SUMMARY Sepsis and the acute respiratory distress syndrome (ARDS) are major causes of mortality without targeted therapies. Although many experimental and clinical observations have implicated gut microbiota in the pathogenesis of these diseases, culture-based studies have failed to demonstrate translocation of bacteria to the lungs in critically ill patients. Here we report culture-independent evidence that the lung microbiome is enriched with gut bacteria both in a murine model of sepsis and in humans with established ARDS. Following experimental sepsis, lung communities were dominated by viable gut-associated bacteria. Ecologic analysis identified the lower gastrointestinal tract, rather than the upper respiratory tract, as the likely source community of post-sepsis lung bacteria. In bronchoalveolar lavage fluid from humans with ARDS, gut-specific bacteria (Bacteroides spp.) were common and abundant, undetected by culture, and correlated with the intensity of systemic inflammation. Alveolar TNF-α, a key mediator of alveolar inflammation in ARDS, was significantly correlated with altered lung microbiota. Our results demonstrate that the lung microbiome is enriched with gut-associated bacteria in sepsis and ARDS, potentially representing a shared mechanism of pathogenesis in these common and lethal diseases.

Although culture-independent techniques have refuted lung sterility in health, controversy about contamination during bronchoscope passage through the upper respiratory tract (URT) has impeded research progress. We sought to establish whether bronchoscopic sampling accurately reflects the lung microbiome in health and to distinguish between two proposed routes of authentic microbial immigration, (i) dispersion along contiguous respiratory mucosa and (ii) subclinical microaspiration. During bronchoscopy of eight adult volunteers without lung disease, we performed seven protected specimen brushings (PSB) and bilateral bronchoalveolar lavages (BALs) per subject. We amplified, sequenced, and analyzed the bacterial 16S rRNA gene V4 regions by using the Illumina MiSeq platform. Rigorous attention was paid to eliminate potential sources of error or contamination, including a randomized processing order and the inclusion and analysis of exhaustive procedural and sequencing control specimens. Indices of mouth-lung immigration (mouth-lung community similarity, bacterial burden, and community richness) were all significantly greater in airway and alveolar specimens than in bronchoscope contamination control specimens, indicating minimal evidence of pharyngeal contamination. Ecological indices of mouth-lung immigration peaked at or near the carina, as predicted for a primary immigration route of microaspiration. Bacterial burden, diversity, and mouth-lung similarity were greater in BAL than PSB samples, reflecting differences in the sampled surface areas. (This study has been registered at ClinicalTrials.gov under registration no. NCT02392182.)

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

ABSTRACTCandida albicansis a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota,C. albicanscan disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role ofC. albicansin shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventingC. albicanscolonization. Histological analysis demonstrated that colonization withC. albicansduring antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence ofC. albicansduring antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishesBacteroidetespopulations long term in the ceca of mice, but the presence ofC. albicansduring cefoperazone recovery promotedBacteroidetespopulation recovery. However, the presence ofC. albicansresulted in a long-term reduction inLactobacillusspp. and promotedEnterococcus faecalispopulations. Previous studies have focused on the ability of bacteria to alterC. albicans; this study addresses the ability ofC. albicansto alter the bacterial microbiota during nonpathogenic colonization.

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Rationale: The "gut-lung axis" is commonly invoked to explain the microbiome's influence on lung inflammation. Yet the lungs harbor their own microbiome, which is altered in respiratory disease. The relative influence of gut and lung bacteria on lung inflammation is unknown. Objectives: To determine whether baseline lung immune tone reflects local (lung-lung) or remote (gut-lung) microbe-host interactions. Methods: We compared lung, tongue, and cecal bacteria in 40 healthy, genetically identical, 10-week-old mice, using 16S ribosomal RNA gene quantification and sequencing. We measured inflammatory cytokines, using a multiplex assay of homogenized lung tissue. We compared lung bacteria in healthy mice treated with varied durations of systemic antibiotics. Measurements and Main Results: Lung bacterial communities are highly variable among mice, cluster strongly by cage, shipment, and vendor, and are altered by antibiotics in a microbiologically predictable manner. Baseline lung concentrations of two key inflammatory cytokines (IL-1a and IL-4) are correlated with the diversity and community composition of lung bacterial communities. Lung concentrations of these inflammatory cytokines correlate more strongly with variation in lung bacterial communities than with that of the gut or mouth. Conclusions: In the lungs of healthy mice, baseline innate immune tone more strongly reflects local (lung-lung) microbe-host interactions than remote (gut-lung) microbe-host interactions. Our results independently confirm the existence and immunologic significance of the murine lung microbiome, even in health. Variation in lung microbiota is likely an important, underappreciated source of experimental and clinical variability. The lung microbiome is an unexplored therapeutic target for the prevention and treatment of inflammatory lung disease.

Lending support to the hygiene hypothesis, epidemiological studies have demonstrated that allergic disease correlates with widespread use of antibiotics and alterations in fecal microbiota ("microflora"). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators, from the microbiota. We have recently developed a mouse model of antibiotic-induced gastrointestinal microbiota disruption that is characterized by stable increases in levels of gastrointestinal enteric bacteria and Candida. Using this model, we have previously demonstrated that microbiota disruption can drive the development of a CD4 T-cell-mediated airway allergic response to mold spore challenge in immunocompetent C57BL/6 mice without previous systemic antigen priming. The studies presented here address important questions concerning the universality of the model. To investigate the role of host genetics, we tested BALB/c mice. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required interleukin-13 (IL-13) (the response was absent in IL-13 ؊/؊ mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption.

The indigenous bacterial microbiome of the stomach, including lactobacilli, is vital in promoting colonization resistance against Candida albicans. However, there are gaps in our understanding about C. albicans gastric colonization versus disease, especially during the postantibiotic recovery phase. This study compared the gastric responses to C. albicans strains CHN1 and SC5314 in microbiome-disturbed and germfree mice to elucidate the contribution of the indigenous microbiota in C. albicans colonization versus disease and yeast-bacterium antagonism during the post-cefoperazone recolonization period. C. albicans can prevent the regrowth of Lactobacillus spp. in the stomach after cefoperazone and promote increased colonization by Enterococcus spp. Using a culture-independent analysis, the effects of oral cefoperazone on the gastric bacterial microbiota were observed to last at least 3 weeks after the cessation of the antibiotic. Disturbance of the gastric bacterial community by cefoperazone alone was not sufficient to cause gastritis, C. albicans colonization was also needed. Gastritis was not evident until after day 7 in cefoperazonetreated infected mice. In contrast, in germfree mice which lack a gastric microbiota, C. albicans induced gastric inflammation within 1 week of inoculation. Therefore, the gastric bacterial community in cefoperazone-treated mice during the first week of postantibiotic recolonization was sufficient to prevent the development of gastritis, despite being ineffective at conferring colonization resistance against C. albicans. Altogether, these data implicate a dichotomy between C. albicans colonization and gastric disease that is bacterial microbiome dependent.