 3,584 (44%) underwent it within 90 days after diagnosis. There was notable regional variation in MRI use. The MRIs were ordered most frequently by primary care specialties and for younger, female, commercially insured, and patients with more comorbidities. Performance of an MRI was associated with an increase in downstream treatments, including surgery (odds ratio [OR], 9.58 [9.12-10.07]), injection (OR, 2.90 [2.77-3.04]), therapy (OR, 1.81 [1.72-1.91]), and cost ($134 per patient). CONCLUSIONS: Although there is variation in the use of MRI for lateral epicondylitis and its use is associated with downstream effects, the routine use of MRI for the diagnosis of lateral epicondylitis is low. CLINICAL RELEVANCE: The routine use of MRI for lateral epicondylitis is low. Understanding interventions to minimize such low-value care in lateral epicondylitis can be used to inform improvement efforts to minimize low-value care for other conditions.

Subscapularis repair for reverse shoulder arthroplasty: a systematic review and meta-analysis.

BACKGROUND: The purpose of this study is to perform a systematic review of the literature evaluating the importance of subscapularis repair in patients who underwent reverse shoulder arthroplasty (RSA). METHODS: A systematic search of articles in PubMed, EMBASE, and the Cochrane Library databases was carried out according to the PRISMA guidelines. Cohort studies comparing RSA with subscapularis repair vs. RSA without subscapularis repair were included. All statistical analysis was performed using Review Manager. A P value of <.05 was considered to be statistically significant. RESULTS: Seventeen studies with 2620 patients were included. Subscapularis repair resulted in less instability compared to without subscapularis repair (0.8% vs. 4.2%, P = .04), and there were no significant differences in rate of instability with lateralization (0.6% vs. 1.6%, P = .40), revision rates (2.6% vs. 3.9%, P = .62), and complication rates (7.7% vs. 4.9%, P = .21). Subscapularis repair had improved American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES) scores (83.6 vs. 80.2, P = .02) and Constant scores (72.6 vs. 68.9, P < .01) but there was not a significant difference in visual analog scale pain scores (1.2 vs. 1.6, P = .11). Subscapularis repair had a significant difference in forward flexion (140° vs. 137°, P = .04) and internal rotation score (5.5 vs. 4.6, P = .001); however, there was no significant difference in external rotation (35° vs. 35°, P = .80) and abduction (117° vs. 123°, P = .13). CONCLUSION: This study found that RSA with subscapularis repair demonstrated a reduction in the occurrence of implant instability with medialized implants. However, subscapularis repair did not yield improvements in revision rates, complications, or instability with lateralized implants. Additionally, other outcome measures such as ASES, Constant score, and range of motion exhibited statistically significant improvements with subscapularis repair but did not surpass clinically significant thresholds.

Is Low-value Testing Before Low-risk Hand Surgery Associated With Increased Downstream Healthcare Use and Reimbursements? A National Claims Database Analysis.

BACKGROUND: Minor hand procedures can often be completed in the office without any laboratory testing. Preoperative screening tests before minor hand procedures are unnecessary and considered low value because they can lead to preventable invasive confirmatory tests and/or procedures. Prior studies have shown that low-value testing before low-risk hand surgery is still common, yet little is known about their downstream effects and associated costs. Assessing these downstream events can elucidate the consequences of obtaining a low-value test and inform context-specific interventions to reduce their use. QUESTIONS/PURPOSES: (1) Among healthy adults undergoing low-risk hand surgery, are patients who receive a preoperative low-value test more likely to have subsequent diagnostic tests and procedures than those who do not receive a low-value test? (2) What is the increased 90-day reimbursement associated with subsequent diagnostic tests and procedures in patients who received a low-value test compared with those who did not? METHODS: In this retrospective, comparative study using a large national database, we queried a large health insurance provider's administrative claims data to identify adult patients undergoing low-risk hand surgery (carpal tunnel release, trigger finger release, Dupuytren fasciectomy, de Quervain release, thumb carpometacarpal arthroplasty, wrist ganglion cyst, or mass excision) between 2011 and 2017. This database was selected for its ability to track patient claims longitudinally with direct provision of reimbursement data in a large, geographically diverse patient population. Patients who received at least one preoperative low-value test, including complete blood count, basic metabolic panel, electrocardiogram, chest radiography, pulmonary function test, and urinalysis within the 30-day preoperative period, were matched with propensity scores to those who did not. Among the 73,112 patients who met our inclusion criteria (mean age 57 ± 14 years; 68% [49,847] were women), 27% (19,453) received at least one preoperative low-value test and were propensity score-matched to those who did not. Multivariable regression analyses were performed to assess the frequency and reimbursements of subsequent diagnostic tests and procedures in the 90 days after surgery while controlling for potentially confounding variables such as age, sex, comorbidities, and baseline healthcare use. RESULTS: When controlling for covariates such as age, sex, comorbidities, and baseline healthcare use, patients in the low-value test cohort had an adjusted odds ratio of 1.57 (95% confidence interval [CI] 1.50 to 1.64; p < 0.001) for a postoperative use event (a downstream diagnostic test or procedure) compared with those who did not have a low-value test. The median (IQR) per-patient reimbursements associated with downstream utilization events in patients who received a low-value test was USD 231.97 (64.37 to 1138.84), and those who did not receive a low-value test had a median of USD 191.52 (57.1 to 899.42) (adjusted difference when controlling for covariates: USD 217.27 per patient [95% CI 59.51 to 375.03]; p = 0.007). After adjusting for inflation, total additional reimbursements for patients in the low-value test cohort increased annually. CONCLUSION: Low-value tests generate downstream tests and procedures that are known to provide minimal benefit to healthy patients and may expose patients to potential harms associated with subsequent, unnecessary invasive tests and procedures in response to false positives. Nevertheless, low-value testing remains common and the rising trend in low-value test-associated spending demonstrates the need for multicomponent interventions that target change at both the payer and health system level. Such interventions should disincentivize the initial low-value test and the cascade that may follow. Future work to identify the barriers and facilitators to reduce low-value testing in hand surgery can inform the development and revision of deimplementation strategies. LEVEL OF EVIDENCE: Level III, therapeutic study.

