The Microbiome of Complicated Diverticulitis: An Imbalance of Sulfur-Metabolizing Bacteria.

BACKGROUND: The progression to acute diverticulitis from the relatively benign condition of colonic diverticulosis is not well characterized. A smaller subset may even develop complicated (perforated) diverticulitis resulting in sepsis and/or death. Characterizing the differences between recurrent, uncomplicated diverticulitis, and the more virulent, complicated diverticulitis is necessary to guide clinical decision-making. Alterations to the microbiome offer a possible explanation for local inflammation and the pathophysiology of diverticular disease. OBJECTIVE: This study aimed to characterize the mucosal-associated microbiome in patients with recurrent uncomplicated diverticulitis and complicated (perforated) diverticulitis. DESIGN: Microbial DNA was extracted from full-thickness surgical specimens for 16S rRNA gene sequencing, targeting the V4 hypervariable region. Sequences were analyzed and a quantitative characterization based on taxonomic classification was performed. SETTING: A tertiary care academic medical center. PATIENTS: This study compared 48 patients with recurrent, uncomplicated diverticulitis and 35 patients with radiographically confirmed perforated (complicated) diverticulitis. Tissues were harvested from surgical resection specimens to include both diseased regions and nondiseased (adjacent normal) regions. MAIN OUTCOME MEASURES: We assessed differences in relative abundance and taxonomic classification of mucosal-associated microbes in surgical resection specimens from diverticular disease. RESULTS: When analyzing the tissue of diverticular resection specimens, the complicated diseased segments demonstrated an increased abundance of sulfur-reducing and sulfur-oxidizing bacteria compared to nondiseased, adjacent normal regions. When comparing diseased segments, tissues of patients with complicated diverticulitis had a marked increase in sulfur-reducing microbes. LIMITATIONS: We characterized the mucosal-associated microbiome present at the time of surgical resection, limiting conclusions on its role in pathophysiology. Furthermore, antibiotic usage and bowel preparation before surgery may result in perturbations to microbial flora. CONCLUSIONS: The microbiome of complicated diverticulitis is marked by a localized imbalance of sulfur-metabolizing microbes. The abundance of sulfur-reducing microbes may lead to an excess of hydrogen sulfide and subsequent inflammation. See Video Abstract at http://links.lww.com/DCR/C175 . LA MICROBIOMA DE LA DIVERTICULITIS COMPLICADA UN DESEQUILIBRIO DE LAS BACTERIAS METABOLIZADORAS DE AZUFRE: ANTECEDENTES: La progresión a diverticulitis aguda de la condición relativamente benigna de diverticulosis colónica no está bien caracterizada. Un subgrupo más pequeño puede incluso desarrollar diverticulitis complicada (perforada) que resulta en sepsis y/o muerte. Es necesario caracterizar las diferencias entre la diverticulitis recurrente no complicada y la diverticulitis complicada más virulenta para guiar la toma de decisiones clínicas. Las alteraciones del microbioma ofrecen una posible explicación de la inflamación local y la fisiopatología de la enfermedad diverticular.OBJETIVO: Caracterizar el microbioma asociado a la mucosa en pacientes con diverticulitis no complicada recurrente y diverticulitis complicada (perforada).DISEÑO: El ADN microbiano se extrajo de especímenes quirúrgicos de espesor completo para la secuenciación del gen 16S rRNA, dirigido a la región hipervariable V4. Se analizaron las secuencias y se realizó una caracterización cuantitativa basada en la clasificación taxonómica.AJUSTE: Un centro médico académico de atención terciaria.PACIENTES: Este estudio comparó 48 pacientes con diverticulitis recurrente no complicada y 35 pacientes con diverticulitis perforada (complicada) confirmada radiográficamente. Se recogieron tejidos de especímenes de resección quirúrgica para incluir tanto regiones enfermas como regiones no enfermas (normales adyacentes).PRINCIPALES MEDIDAS DE RESULTADO: Evaluamos las diferencias en la abundancia relativa y la clasificación taxonómica de los microbios asociados a la mucosa en muestras de resección quirúrgica de enfermedad diverticular.RESULTADOS: Al analizar el tejido de las muestras de resección diverticular, los segmentos enfermos complicados demostraron una mayor abundancia de bacterias reductoras de azufre y oxidantes de azufre en comparación con las regiones normales adyacentes no enfermas. Al comparar segmentos enfermos, los tejidos de pacientes complicados tenían un marcado aumento de microbios reductores de azufre.LIMITACIONES: Caracterizamos el microbioma asociado a la mucosa presente en el momento de la resección quirúrgica, lo que limita las conclusiones sobre su papel en la fisiopatología. Además, el uso de antibióticos y la preparación intestinal antes de la cirugía pueden provocar alteraciones en la flora microbiana.CONCLUSIONES: El microbioma de la diverticulitis complicada está marcado por un desequilibrio localizado de microbios metabolizadores de azufre. La abundancia de microbios reductores de azufre puede provocar un exceso de sulfuro de hidrógeno y la consiguiente inflamación. Consulte Video Resumen en http://links.lww.com/DCR/C175 . (Traducción-Dr. Ingrid Melo ).

