Reverse-engineered exclusive enteral nutrition in pediatric Crohn's disease: A pilot trial.

BACKGROUND: In pediatric Crohn's disease (CD), commercial formulas used as exclusive enteral nutrition (EEN) are effective at inducing remission. This study aims to assess the impact of a whole-food blended smoothie as EEN on CD activity and the intestinal microbiome. METHODS: A 4-week prospective trial assessed the impact of EEN with a whole-food smoothie on newly diagnosed mild-to-moderate active pediatric CD. The smoothie with a multivitamin were developed to meet age-appropriate nutritional requirements. Assessment over 4 weeks included Pediatric Crohn's Disease Activity Index (PCDAI), serum laboratories, fecal calprotectin (FCP), and stool collection for metagenomic shotgun sequencing and microbiota composition analysis. Clinical remission was defined as PCDAI ≤ 10 at week 4. RESULTS: Ten participants were enrolled with median age 14.5 years, and 8 completed the trial. Baseline mean PCDAI was 26.3 ± 9.1 and mean FCP 1149 ± 718 µg/g. At week 4, 80% of participants achieved clinical remission. FCP decreased by over half in 60% of participants, with FCP below 250 µg/g in 60% and below 100 µg/g in 40%. Microbiome analysis showed a significant increase in species richness over 4 weeks (p = 0.01). Compared to baseline, the relative abundance at week 2 and at week 4 was significantly increased for Bifidobacterium and Streptococcus and decreased for Blautia (p < 0.05 for all). CONCLUSION: A whole-food blended smoothie was effective for inducing clinical remission and decreasing FCP in pediatric CD similar to commercial EEN formulas. Further research may give insight into data-driven whole-food dietary approaches for CD management. CLINICALTRIALS: gov NCT03508193.

Alterations in the fecal microbiota in patients with advanced cystic fibrosis liver disease after 6 months of elexacaftor/tezacaftor/ivacaftor.

BACKGROUND: Cystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI. METHODS: This is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI. RESULTS: We analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula. CONCLUSIONS: These results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD.

Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

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.

Burkholderia thailandensis Methylated Hydroxyalkylquinolines: Biosynthesis and Antimicrobial Activity in Cocultures.

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In coculture experiments, B. thailandensis strain E264 secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis strain 168. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC, and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576 ("hmqL"). The results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NOs). Synthetic HMAQ-NO is active against B. subtilis 168, showing ∼50-fold more activity than HMAQ. Both the methyl group and the length of the carbon side chain account for the high activity of HMAQ-NO. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensisIMPORTANCE The soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones that are mostly methylated 4-hydroxyalkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa Several of the methylated 4-hydroxyalkenylquinolines have antimicrobial activity against other species. We show that Bacillus subtilis strain 168 is particularly susceptible to N-oxidated methylalkenylquinolines (HMAQ-NOs). We confirmed that HMAQ-NO biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxyalkenylquinolines, which are unique to B. thailandensis This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

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.

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.

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.

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.

Multi-drug resistant non-typhoidal Salmonella associated with invasive disease in western Kenya.

Non-typhoidal Salmonella (NTS) is a leading cause of bloodstream infections in Africa, but the various contributions of host susceptibility versus unique pathogen virulence factors are unclear. We used data from a population-based surveillance platform (population ~25,000) between 2007-2014 and NTS genome-sequencing to compare host and pathogen-specific factors between individuals presenting with NTS bacteremia and those presenting with NTS diarrhea. Salmonella Typhimurium ST313 and Salmonella Enteritidis ST11 were the most common isolates. Multi-drug resistant strains of NTS were more commonly isolated from patients presenting with NTS bacteremia compared to NTS diarrhea. This relationship was observed in patients under age five [aOR = 15.16, 95% CI (2.84-81.05), P = 0.001], in patients five years and older, [aOR = 6.70 95% CI (2.25-19.89), P = 0.001], in HIV-uninfected patients, [aOR = 21.61, 95% CI (2.53-185.0), P = 0.005], and in patients infected with Salmonella serogroup B [aOR = 5.96, 95% CI (2.28-15.56), P < 0.001] and serogroup D [aOR = 14.15, 95% CI (1.10-182.7), P = 0.042]. Thus, multi-drug-resistant NTS was strongly associated with bacteremia compared to diarrhea among children and adults. This association was seen in HIV-uninfected individuals infected with either S. Typhimurium or S. Enteritidis. Risk of developing bacteremia from NTS infection may be driven by virulence properties of the Salmonella pathogen.