Prevalence, Burden, and Sources of Out-of-Network Billing in Elective Hand Surgery: A National Claims Database Analysis.

PURPOSE: Surprise out-of-network (OON) bills can represent a considerable cost burden on patients. However, OON billing remains underexplored in elective, outpatient surgery procedures, which have greater latitude for patient choice. We aimed to answer the following questions: (1) What is the prevalence and magnitude of OON charges in hand surgery? (2) What are the sources of OON charges? and (3) What factors are associated with OON charges? METHODS: We analyzed patient-level data from the Clinformatics Data Mart database. We identified patients undergoing carpal tunnel release, trigger finger release, wrist ganglion removal, de Quervain release, limited palmar fasciectomy, or thumb carpometacarpal arthroplasty at in-network facilities with an in-network primary surgeon. The primary outcome was the proportion of surgical episodes with at least 1 OON charge. Secondary outcomes included the magnitude of potential balance bills (portion of OON bill exclusive of the standardized payment and expected patient cost-sharing), sources of OON charges, and factors associated with OON charges. RESULTS: Of 112,211 elective hand surgery episodes, 8% (9,158) had at least 1 OON charge. OON charges ranged from $1,154 (95% confidence interval, $1,018-$1,289) for wrist ganglion removal to $3,162 (95% confidence interval, $2,902-$3,423) for thumb carpometacarpal arthroplasty. In episodes with OON charges, the major sources of OON charges were anesthesiologists (75% of episodes), durable medical equipment (10% of episodes), and pathologists (9% of episodes). Site of service, geographic region, and health exchange-purchased plans were highly associated with OON charges. CONCLUSIONS: Out-of-network billing can represent a substantial cost burden to patients and should be considered in perioperative decision-making in elective hand surgery. CLINICAL RELEVANCE: Understanding the potential costs related to OON services during a surgical episode, and its drivers, allows surgeons to consider detailed cost discussions during perioperative decision making.

Quality Measures for Pediatric Orthopaedic Surgery: A Systematic Review.