Bacterial Swarmers Enriched During Intestinal Stress Ameliorate Damage.

BACKGROUND AND AIMS: Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. METHODS: We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. RESULTS: We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. CONCLUSIONS: Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

Synergistic effects of exercise and catalase overexpression on gut microbiome.

Exercise influences metabolic parameters in part by modulating redox stress and as recently suggested, by affecting the gut microbiome. However, whether excess endogenous antioxidant potentiates or interferes with the beneficial effects of exercise on the gut microbiome is not known. A comparison of the gut microbiome of C57Bl6 (C57/WT) mice to the 'stress-less' catalase overexpressing mice models ([Tg(CAT)± ] and Bob-Cat), that were either exercised or remained sedentary, showed differences in both alpha and beta diversity. The significant variation was explained by genotypes along with exercise, suggesting a synergistic relationship between exercise and genotypic traits. Linear discriminant analysis effect size (LEfSe) analysis also revealed differential taxa within the exercised/genotype cohorts in contrast to those within sedentary/genotype cohorts. Functional pathway predictions from PICRUSt2 showed enrichment for the metabolism of short-chain fatty acids, butanoate and propanoate pathways in exercised groups. Spearman correlations between enriched taxa and metabolic parameters showed correlations with body or fat weight in some of the cohorts. However, there were significant correlations of differential taxa among all cohorts against parameters that predict energy metabolism, such as respiratory exchange ratio and energy expenditure. Overall, our study showed that there was a synergistic beneficial influence of antioxidant overexpression and exercise on the gut microbiome.

Herbs and Spices Modulate Gut Bacterial Composition in Adults at Risk for CVD: Results of a Prespecified Exploratory Analysis from a Randomized, Crossover, Controlled-Feeding Study.