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.

Clinical and Fecal Microbial Changes With Diet Therapy in Active Inflammatory Bowel Disease.

GOAL: To determine the effect of the specific carbohydrate diet (SCD) on active inflammatory bowel disease (IBD). BACKGROUND: IBD is a chronic idiopathic inflammatory intestinal disorder associated with fecal dysbiosis. Diet is a potential therapeutic option for IBD based on the hypothesis that changing the fecal dysbiosis could decrease intestinal inflammation. STUDY: Pediatric patients with mild to moderate IBD defined by pediatric Crohn's disease activity index (PCDAI 10-45) or pediatric ulcerative colitis activity index (PUCAI 10-65) were enrolled into a prospective study of the SCD. Patients started SCD with follow-up evaluations at 2, 4, 8, and 12 weeks. PCDAI/PUCAI, laboratory studies were assessed. RESULTS: Twelve patients, ages 10 to 17 years, were enrolled. Mean PCDAI decreased from 28.1±8.8 to 4.6±10.3 at 12 weeks. Mean PUCAI decreased from 28.3±23.1 to 6.7±11.6 at 12 weeks. Dietary therapy was ineffective for 2 patients while 2 individuals were unable to maintain the diet. Mean C-reactive protein decreased from 24.1±22.3 to 7.1±0.4 mg/L at 12 weeks in Seattle Cohort (nL<8.0 mg/L) and decreased from 20.7±10.9 to 4.8±4.5 mg/L at 12 weeks in Atlanta Cohort (nL<4.9 mg/L). Stool microbiome analysis showed a distinctive dysbiosis for each individual in most prediet microbiomes with significant changes in microbial composition after dietary change. CONCLUSIONS: SCD therapy in IBD is associated with clinical and laboratory improvements as well as concomitant changes in the fecal microbiome. Further prospective studies are required to fully assess the safety and efficacy of dietary therapy in patients with IBD.

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.

Staphylococcus aureus Protein A Mediates Interspecies Interactions at the Cell Surface of Pseudomonas aeruginosa.

UNLABELLED: While considerable research has focused on the properties of individual bacteria, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. Staphylococcus aureus frequently coinfects with other pathogens in a range of different infectious diseases. For example, coinfection by S. aureus with Pseudomonas aeruginosa occurs commonly in people with cystic fibrosis and is associated with higher lung disease morbidity and mortality. S. aureus secretes numerous exoproducts that are known to interact with host tissues, influencing inflammatory responses. The abundantly secreted S. aureus staphylococcal protein A (SpA) binds a range of human glycoproteins, immunoglobulins, and other molecules, with diverse effects on the host, including inhibition of phagocytosis of S. aureus cells. However, the potential effects of SpA and other S. aureus exoproducts on coinfecting bacteria have not been explored. Here, we show that S. aureus-secreted products, including SpA, significantly alter two behaviors associated with persistent infection. We found that SpA inhibited biofilm formation by specific P. aeruginosa clinical isolates, and it also inhibited phagocytosis by neutrophils of all isolates tested. Our results indicate that these effects were mediated by binding to at least two P. aeruginosa cell surface structures-type IV pili and the exopolysaccharide Psl-that confer attachment to surfaces and to other bacterial cells. Thus, we found that the role of a well-studied S. aureus exoproduct, SpA, extends well beyond interactions with the host immune system. Secreted SpA alters multiple persistence-associated behaviors of another common microbial community member, likely influencing cocolonization and coinfection with other microbes. IMPORTANCE: Bacteria rarely exist in isolation, whether on human tissues or in the environment, and they frequently coinfect with other microbes. However, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. We identified a novel interaction between two bacterial species that frequently infect together-Staphylococcus aureus and Pseudomonas aeruginosa We show that the S. aureus-secreted protein staphylococcal protein A (SpA), which is well-known for interacting with host targets, also binds to specific P. aeruginosa cell surface molecules and alters two persistence-associated P. aeruginosa behaviors: biofilm formation and uptake by host immune cells. Because S. aureus frequently precedes P. aeruginosa in chronic infections, these findings reveal how microbial community interactions can impact persistence and host interactions during coinfections.

Metagenomic evidence for taxonomic dysbiosis and functional imbalance in the gastrointestinal tracts of children with cystic fibrosis.

