The Gut Microbiome Modulates Body Temperature Both in Sepsis and Health.

Rationale: Among patients with sepsis, variation in temperature trajectories predicts clinical outcomes. In healthy individuals, normal body temperature is variable and has decreased consistently since the 1860s. The biologic underpinnings of this temperature variation in disease and health are unknown. Objectives: To establish and interrogate the role of the gut microbiome in calibrating body temperature. Methods: We performed a series of translational analyses and experiments to determine whether and how variation in gut microbiota explains variation in body temperature in sepsis and in health. We studied patient temperature trajectories using electronic medical record data. We characterized gut microbiota in hospitalized patients using 16S ribosomal RNA gene sequencing. We modeled sepsis using intraperitoneal LPS in mice and modulated the microbiome using antibiotics, germ-free, and gnotobiotic animals. Measurements and Main Results: Consistent with prior work, we identified four temperature trajectories in patients hospitalized with sepsis that predicted clinical outcomes. In a separate cohort of 116 hospitalized patients, we found that the composition of patients' gut microbiota at admission predicted their temperature trajectories. Compared with conventional mice, germ-free mice had reduced temperature loss during experimental sepsis. Among conventional mice, heterogeneity of temperature response in sepsis was strongly explained by variation in gut microbiota. Healthy germ-free and antibiotic-treated mice both had lower basal body temperatures compared with control animals. The Lachnospiraceae family was consistently associated with temperature trajectories in hospitalized patients, experimental sepsis, and antibiotic-treated mice. Conclusions: The gut microbiome is a key modulator of body temperature variation in both health and critical illness and is thus a major, understudied target for modulating physiologic heterogeneity in sepsis.

In critically ill patients, anti-anaerobic antibiotics increase risk of adverse clinical outcomes.

BACKGROUND: Critically ill patients routinely receive antibiotics with activity against anaerobic gut bacteria. However, in other disease states and animal models, gut anaerobes are protective against pneumonia, organ failure and mortality. We therefore designed a translational series of analyses and experiments to determine the effects of anti-anaerobic antibiotics on the risk of adverse clinical outcomes among critically ill patients. METHODS: We conducted a retrospective single-centre cohort study of 3032 critically ill patients, comparing patients who did and did not receive early anti-anaerobic antibiotics. We compared intensive care unit outcomes (ventilator-associated pneumonia (VAP)-free survival, infection-free survival and overall survival) in all patients and changes in gut microbiota in a subcohort of 116 patients. In murine models, we studied the effects of anaerobe depletion in infectious (Klebsiella pneumoniae and Staphylococcus aureus pneumonia) and noninfectious (hyperoxia) injury models. RESULTS: Early administration of anti-anaerobic antibiotics was associated with decreased VAP-free survival (hazard ratio (HR) 1.24, 95% CI 1.06-1.45), infection-free survival (HR 1.22, 95% CI 1.09-1.38) and overall survival (HR 1.14, 95% CI 1.02-1.28). Patients who received anti-anaerobic antibiotics had decreased initial gut bacterial density (p=0.00038), increased microbiome expansion during hospitalisation (p=0.011) and domination by Enterobacteriaceae spp. (p=0.045). Enterobacteriaceae were also enriched among respiratory pathogens in anti-anaerobic-treated patients (p<2.2×10-16). In murine models, treatment with anti-anaerobic antibiotics increased susceptibility to Enterobacteriaceae pneumonia (p<0.05) and increased the lethality of hyperoxia (p=0.0002). CONCLUSIONS: In critically ill patients, early treatment with anti-anaerobic antibiotics is associated with increased mortality. Mechanisms may include enrichment of the gut with respiratory pathogens, but increased mortality is incompletely explained by infections alone. Given consistent clinical and experimental evidence of harm, the widespread use of anti-anaerobic antibiotics should be reconsidered.

Role of interferon-γ and inflammatory monocytes in driving colonic inflammation during acute Clostridium difficile infection in mice.

The inflammatory response to the colonic pathogen Clostridium difficile is characterized by the induction of inflammatory cytokines including Interleukin-23 (IL-23) and interferon-γ (IFN-γ) and the recruitment of myeloid cells including Ly6CHigh monocytes. IL-23 knockout mice showed reduced expression of the monocyte chemokines Ccl4 and Ccl7, but not Ccl2, as well as reduced Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile colitis. Clostridium difficile-infected CCR2-/- (CCR2 KO) mice showed a significant defect in Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile. Although there was no decrease in expression of the inflammatory cytokines Il1b, Il6 or Tnf or reduction in the severity of colonic histopathology associated with ablation of monocyte recruitment, Slpi and Inos expression was significantly reduced in the colons of these animals. Additionally, neutralization of IFN-γ through the administration of anti-IFN-γ monoclonal antibody resulted in a significant reduction in the expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10, but not a reduction in the neutrophil chemokines Cxcl1, Cxcl2 and Ccl3 or the monocyte chemokine Ccl2. Consistently, monocyte and neutrophil recruitment were unchanged following anti-IFN-γ treatment. Additionally, Inos and Slpi expression were unchanged following anti-IFN-γ treatment, suggesting that Inos and Slpi regulation is independent of IFN-γ during C. difficile colitis. Taken together, these data strongly suggest that IL-23 and CCR2 signalling are required for monocyte recruitment during C. difficile colitis. Additionally, these studies also suggest that monocytes, but not IFN-γ, are necessary for full expression of Inos and Slpi in the colon.

Interleukin-23 (IL-23), independent of IL-17 and IL-22, drives neutrophil recruitment and innate inflammation during Clostridium difficile colitis in mice.

Our objective was to determine the role of the inflammatory cytokine interleukin-23 (IL-23) in promoting neutrophil recruitment, inflammatory cytokine expression and intestinal histopathology in response to Clostridium difficile infection. Wild-type (WT) and p19(-/-) (IL-23KO) mice were pre-treated with cefoperazone in their drinking water for 5 days, and after a 2-day recovery period were challenged with spores from C. difficile strain VPI 10463. Interleukin-23 deficiency was associated with significant defects in both the recruitment of CD11b(High) Ly6G(H) (igh) neutrophils to the colon and the expression of neutrophil chemoattractants and stabilization factors including Cxcl1, Cxcl2, Ccl3 and Csf3 within the colonic mucosa as compared with WT animals. Furthermore, the expression of inflammatory cytokines including Il33, Tnf and Il6 was significantly reduced in IL-23-deficient animals. There was also a trend towards less severe colonic histopathology in the absence of IL-23. The induction of Il17a and Il22 was also significantly abrogated in IL-23KO mice. Inflammatory cytokine expression and neutrophilic inflammation were not reduced in IL-17a-deficient mice or in mice treated with anti-IL-22 depleting monoclonal antibody. However, induction of RegIIIg was significantly reduced in animals treated with anti-IL-22 antibody. Taken together, these data indicate that IL-23, but not IL-17a or IL-22, promotes neutrophil recruitment and inflammatory cytokine and chemokine expression in the colon in response to C. difficile infection.

The role of Gr-1(+) cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice.

The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1(+) cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1(+) cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C(Mid)  Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C(Mid) Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1(+) cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.

Interleukin-22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice.

Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.

Interleukin-17 drives pulmonary eosinophilia following repeated exposure to Aspergillus fumigatus conidia.

Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper T(H)2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the T(H)2 response has begun to resolve. T(H)17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4(+) CD25(+) FoxP3(+) cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving T(H)2-type inflammation to repeated inhalation of fungal conidia.

The nature and structure of psychological distress in people at high risk for melanoma: a factor analytic study.

OBJECTIVE: This study examined the psychometric properties of two commonly used measures of psychological distress, the Hospital Anxiety and Depression Scale (HADS) and the Impact of Events Scale (IES) in a sample of individuals at high risk of developing melanoma due to strong family history. METHODS: One hundred thirty-two individuals with a known family-specific CDKN2A mutation (74% response rate) completed a mailed, self-administered questionnaire including the HADS and the IES. Initial correlational analyses were followed by both exploratory and confirmatory factor analyses, according to a predetermined procedure for order of analyses. RESULTS: Exploratory factor analyses found that neither a two-, three- or four-factor solution satisfactorily accounted for all IES items in the present sample. By contrast, a unidimensional account of the data emerged to best account for all IES items, leaving no items unaccounted for. In contrast, the traditional two-factor (anxiety and depression) structure of the HADS appeared to fit the data well. CONCLUSIONS: The traditional, two-factor (intrusion and avoidance) structure of the IES was not borne out within this familial melanoma cohort. Assessment of a single dimension of emotional distress in response to melanoma risk may facilitate more meaningful explorations of psychological adjustment in this context. These findings also raise questions about whether a post-traumatic stress framework is indeed the most appropriate framework to capture the unique nature of melanoma- or cancer-related distress.

Contribution of the Microbiome, Environment, and Genetics to Mucosal Type 2 Immunity and Anaphylaxis in a Murine Food Allergy Model.

There is heterogeneity inherent in the immune responses of individual mice in murine models of food allergy, including anaphylaxis, similar to the clinical heterogeneity observed in humans with food allergies to a defined food. One major driver of this heterogeneity may be differences in the microbiome between sensitized individuals. Our laboratory and others have reported that disruption of the microbiome (dysbiosis) by broad spectrum antibiotics and/or yeast colonization can alter systemic immunity and favor the development of mucosal Type 2 immunity to aeroallergens. Our objective was to use a well-characterized murine model (Balb/c mice) of food allergies (chicken egg ovalbumin, OVA) and determine if antibiotic-mediated dysbiosis (including C. albicans colonization) could enhance the manifestation of food allergies. Furthermore, we sought to identify elements of the microbiome and host response that were associated with this heterogeneity in the anaphylactic reaction between individual food allergen-sensitized mice. In our dataset, the intensity of the anaphylactic reactions was most strongly associated with a disrupted microbiome that included colonization by C. albicans, loss of a specific Lachnoclostridium species (tentatively, Lachnoclostridium YL32), development of a highly polarized Type 2 response in the intestinal mucosa and underlying tissue, and activation of mucosal mast cells. Serum levels of allergen-specific IgE were not predictive of the response and a complete absence of a microbiome did not fully recapitulate the response. Conventionalization of germ-free mice resulted in Akkermansia muciniphila outgrowth and a higher degree of heterogeneity in the allergic response. C57BL/6 mice remained resistant even under the same dysbiosis-inducing antibiotic regimens, while changes in the microbiome markedly altered the reactivity of Balb/c mice to OVA, as noted above. Strikingly, we also observed that genetically identical mice from different rooms in our vivarium develop different levels of a Type 2 response, as well as anaphylactic reactions. The intestinal microbiome in these mice also differed between rooms. Thus, our data recapitulate the heterogeneity in anaphylactic reactions, ranging from severe to none, seen in patients that have circulating levels of food allergen-reactive IgE and support the concept that alterations in the microbiome can be one factor underlying this heterogeneity.

Antibiotics cause metabolic changes in mice primarily through microbiome modulation rather than behavioral changes.

BACKGROUND: The microbiome is an important and increasingly-studied mediator of organismal metabolism, although how the microbiome affects metabolism remains incompletely understood. Many investigators use antibiotics to experimentally perturb the microbiome. However, antibiotics have poorly understood yet profound off-target effects on behavior and diet, including food and water aversion, that can confound experiments and limit their applicability. We thus sought to determine the relative influence of microbiome modulation and off-target antibiotic effects on the behavior and metabolic activity of mice. RESULTS: Mice treated with oral antibiotics via drinking water exhibited significant weight loss in fat, liver, and muscle tissue. These mice also exhibited a reduction in water and food consumption, with marked variability across antibiotic regimens. While administration of bitter-tasting but antimicrobially-inert compounds caused a similar reduction in water consumption, this did not cause tissue weight loss or reduced food consumption. Mice administered intraperitoneal antibiotics (bypassing the gastrointestinal tract) exhibited reduced tissue weights and oral intake, comparable to the effects of oral antibiotics. Antibiotic-treated germ-free mice did not have reduced tissue weights, providing further evidence that direct microbiome modulation (rather than behavioral effects) mediates these metabolic changes. CONCLUSIONS: While oral antibiotics cause profound effects on food and water consumption, antibiotic effects on organismal metabolism are primarily mediated by microbiome modulation. We demonstrate that tissue-specific weight loss following antibiotic administration is due primarily to microbiome effects rather than food and water aversion, and identify antibiotic regimens that effectively modulate gut microbiota while minimizing off-target behavioral effects.

Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.

The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4(-/-)), and gamma interferon-deficient (IFN-γ(-/-)) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic "steady-state" fungal infection and the resultant ABPM pathology.

Coevolution of TH1, TH2, and TH17 responses during repeated pulmonary exposure to Aspergillus fumigatus conidia.

Aspergillus fumigatus, a ubiquitous airborne fungus, can cause invasive infection in immunocompromised individuals but also triggers allergic bronchopulmonary aspergillosis in a subset of otherwise healthy individuals repeatedly exposed to the organism. This study addresses a critical gap in our understanding of the immunoregulation in response to repeated exposure to A. fumigatus conidia. C57BL/6 mice were challenged intranasally with A. fumigatus conidia weekly, and leukocyte composition, activation, and cytokine production were examined after two, four, and eight challenges. Approximately 99% of A. fumigatus conidia were cleared within 24 h after inoculation, and repeated exposure to A. fumigatus conidia did not result in hyphal growth or accumulation of conidia with time. After 2 challenges, there was an early influx of neutrophils and regulatory T (T(reg)) cells into the lungs but minimal inflammation. Repeated exposure promoted sustained expansion of the draining lymph nodes, while the influx of eosinophils and other myeloid cells into the lungs peaked after four exposures and then decreased despite continued A. fumigatus challenges. Goblet cell metaplasia and low-level fibrosis were evident during the response. Repeated exposure to A. fumigatus conidia induced T cell activation in the lungs and the codevelopment by four exposures of T(H)1, T(H)2, and T(H)17 responses in the lungs, which were maintained through eight exposures. Changes in CD4 T cell polarization or T(reg) numbers did not account for the reduction in myeloid cell numbers later in the response, suggesting a non-T-cell regulatory pathway involved in dampening inflammation during repeated exposure to A. fumigatus conidia.

Dual roles of CD40 on microbial containment and the development of immunopathology in response to persistent fungal infection in the lung.

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4+ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.

CD11c+ cells are required to prevent progression from local acute lung injury to multiple organ failure and death.

To investigate the role of CD11c(+) cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 microg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F(2alpha), a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c(+) cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge.

Measuring Latent Quantities.

A distinction is proposed between measures and predictors of latent variables. The discussion addresses the consequences of the distinction for the true-score model, the linear factor model, Structural Equation Models, longitudinal and multilevel models, and item-response models. A distribution-free treatment of calibration and error-of-measurement is given, and the contrasting properties of measures and predictors are examined.

Whole lung tissue is the preferred sampling method for amplicon-based characterization of murine lung microbiota.

