Publications by authors named "Mitchell Brittnacher"

49 Publications

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

BMC Microbiol 2021 09 15;21(1):247. Epub 2021 Sep 15.

Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel.

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.
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http://dx.doi.org/10.1186/s12866-021-02305-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8444586PMC
September 2021

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

Thorax 2020 09 6;75(9):780-790. Epub 2020 Jul 6.

Pediatrics, University of Washington School of Medicine, Seattle, Washington, USA

Rationale: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic lung infections. While the effects of inhaled tobramycin on 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.
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http://dx.doi.org/10.1136/thoraxjnl-2019-214187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875198PMC
September 2020

CFTR dysregulation drives active selection of the gut microbiome.

PLoS Pathog 2020 01 21;16(1):e1008251. Epub 2020 Jan 21.

Department of Comparative Medicine, University of Washington, Seattle, WA, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994172PMC
January 2020

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

Nat Med 2020 02 20;26(2):215-221. Epub 2020 Jan 20.

Department of Microbiology, University of Washington, Seattle, WA, USA.

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 age. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life. Although this early linear growth failure is associated with worse long-term respiratory function and survival, 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 dysbiosis. As GI microbiome activities are known to affect endocrine functions, 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.
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http://dx.doi.org/10.1038/s41591-019-0714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018602PMC
February 2020

Oral health and plaque microbial profile in juvenile idiopathic arthritis.

Pediatr Rheumatol Online J 2019 Dec 16;17(1):81. Epub 2019 Dec 16.

Seattle Children's Hospital, 4800 Sand Point Way NE, Seattle, WA, 98105, USA.

Background: The oral microbiota has been implicated in the pathogenesis of rheumatoid arthritis through activation of mucosal immunity. This study tested for associations between oral health, microbial communities and juvenile idiopathic arthritis (JIA).

Methods: A cross-sectional exploratory study of subjects aged 10-18 years with oligoarticular, extended oligoarticular and polyarticular JIA was conducted. Control groups included pediatric dental clinic patients and healthy volunteers. The primary aim was to test for an association between dental health indices and JIA; the secondary aim was to characterize the microbial profile of supragingival plaque using 16S rRNA gene sequencing.

Results: The study included 85 patients with JIA, 62 dental patients and 11 healthy child controls. JIA patients overall had significantly more gingival inflammation compared to dental patients, as evidenced by bleeding on probing of the gingiva, the most specific sign of active inflammation (p = 0.02). Overall, however, there was a trend towards better dental hygiene in the JIA patients compared to dental patients, based on indices for plaque, decay, and periodontitis. In the JIA patients, plaque microbiota analysis revealed bacteria belonging to genera Haemophilus or Kingella elevated, and Corynebacterium underrepresented. In poly JIA, bacteria belonging to the genus Porphyromonas was overrepresented and Prevotella was underrepresented.

Conclusion: Increased gingival inflammation in JIA was independent of general oral health, and thus cannot be attributed to poor dental hygiene secondary to disability. The variation of microbial profile in JIA patients could indicate a possible link between gingivitis and synovial inflammation.
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http://dx.doi.org/10.1186/s12969-019-0387-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916162PMC
December 2019

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

Cell Rep 2019 02;26(8):2227-2240.e5

Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98105, USA; Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98105, USA; Pulmonary and Sleep Medicine, Seattle Children's Hospital, Seattle, WA 98105, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.01.091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435281PMC
February 2019

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

Proc Natl Acad Sci U S A 2018 02 29;115(7):1605-1610. Epub 2018 Jan 29.

Department of Microbiology, University of Washington, Seattle, WA 98195;

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. 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 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 isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with from healthy controls, and unrelated CF strains have independently acquired this growth trait. Furthermore, CF and control 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.
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http://dx.doi.org/10.1073/pnas.1714373115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816161PMC
February 2018

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

J Clin Gastroenterol 2018 02;52(2):155-163

Departments of Microbiology.

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.
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http://dx.doi.org/10.1097/MCG.0000000000000772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484760PMC
February 2018

In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

Nat Commun 2016 11 11;7:13414. Epub 2016 Nov 11.