Cystic fibrosis (CF) results in inflammation, malabsorption of fats and other nutrients, and obstruction in the gastrointestinal (GI) tract, yet the mechanisms linking these disease manifestations to microbiome composition remain largely unexplored. Here we used metagenomic analysis to systematically characterize fecal microbiomes of children with and without CF, demonstrating marked CF-associated taxonomic dysbiosis and functional imbalance. We further showed that these taxonomic and functional shifts were especially pronounced in young children with CF and diminished with age. Importantly, the resulting dysbiotic microbiomes had significantly altered capacities for lipid metabolism, including decreased capacity for overall fatty acid biosynthesis and increased capacity for degrading anti-inflammatory short-chain fatty acids. Notably, these functional differences correlated with fecal measures of fat malabsorption and inflammation. Combined, these results suggest that enteric fat abundance selects for pro-inflammatory GI microbiota in young children with CF, offering novel strategies for improving the health of children with CF-associated fat malabsorption.

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.

Low Level Engraftment and Improvement following a Single Colonoscopic Administration of Fecal Microbiota to Patients with Ulcerative Colitis.

OBJECTIVE: Fecal microbiota transplantation (FMT) is an investigational treatment for diseases thought to involve alterations in the intestinal microbiota including ulcerative colitis (UC). Case reports have described therapeutic benefit of FMT in patients with UC, possibly due to changes in the microbiota. We measured the degree to which the transplanted microbiota engraft following FMT in patients with UC using a donor similarity index (DSI). METHODS: Seven patients with mild to moderate UC (UC disease activity index scores 3-10) received a single colonoscopic administration of FMT. Metagenomic sequence data from stool were analyzed using an alignment-free comparison tool, to measure the DSI, and a phylogenetic analysis tool, to characterize taxonomic changes. Clinical, endoscopic, histologic, and fecal calprotectin outcome measures were also collected. RESULTS: One of 5 patients from whom sequencing data were available achieved the primary endpoint of 50% donor similarity at week 4; an additional 2 patients achieved 40% donor similarity. One patient with 40% donor similarity achieved clinical and histologic remission 1 month after FMT. However, these were lost by 2-3 months, and loss correlated with a decrease in DSI. The remaining patients did not demonstrate clinical response or remission. Histology scores improved in all but 1 patient. No patients remained in remission at 3 months after FMT. CONCLUSIONS: Following a single colonoscopic fecal transplant, a DSI of 40-50% is achieved in about two-thirds of recipients. This level of engraftment correlated with a temporary clinical improvement in only 1/5 patients. Larger sample sizes could further validate this method for measuring engraftment, and changes in transplant frequency or method might improve microbiota engraftment and efficacy. TRIAL REGISTRATION: ClinicalTrials.gov NCT01742754.

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.

Fecal microbial transplant effect on clinical outcomes and fecal microbiome in active Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a chronic idiopathic inflammatory intestinal disorder associated with fecal dysbiosis. Fecal microbial transplant (FMT) is a potential therapeutic option for individuals with CD based on the hypothesis that changing the fecal dysbiosis could promote less intestinal inflammation. METHODS: Nine patients, aged 12 to 19 years, with mild-to-moderate symptoms defined by Pediatric Crohn's Disease Activity Index (PCDAI of 10-29) were enrolled into a prospective open-label study of FMT in CD (FDA IND 14942). Patients received FMT by nasogastric tube with follow-up evaluations at 2, 6, and 12 weeks. PCDAI, C-reactive protein, and fecal calprotectin were evaluated at each study visit. RESULTS: All reported adverse events were graded as mild except for 1 individual who reported moderate abdominal pain after FMT. All adverse events were self-limiting. Metagenomic evaluation of stool microbiome indicated evidence of FMT engraftment in 7 of 9 patients. The mean PCDAI score improved with patients having a baseline of 19.7 ± 7.2, with improvement at 2 weeks to 6.4 ± 6.6 and at 6 weeks to 8.6 ± 4.9. Based on PCDAI, 7 of 9 patients were in remission at 2 weeks and 5 of 9 patients who did not receive additional medical therapy were in remission at 6 and 12 weeks. No or modest improvement was seen in patients who did not engraft or whose microbiome was most similar to their donor. CONCLUSIONS: This is the first study to demonstrate that FMT for CD may be a possible therapeutic option for CD. Further prospective studies are required to fully assess the safety and efficacy of the FMT in patients with CD.

An underlying mechanism for the increased mutagenesis of lagging-strand genes in Bacillus subtilis.

We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand in Bacillus subtilis. Although we proposed that orientation-specific encounters between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require the B. subtilis Y-family polymerase, PolY1 (yqjH). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses of B. subtilis genomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes.