Infants with cystic fibrosis have altered fecal functional capacities with potential clinical and metabolic consequences.

BACKGROUND: Infants with cystic fibrosis (CF) suffer from gastrointestinal (GI) complications, including pancreatic insufficiency and intestinal inflammation, which have been associated with impaired nutrition and growth. Recent evidence identified altered fecal microbiota taxonomic compositions in infants with CF relative to healthy infants that were characterized by differences in the abundances of taxa associated with GI health and nutrition. Furthermore, these taxonomic differences were more pronounced in low length infants with CF, suggesting a potential link to linear growth failure. We hypothesized that these differences would entail shifts in the microbiome's functional capacities that could contribute to inflammation and nutritional failure in infants with CF. RESULTS: To test this hypothesis, we compared fecal microbial metagenomic content between healthy infants and infants with CF, supplemented with an analysis of fecal metabolomes in infants with CF. We identified notable differences in CF fecal microbial functional capacities, including metabolic and environmental response functions, compared to healthy infants that intensified during the first year of life. A machine learning-based longitudinal metagenomic age analysis of healthy and CF fecal metagenomic functional profiles further demonstrated that these differences are characterized by a CF-associated delay in the development of these functional capacities. Moreover, we found metagenomic differences in functions related to metabolism among infants with CF that were associated with diet and antibiotic exposure, and identified several taxa as potential drivers of these functional differences. An integrated metagenomic and metabolomic analysis further revealed that abundances of several fecal GI metabolites important for nutrient absorption, including three bile acids, correlated with specific microbes in infants with CF. CONCLUSIONS: Our results highlight several metagenomic and metabolomic factors, including bile acids and other microbial metabolites, that may impact nutrition, growth, and GI health in infants with CF. These factors could serve as promising avenues for novel microbiome-based therapeutics to improve health outcomes in these infants.

Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.

RATIONALE: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments. OBJECTIVES: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics. METHODS AND MEASUREMENTS: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories. MAIN RESULTS: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. CONCLUSIONS: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.

CFTR dysregulation drives active selection of the gut microbiome.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.

Fecal dysbiosis in infants with cystic fibrosis is associated with early linear growth failure.

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age1. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life1. Although this early linear growth failure is associated with worse long-term respiratory function and survival2,3, the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis4,5. As GI microbiome activities are known to affect endocrine functions6,7, the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.

Adaptation of commensal proliferating Escherichia coli to the intestinal tract of young children with cystic fibrosis.

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.

GUTSS: An Alignment-Free Sequence Comparison Method for Use in Human Intestinal Microbiome and Fecal Microbiota Transplantation Analysis.

BACKGROUND: Comparative analysis of gut microbiomes in clinical studies of human diseases typically rely on identification and quantification of species or genes. In addition to exploring specific functional characteristics of the microbiome and potential significance of species diversity or expansion, microbiome similarity is also calculated to study change in response to therapies directed at altering the microbiome. Established ecological measures of similarity can be constructed from species abundances, however methods for calculating these commonly used ecological measures of similarity directly from whole genome shotgun (WGS) metagenomic sequence are lacking. RESULTS: We present an alignment-free method for calculating similarity of WGS metagenomic sequences that is analogous to the Bray-Curtis index for species, implemented by the General Utility for Testing Sequence Similarity (GUTSS) software application. This method was applied to intestinal microbiomes of healthy young children to measure developmental changes toward an adult microbiome during the first 3 years of life. We also calculate similarity of donor and recipient microbiomes to measure establishment, or engraftment, of donor microbiota in fecal microbiota transplantation (FMT) studies focused on mild to moderate Crohn's disease. We show how a relative index of similarity to donor can be calculated as a measure of change in a patient's microbiome toward that of the donor in response to FMT. CONCLUSION: Because clinical efficacy of the transplant procedure cannot be fully evaluated without analysis methods to quantify actual FMT engraftment, we developed a method for detecting change in the gut microbiome that is independent of species identification and database bias, sensitive to changes in relative abundance of the microbial constituents, and can be formulated as an index for correlating engraftment success with clinical measures of disease. More generally, this method may be applied to clinical evaluation of human microbiomes and provide potential diagnostic determination of individuals who may be candidates for specific therapies directed at alteration of the microbiome.

Genomic Analysis of Salmonella enterica Serovar Typhimurium Characterizes Strain Diversity for Recent U.S. Salmonellosis Cases and Identifies Mutations Linked to Loss of Fitness under Nitrosative and Oxidative Stress.

UNLABELLED: Salmonella enterica serovar Typhimurium is one of the most common S. enterica serovars associated with U.S. foodborne outbreaks. S. Typhimurium bacteria isolated from humans exhibit wide-ranging virulence phenotypes in inbred mice, leading to speculation that some strains are more virulent in nature. However, it is unclear whether increased virulence in humans is related to organism characteristics or initial treatment failure due to antibiotic resistance. Strain diversity and genetic factors contributing to differential human pathogenicity remain poorly understood. We reconstructed phylogeny, resolved genetic population structure, determined gene content and nucleotide variants, and conducted targeted phenotyping assays for S. Typhimurium strains collected between 1946 and 2012 from humans and animals in the United States and abroad. Strains from recent U.S. salmonellosis cases were associated with five S. Typhimurium lineages distributed within three phylogenetic clades, which are not restricted by geography, year of acquisition, or host. Notably, two U.S. strains and four Mexican strains are more closely related to strains associated with human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa than to other North American strains. Phenotyping studies linked variants specific to these strains in hmpA and katE to loss of fitness under nitrosative and oxidative stress, respectively. These results suggest that U.S. salmonellosis is caused by diverse S. Typhimurium strains circulating worldwide. One lineage has mutations in genes affecting fitness related to innate immune system strategies for fighting pathogens and may be adapting to immunocompromised humans by a reduction in virulence capability, possibly due to a lack of selection for its maintenance as a result of the worldwide HIV epidemic. IMPORTANCE: Nontyphoidal Salmonella bacteria cause an estimated 1.2 million illnesses annually in the United States, 80 million globally, due to ingestion of contaminated food or water. Salmonella Typhimurium is one of the most common serovars associated with foodborne illness, causing self-limiting gastroenteritis and, in approximately 5% of infected patients, systemic infection. Although some S. Typhimurium strains are speculated to be more virulent than others, it is unknown how strain diversity and genetic factors contribute to differential human pathogenicity. Ours is the first study to examine the diversity of S. Typhimurium associated with recent cases of U.S. salmonellosis and to provide some initial correlation between observed genotypes and phenotypes. Definition of specific S. Typhimurium lineages based on such phenotype/genotype correlations may identify strains with greater capability of associating with specific food sources, allowing outbreaks to be more quickly identified. Additionally, defining simple correlates of pathogenesis may have predictive value for patient outcome.

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii.

UNLABELLED: Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCE: Acinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.

Genomic analysis of the emergence of 20th century epidemic dysentery.

BACKGROUND: Shigella dysenteriae type 1 (Sd1) causes recurrent epidemics of dysentery associated with high mortality in many regions of the world. Sd1 infects humans at very low infectious doses (10 CFU), and treatment is complicated by the rapid emergence of antibiotic resistant Sd1 strains. Sd1 is only detected in the context of human infections, and the circumstances under which epidemics emerge and regress remain unknown. RESULTS: Phylogenomic analyses of 56 isolates collected worldwide over the past 60 years indicate that the Sd1 clone responsible for the recent pandemics emerged at the turn of the 20th century, and that the two world wars likely played a pivotal role for its dissemination. Several lineages remain ubiquitous and their phylogeny indicates several recent intercontinental transfers. Our comparative genomics analysis reveals that isolates responsible for separate outbreaks, though closely related to one another, have independently accumulated antibiotic resistance genes, suggesting that there is little or no selection to retain these genes in-between outbreaks. The genomes appear to be subjected to genetic drift that affects a number of functions currently used by diagnostic tools to identify Sd1, which could lead to the potential failure of such tools. CONCLUSIONS: Taken together, the Sd1 population structure and pattern of evolution suggest a recent emergence and a possible human carrier state that could play an important role in the epidemic pattern of infections of this human-specific pathogen. This analysis highlights the important role of whole-genome sequencing in studying pathogens for which epidemiological or laboratory investigations are particularly challenging.