Department of Genome Sciences, University of Washington, 850 Republican Street, Brotman Building Room 154, Seattle, Washington 98109, USA.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.
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http://dx.doi.org/10.1038/ncomms13414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114622PMC
November 2016

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

PLoS One 2016 8;11(7):e0158897. Epub 2016 Jul 8.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938407PMC
July 2017

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.

mBio 2016 Mar 8;7(2):e00154. Epub 2016 Mar 8.

Department of Microbiology, University of Washington, Seattle, Washington, USA Department of Medicine, University of Washington, Seattle, Washington, USA Department of Genome Sciences, University of Washington, Seattle, Washington, USA

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.
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http://dx.doi.org/10.1128/mBio.00154-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810482PMC
March 2016

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

Sci Rep 2016 Mar 4;6:22493. Epub 2016 Mar 4.

Department of Genome Sciences, University of Washington, Seattle, US.

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.
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http://dx.doi.org/10.1038/srep22493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778032PMC
March 2016

Regional Isolation Drives Bacterial Diversification within Cystic Fibrosis Lungs.

Cell Host Microbe 2015 Sep 20;18(3):307-19. Epub 2015 Aug 20.

Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA. Electronic address:

Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting ten CF lung pairs and studying ∼12,000 regional isolates, we were able to investigate whether clonally related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely diseased lung regions and found evolved differences in bacterial nutritional requirements, host defense and antibiotic resistance, and virulence due to hyperactivity of the type 3 secretion system. These findings suggest that bacterial intermixing is limited in CF lungs and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.
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http://dx.doi.org/10.1016/j.chom.2015.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589543PMC
September 2015

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

PLoS One 2015 19;10(8):e0133925. Epub 2015 Aug 19.

Department of Medicine, Division of Gastroenterology, University of Washington, Seattle, Washington, 98195, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544847PMC
May 2016

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

J Bacteriol 2015 Jun 6;197(12):2027-35. Epub 2015 Apr 6.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

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.
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http://dx.doi.org/10.1128/JB.00131-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438207PMC
June 2015

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

Proc Natl Acad Sci U S A 2015 Mar 23;112(10):E1096-105. Epub 2015 Feb 23.

Departments of Microbiology and

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.
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http://dx.doi.org/10.1073/pnas.1416651112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364195PMC
March 2015

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

Inflamm Bowel Dis 2015 Mar;21(3):556-63

*Department of Pediatrics, Division of Gastroenterology, Seattle Children's Hospital, University of Washington, Seattle, Washington; Departments of †Microbiology, and ‡Laboratory Medicine, University of Washington, Seattle, Washington; §Department of Pediatrics, Division of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, California; and Departments of ‖Medicine, ¶Immunology, and **Genome Sciences, University of Washington, Seattle, Washington.

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.
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http://dx.doi.org/10.1097/MIB.0000000000000307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329080PMC
March 2015

Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

J Bacteriol 2014 Nov 2;196(22):3862-71. Epub 2014 Sep 2.

Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA

Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei.
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http://dx.doi.org/10.1128/JB.01974-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248825PMC
November 2014

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

BMC Genomics 2014 May 10;15:355. Epub 2014 May 10.

Department of Microbiology, University of Washington, Seattle, WA, USA.

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.
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http://dx.doi.org/10.1186/1471-2164-15-355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038718PMC
May 2014

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Am J Pathol 2014 May 15;184(5):1309-22. Epub 2014 Mar 15.

Department of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.
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http://dx.doi.org/10.1016/j.ajpath.2014.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005986PMC
May 2014

Global analysis of the Burkholderia thailandensis quorum sensing-controlled regulon.

J Bacteriol 2014 Apr 24;196(7):1412-24. Epub 2014 Jan 24.

Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA.

Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei.
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http://dx.doi.org/10.1128/JB.01405-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993349PMC
April 2014

Escherichia coli dysbiosis correlates with gastrointestinal dysfunction in children with cystic fibrosis.

Clin Infect Dis 2014 Feb 30;58(3):396-9. Epub 2013 Oct 30.

Departments of Pediatrics.

Cystic fibrosis gastrointestinal disease includes nutrient malabsorption and intestinal inflammation. We show that the abundances of Escherichia coli in fecal microbiota were significantly higher in young children with cystic fibrosis than in controls and correlated with fecal measures of nutrient malabsorption and inflammation, suggesting that E. coli could contribute to cystic fibrosis gastrointestinal dysfunction.
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http://dx.doi.org/10.1093/cid/cit715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890337PMC
February 2014

Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

J Biol Chem 2013 Sep 22;288(37):26616-24. Epub 2013 Jul 22.