BACKGROUND: Quality measures provide a way to assess health care delivery and to identify areas for improvement that can inform patient care delivery. When operationalized by a hospital or a payer, quality measures can also be tied to physician or hospital reimbursement. Prior work on quality measures in orthopaedic surgery have identified substantial gaps in measurement portfolios and have highlighted areas for future measure development. This study aims to identify the portfolio of quality measures in pediatric orthopaedic surgery. METHODS: We used methodology of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and reviewed PubMed/ MEDLINE and EMBASE, the American Academy of Orthopaedic Surgery (AAOS), National Quality Forum (NQF), and the Agency for Healthcare Research and Quality (AHRQ), for quality measures and candidate quality measures. Quality measure and candidate quality measures were categorized as structure, process, or outcome. Measures were also classified into 1 of the 6 National Quality Strategy priorities (safety, effective, patient centered, timely, efficient, and equitable). RESULTS: A review of PubMed/EMBASE returned 1640 potential quality measures and articles. A review of AAOS, NQF, and AHRQ databases found 80 potential quality measures. After screening we found a total of 18 quality measures and candidate quality measures specifically for pediatric orthopaedic surgery. Quality measures addressed conditions such as supracondylar humerus fractures, developmental dysplasia of the hip, and osteochondritis dissecans. There were 10 process measures, 8 outcome measure, and 0 structure measures. When we categorized by National Quality Strategy priorities and found 50% (9/18) were effective clinical care, 44% (8/18) were person and care-giver centered experience and outcomes, 6% (1/18) were efficient use of resources. CONCLUSIONS: There are few quality measures and candidate quality measures to assess pediatric orthopaedic surgery. Of the quality measure available, process measures are relatively over-represented. Pediatric orthopaedic surgeons can lead the development of outcome (eg, patient-reported outcomes after surgery) and structure measures (eg, subspecialty training certification) to assess quality of care in pediatric orthopaedic surgery. LEVEL OF EVIDENCE: Level II-systematic review.

Semi-blind sparse affine spectral unmixing of autofluorescence-contaminated micrographs.

MOTIVATION: Spectral unmixing methods attempt to determine the concentrations of different fluorophores present at each pixel location in an image by analyzing a set of measured emission spectra. Unmixing algorithms have shown great promise for applications where samples contain many fluorescent labels; however, existing methods perform poorly when confronted with autofluorescence-contaminated images. RESULTS: We propose an unmixing algorithm designed to separate fluorophores with overlapping emission spectra from contamination by autofluorescence and background fluorescence. First, we formally define a generalization of the linear mixing model, called the affine mixture model (AMM), that specifically accounts for background fluorescence. Second, we use the AMM to derive an affine nonnegative matrix factorization method for estimating fluorophore endmember spectra from reference images. Lastly, we propose a semi-blind sparse affine spectral unmixing (SSASU) algorithm that uses knowledge of the estimated endmembers to learn the autofluorescence and background fluorescence spectra on a per-image basis. When unmixing real-world spectral images contaminated by autofluorescence, SSASU greatly improved proportion indeterminacy as compared to existing methods for a given relative reconstruction error. AVAILABILITY AND IMPLEMENTATION: The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.

Longitudinal Changes in Parkinson's Disease Symptoms with and Without Rapid Eye Movement Sleep Behavior Disorder: The Oxford Discovery Cohort Study.

BACKGROUND: Parkinson's disease (PD) comorbid with rapid eye movement sleep behavior disorder (RBD) may show more severe motor and nonmotor symptoms, suggesting a distinct PD subtype. OBJECTIVE: The aim of this study was to investigate the impact of RBD on the longitudinal change of motor and nonmotor symptoms in patients with PD. METHODS: Patients with early PD (diagnosed within 3.5 years) recruited from 2010 to 2019 were followed every 18 months in the Oxford Parkinson's Disease Centre Discovery cohort. At each visit, we used standard questionnaires and measurements to assess demographic features and motor and nonmotor symptoms (including RBD, daytime sleepiness, mood, autonomic symptoms, cognition, and olfaction). Data were analyzed with linear mixed effects and Cox regression models. Possible RBD (pRBD) was longitudinally determined according to RBD Screening Questionnaire scores. RESULTS: A total of 923 patients were recruited (mean age: 67.1 ± 9.59 years; 35.9% female), and 788 had follow-up assessment(s) (mean: 4.8 ± 1.98 years, range: 1.3-8.3). Among them, 33.3% were identified as pRBD (PD + pRBD). Patients with PD + pRBD had more severe baseline symptoms and showed faster progression on Movement Disorder Society-Unified Parkinson's Disease Rating Scale parts I and III, Purdue Pegboard test, and Beck Depression Inventory scores. Moreover, PD + pRBD was associated with an increased level of risk for mild cognitive impairment (hazard ratio [HR] = 1.36, 95% confidence interval [CI]: 1.01-1.83), freezing of gait (HR = 1.42, 95% CI: 1.10-1.86), and frequent falling (HR = 1.62, 95% CI: 1.02-2.60). CONCLUSIONS: Patients with PD + pRBD progress faster on motor, mood, and cognitive symptoms, confirming a more aggressive PD subtype that can be identified at baseline and has major clinical implications. © 2021 International Parkinson and Movement Disorder Society.

Spatial organization of a model 15-member human gut microbiota established in gnotobiotic mice.