BACKGROUND: Low-biomass microbiome studies (such as those of the lungs, placenta, and skin) are vulnerable to contamination and sequencing stochasticity, which obscure legitimate microbial signal. While human lung microbiome studies have rigorously identified sampling strategies that reliably capture microbial signal from these low-biomass microbial communities, the optimal sampling strategy for characterizing murine lung microbiota has not been empirically determined. Performing accurate, reliable characterization of murine lung microbiota and distinguishing true microbial signal from noise in these samples will be critical for further mechanistic microbiome studies in mice. RESULTS: Using an analytic approach grounded in microbial ecology, we compared bacterial DNA from the lungs of healthy adult mice collected via two common sampling approaches: homogenized whole lung tissue and bronchoalveolar lavage (BAL) fluid. We quantified bacterial DNA using droplet digital PCR, characterized bacterial communities using 16S rRNA gene sequencing, and systematically assessed the quantity and identity of bacterial DNA in both specimen types. We compared bacteria detected in lung specimens to each other and to potential source communities: negative (background) control specimens and paired oral samples. By all measures, whole lung tissue in mice contained greater bacterial signal and less evidence of contamination than did BAL fluid. Relative to BAL fluid, whole lung tissue exhibited a greater quantity of bacterial DNA, distinct community composition, decreased sample-to-sample variation, and greater biological plausibility when compared to potential source communities. In contrast, bacteria detected in BAL fluid were minimally different from those of procedural, reagent, and sequencing controls. CONCLUSIONS: An ecology-based analytical approach discriminates signal from noise in this low-biomass microbiome study and identifies whole lung tissue as the preferred specimen type for murine lung microbiome studies. Sequencing, analysis, and reporting of potential source communities, including negative control specimens and contiguous biological sites, are crucial for biological interpretation of low-biomass microbiome studies, independent of specimen type. Video abstract.

Critical Relevance of Stochastic Effects on Low-Bacterial-Biomass 16S rRNA Gene Analysis.

The bacterial microbiome of human body sites, previously considered sterile, remains highly controversial because it can be challenging to isolate signal from noise when low-biomass samples are being analyzed. We tested the hypothesis that stochastic sequencing noise, separable from reagent contamination, is generated during sequencing on the Illumina MiSeq platform when DNA input is below a critical threshold. We first purified DNA from serial dilutions of Pseudomonas aeruginosa and from negative controls using three DNA purification kits, quantified input using droplet digital PCR, and then sequenced the 16S rRNA gene in four technical replicates. This process identified reproducible contaminant signal that was separable from an irreproducible stochastic noise, which occurred as bacterial biomass of samples decreased. This approach was then applied to authentic respiratory samples from healthy individuals (n = 22) that ranged from high to ultralow bacterial biomass. Using oral rinse, bronchoalveolar lavage (BAL) fluid, and exhaled breath condensate (EBC) samples and matched controls, we were able to demonstrate (i) that stochastic noise dominates sequencing in real-world low-bacterial-biomass samples that contain fewer than 104 copies of the 16S rRNA gene per sample, (ii) that critical examination of the community composition of technical replicates can be used to separate signal from noise, and (iii) that EBC is an irreproducible sampling modality for sampling the microbiome of the lower airways. We anticipate that these results combined with suggested methods for identifying and dealing with noisy communities will facilitate increased reproducibility while simultaneously permitting characterization of potentially important low-biomass communities.IMPORTANCE DNA contamination from external sources (reagents, environment, operator, etc.) has long been assumed to be the main cause of spurious signals that appear under low-bacterial-biomass conditions. Here, we demonstrate that contamination can be separated from another, random signal generated during low-biomass-sample sequencing. This stochastic noise is not reproduced between technical replicates; however, results for any one replicate taken alone could look like a microbial community different from the controls. Using this information, we investigated respiratory samples from healthy humans and determined the narrow range of bacterial biomass where samples transition from producing reproducible microbial sequences to ones dominated by noise. We present a rigorous approach to studies involving low-bacterial-biomass samples to detect this source of noise and provide a framework for deciding if a sample is likely to be dominated by noise. We anticipate that this work will facilitate increased reproducibility in the characterization of potentially important low-biomass communities.

A five-year review of ground-to-air emergency medical advice.

We reviewed five years' experience of providing ground-to-air medical advice for commercial passenger aircraft. A total of 273 events occurred in-flight where further advice was sought. The most common age group resulting in calls were from those aged 21-30 years, who generated 38 calls (16%). The 273 cases included gastrointestinal conditions (26%), neurological (22%) and cardiovascular events (14%). Most incidents were managed conservatively in-flight with oxygen and/or other medications. Aircraft diversion occurred on 9% of occasions; cardiovascular and neurological emergencies were the major cause of this. Collaboration between aviation providers should be considered to develop a common database of in-flight emergencies so that contents of medical kits can be standardized in order to improve the quality of care in the air.