From the Departments of Microbiology and.

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.
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http://dx.doi.org/10.1074/jbc.M113.488320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772208PMC
September 2013

Genome sequence of Francisella tularensis subspecies holarctica strain FSC200, isolated from a child with tularemia.

J Bacteriol 2012 Dec;194(24):6965-6

CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden.

Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.
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http://dx.doi.org/10.1128/JB.01040-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510583PMC
December 2012

Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon.

Proc Natl Acad Sci U S A 2012 Oct 17;109(41):E2823-31. Epub 2012 Sep 17.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing-regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.
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http://dx.doi.org/10.1073/pnas.1214128109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478653PMC
October 2012

Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

Proc Natl Acad Sci U S A 2012 Aug 25;109(35):E2343-52. Epub 2012 Jul 25.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.
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http://dx.doi.org/10.1073/pnas.1206701109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435171PMC
August 2012

Identification of a p-coumarate degradation regulon in Rhodopseudomonas palustris by Xpression, an integrated tool for prokaryotic RNA-seq data processing.

Appl Environ Microbiol 2012 Oct 13;78(19):6812-8. Epub 2012 Jul 13.

Department of Microbiology, University of Washington, Seattle, Washington, USA.

High-throughput sequencing of cDNA prepared from RNA, an approach known as RNA-seq, is coming into increasing use as a method for transcriptome analysis. Despite its many advantages, widespread adoption of the technique has been hampered by a lack of easy-to-use, integrated, open-source tools for analyzing the nucleotide sequence data that are generated. Here we describe Xpression, an integrated tool for processing prokaryotic RNA-seq data. The tool is easy to use and is fully automated. It performs all essential processing tasks, including nucleotide sequence extraction, alignment, quantification, normalization, and visualization. Importantly, Xpression processes multiplexed and strand-specific nucleotide sequence data. It extracts and trims specific sequences from files and separately quantifies sense and antisense reads in the final results. Outputs from the tool can also be conveniently used in downstream analysis. In this paper, we show the utility of Xpression to process strand-specific RNA-seq data to identify genes regulated by CouR, a transcription factor that controls p-coumarate degradation by the bacterium Rhodopseudomonas palustris.
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http://dx.doi.org/10.1128/AEM.01418-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457485PMC
October 2012

Evolution of Burkholderia pseudomallei in recurrent melioidosis.

PLoS One 2012 15;7(5):e36507. Epub 2012 May 15.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352902PMC
September 2012

A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach.

Cell Host Microbe 2012 May;11(5):538-49

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.
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http://dx.doi.org/10.1016/j.chom.2012.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358704PMC
May 2012

Genotype-phenotype associations in a nonmodel prokaryote.

mBio 2012 20;3(2). Epub 2012 Mar 20.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

Unlabelled: To help define the biological functions of nonessential genes of Francisella novicida, we measured the growth of arrayed members of a comprehensive transposon mutant library under a variety of nutrition and stress conditions. Mutant phenotypes were identified for 37% of the genes, corresponding to ten carbon source utilization pathways, nine amino acid- and nucleotide-biosynthetic pathways, ten intrinsic antibiotic resistance traits, and six other stress resistance traits. The greatest surprise of the analysis was the large number of genotype-phenotype relationships that were not predictable from studies of Escherichia coli and other model species. The study identified candidate genes for a missing glycolysis function (phosphofructokinase), an unusual proline-biosynthetic pathway, parallel outer membrane lipid asymmetry maintenance systems, and novel antibiotic resistance functions. The analysis provides an evaluation of annotation predictions, identifies cases in which fundamental processes differ from those in model species, and helps create an empirical foundation for understanding virulence and other complex processes.

Importance: The value of genome sequences as foundations for analyzing complex traits in nonmodel organisms is limited by the need to rely almost exclusively on sequence similarities to predict gene functions in annotations. Many genes cannot be assigned functions, and some predictions are incorrect or incomplete. Due to these limitations, genome-scale experimental approaches that test and extend bioinformatics-based predictions are sorely needed. In this study, we describe such an approach based on phenotypic analysis of a comprehensive, sequence-defined transposon mutant library.
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http://dx.doi.org/10.1128/mBio.00001-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312209PMC
June 2012
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