Knowledge of the spatial organization of the gut microbiota is important for understanding the physical and molecular interactions among its members. These interactions are thought to influence microbial succession, community stability, syntrophic relationships, and resiliency in the face of perturbations. The complexity and dynamism of the gut microbiota pose considerable challenges for quantitative analysis of its spatial organization. Here, we illustrate an approach for addressing this challenge, using (i) a model, defined 15-member consortium of phylogenetically diverse, sequenced human gut bacterial strains introduced into adult gnotobiotic mice fed a polysaccharide-rich diet, and (ii) in situ hybridization and spectral imaging analysis methods that allow simultaneous detection of multiple bacterial strains at multiple spatial scales. Differences in the binding affinities of strains for substrates such as mucus or food particles, combined with more rapid replication in a preferred microhabitat, could, in principle, lead to localized clonally expanded aggregates composed of one or a few taxa. However, our results reveal a colonic community that is mixed at micrometer scales, with distinct spatial distributions of some taxa relative to one another, notably at the border between the mucosa and the lumen. Our data suggest that lumen and mucosa in the proximal colon should be conceptualized not as stratified compartments but as components of an incompletely mixed bioreactor. Employing the experimental approaches described should allow direct tests of whether and how specified host and microbial factors influence the nature and functional contributions of "microscale" mixing to the dynamic operations of the microbiota in health and disease.

Social support, loneliness, eating, and activity among parent-adolescent dyads.

We examined associations of social support and loneliness with eating and activity among parent-adolescent dyads (N = 2968) using actor-partner interdependence modeling. Loneliness had several actor associations with health behaviors (adolescents: less physical activity [PA], p < .001, more sedentariness, p < .001; parents: less fruit/vegetable consumption [FVC], p = .029, more hedonic food consumption [HFC], p = .002, and sedentariness, p < .001), but only one dyadic association (adolescent loneliness with less parent FVC, p = .039). Visible support was associated with less HFC, p < .001, and sedentariness, p < .001, but less FVC, p = .008, among adolescents. Invisible support was associated with less HFC, p = .003, but also less PA, p = .028, among adolescents. Both support types were associated with less HFC among parents, p < .001, but invisible support was also associated with less FVC, p = .029, and PA, p = .012, and more sedentariness, p = .013, among parents. When examining health behavior among parents and adolescents, it may be important to consider social support (but perhaps not loneliness) at a dyadic level.

Biogeography of a human oral microbiome at the micron scale.

The spatial organization of complex natural microbiomes is critical to understanding the interactions of the individual taxa that comprise a community. Although the revolution in DNA sequencing has provided an abundance of genomic-level information, the biogeography of microbiomes is almost entirely uncharted at the micron scale. Using spectral imaging fluorescence in situ hybridization as guided by metagenomic sequence analysis, we have discovered a distinctive, multigenus consortium in the microbiome of supragingival dental plaque. The consortium consists of a radially arranged, nine-taxon structure organized around cells of filamentous corynebacteria. The consortium ranges in size from a few tens to a few hundreds of microns in radius and is spatially differentiated. Within the structure, individual taxa are localized at the micron scale in ways suggestive of their functional niche in the consortium. For example, anaerobic taxa tend to be in the interior, whereas facultative or obligate aerobes tend to be at the periphery of the consortium. Consumers and producers of certain metabolites, such as lactate, tend to be near each other. Based on our observations and the literature, we propose a model for plaque microbiome development and maintenance consistent with known metabolic, adherence, and environmental considerations. The consortium illustrates how complex structural organization can emerge from the micron-scale interactions of its constituent organisms. The understanding that plaque community organization is an emergent phenomenon offers a perspective that is general in nature and applicable to other microbiomes.

Sex Differences in the Association of Perceived Ambiguity, Cancer Fatalism, and Health-Related Self-Efficacy with Fruit and Vegetable Consumption.

Adequate fruit and vegetable (FV) consumption is promoted as a means of preventing chronic health conditions, such as cardiovascular disease and cancer. This study investigated whether perceived ambiguity about cancer prevention recommendations, fatalistic beliefs about cancer, and health-related self-efficacy were associated with FV consumption and whether sex moderated these associations. Data from the five most recent waves (spanning 2011 to 2017) of the nationally representative Health Information National Trends Survey (N = 16,965) were used. Participants reported levels of perceived ambiguity, cancer fatalism, health-related self-efficacy, and daily FV consumption. Perceived ambiguity and cancer fatalism were negatively associated with FV consumption, ps <.001, whereas health-related self-efficacy was positively associated with FV consumption, b = 0.34, p < .001. Sex moderated these associations, ps <.05. Perceived ambiguity and cancer fatalism were more strongly associated with less FV consumption for men, bs < -0.31, ps <.001, than women, bs < -0.14, ps <.01. In contrast, health-related self-efficacy was more strongly associated with more FV consumption for women, b = 0.43, p < .001, than men, b = 0.26, p < .001. These results suggest that tailoring health messaging to target sex-specific barriers may improve their effectiveness.

Oligotyping analysis of the human oral microbiome.

The Human Microbiome Project provided a census of bacterial populations in healthy individuals, but an understanding of the biomedical significance of this census has been hindered by limited taxonomic resolution. A high-resolution method termed oligotyping overcomes this limitation by evaluating individual nucleotide positions using Shannon entropy to identify the most information-rich nucleotide positions, which then define oligotypes. We have applied this method to comprehensively analyze the oral microbiome. Using Human Microbiome Project 16S rRNA gene sequence data for the nine sites in the oral cavity, we identified 493 oligotypes from the V1-V3 data and 360 oligotypes from the V3-V5 data. We associated these oligotypes with species-level taxon names by comparison with the Human Oral Microbiome Database. We discovered closely related oligotypes, differing sometimes by as little as a single nucleotide, that showed dramatically different distributions among oral sites and among individuals. We also detected potentially pathogenic taxa in high abundance in individual samples. Numerous oligotypes were preferentially located in plaque, others in keratinized gingiva or buccal mucosa, and some oligotypes were characteristic of habitat groupings such as throat, tonsils, tongue dorsum, hard palate, and saliva. The differing habitat distributions of closely related oligotypes suggest a level of ecological and functional biodiversity not previously recognized. We conclude that the Shannon entropy approach of oligotyping has the capacity to analyze entire microbiomes, discriminate between closely related but distinct taxa and, in combination with habitat analysis, provide deep insight into the microbial communities in health and disease.

Microbiota organization is a distinct feature of proximal colorectal cancers.

Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging.

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

The oral microbiome: diversity, biogeography and human health.

The human oral microbiota is highly diverse and has a complex ecology, comprising bacteria, microeukaryotes, archaea and viruses. These communities have elaborate and highly structured biogeography that shapes metabolic exchange on a local scale and results from the diverse microenvironments present in the oral cavity. The oral microbiota also interfaces with the immune system of the human host and has an important role in not only the health of the oral cavity but also systemic health. In this Review, we highlight recent advances including novel insights into the biogeography of several oral niches at the species level, as well as the ecological role of candidate phyla radiation bacteria and non-bacterial members of the oral microbiome. In addition, we summarize the relationship between the oral microbiota and the pathology of oral diseases and systemic diseases. Together, these advances move the field towards a more holistic understanding of the oral microbiota and its role in health, which in turn opens the door to the study of novel preventive and therapeutic strategies.

Essential genes for Haemophilus parainfluenzae survival and biofilm growth.

Haemophilus parainfluenzae ( Hp ) is a Gram-negative, pleomorphic rod, highly prevalent and abundant as a commensal in the human oral cavity, and an infrequent extraoral opportunistic pathogen. Hp occupies multiple niches in the oral cavity, including the tongue dorsum, keratinized gingiva, and the supragingival plaque biofilm. As a member of the HACEK group, Hp is also known to cause infective endocarditis. Additionally, case reports have identified Hp as the causative agent of meningitis, septic arthritis, chronic osteomyelitis, septicemia, and a variety of other infectious diseases. Little is known about how Hp interacts with its neighbors in the healthy biofilm nor about its mechanisms of pathogenesis as an extraoral opportunistic pathogen. To address these unknowns, we identified the essential genomes of two Hp strains and the conditionally essential genes for their growth in in vitro biofilms aerobically and anaerobically. Using transposon insertion sequencing (TnSeq) with a highly saturated mariner transposon library in two strains, the ATCC33392 type-strain ( Hp 392) and a commensal oral isolate EL1 ( Hp EL1), we show that the essential genome of Hp 392 and Hp EL1 is composed of 395 and 384 genes, respectively. The core essential genome, consisting of 341 essential genes conserved between both strains, was composed of genes associated with genetic information processing, carbohydrate, protein, and energy metabolism. We also identified conditionally essential genes for aerobic and anaerobic biofilm growth, which were associated with carbohydrate and energy metabolism in both strains of Hp . Additionally, RNAseq analysis determined that most genes upregulated during anaerobic growth are not essential for Hp 392 anaerobic biofilm survival. The completion of this library and analysis under these conditions gives us a foundational insight into the basic biology of H. parainfluenzae in differing oxygen conditions, similar to its in vivo oral habitat. Further, the creation of this library presents a valuable tool for further investigation into conditionally essential genes for an organism that lives in close contact with many microbial species in the human oral habitat.