BACKGROUND: Herbs and spices are rich in polyphenolic compounds that may influence gut bacterial composition. The effect of culinary doses of herbs and spices consumed as part of a well-defined dietary pattern on gut bacterial composition has not been previously studied. OBJECTIVES: The aim of this prespecified exploratory analysis was to examine gut bacterial composition following an average American diet (carbohydrate: 50% kcal; protein: 17%; total fat: 33%; saturated fat: 11%) containing herbs and spices at 0.5, 3.3, and 6.6 g.d-1.2100 kcal-1 [low-, moderate-, and high-spice diets, respectively (LSD, MSD, and HSD)] in adults at risk for CVD. METHODS: Fifty-four adults (57% female; mean ± SD age: 45 ± 11 y; BMI: 29.8 ± 2.9 kg/m2; waist circumference: 102.8 ± 7.1 cm) were included in this 3-period, randomized, crossover, controlled-feeding study. Each diet was provided for 4 wk with a minimum 2-wk washout period. At baseline and the end of each diet period, participants provided a fecal sample for 16S rRNA gene (V4 region) sequencing. QIIME2 was used for data filtration, sequence clustering, taxonomy assignment, and statistical analysis. RESULTS: α-diversity assessed by the observed features metric ( P = 0.046) was significantly greater following the MSD as compared with the LSD; no other between-diet differences in α-diversity were detected. Differences in ß-diversity were not observed between the diets ( P = 0.45). Compared with baseline, ß-diversity differed following all diets ( P < .02). Enrichment of the Ruminococcaceae family was observed following the HSD as compared with the MSD (relative abundance = 22.14%, linear discriminant analysis = 4.22, P = 0.03) and the LSD (relative abundance = 24.90%, linear discriminant analysis = 4.47, P = 0.004). CONCLUSIONS: The addition of herbs and spices to an average American diet induced shifts in gut bacterial composition after 4 wk in adults at risk for CVD. The metabolic implications of these changes merit further investigation. This trial was registered at clinicaltrials.gov as NCT03064932.

Plasma Microbiome in COVID-19 Subjects: An Indicator of Gut Barrier Defects and Dysbiosis.

The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) complications. We aimed to investigate whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n = 146) and healthy individuals (n = 47) were collected during hospitalization and routine visits. Plasma microbiome was analyzed using 16S rRNA sequencing and gut permeability markers including fatty acid binding protein 2 (FABP2), peptidoglycan (PGN), and lipopolysaccharide (LPS) in both patient cohorts. Plasma samples of both cohorts contained predominately Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria. COVID-19 subjects exhibit significant dysbiosis (p = 0.001) of the plasma microbiome with increased abundance of Actinobacteria spp. (p = 0.0332), decreased abundance of Bacteroides spp. (p = 0.0003), and an increased Firmicutes:Bacteroidetes ratio (p = 0.0003) compared to healthy subjects. The concentration of the plasma gut permeability marker FABP2 (p = 0.0013) and the gut microbial antigens PGN (p < 0.0001) and LPS (p = 0.0049) were significantly elevated in COVID-19 patients compared to healthy subjects. These findings support the notion that the intestine may represent a source for bacteremia and contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

Exercise Training Reverses Gut Dysbiosis in Patients With Biopsy-Proven Nonalcoholic Steatohepatitis: A Proof of Concept Study.

Nonalcoholic fatty liver disease is the leading cause of liver disease worldwide and can progress to nonalcoholic steatohepatitis (NASH) through physical inactivity and gut dysbiosis.1 Exercise training reverses gut dysbiosis in non-NASH persons with obesity and in NASH animal models.2,3 Consequently, we conducted a proof-of-concept study investigating the effect of exercise training on gut dysbiosis in NASH patients.

Salmonella Genomics and Population Analyses Reveal High Inter- and Intraserovar Diversity in Freshwater.

Freshwater can support the survival of the enteric pathogen Salmonella, though temporal Salmonella diversity in a large watershed has not been assessed. At 28 locations within the Susquehanna River basin, 10-liter samples were assessed in spring and summer over 2 years. Salmonella prevalence was 49%, and increased river discharge was the main driver of Salmonella presence. The amplicon-based sequencing tool, CRISPR-SeroSeq, was used to determine serovar population diversity and detected 25 different Salmonella serovars, including up to 10 serovars from a single water sample. On average, there were three serovars per sample, and 80% of Salmonella-positive samples contained more than one serovar. Serovars Give, Typhimurium, Thompson, and Infantis were identified throughout the watershed and over multiple collections. Seasonal differences were evident: serovar Give was abundant in the spring, whereas serovar Infantis was more frequently identified in the summer. Eight of the ten serovars most commonly associated with human illness were detected in this study. Crucially, six of these serovars often existed in the background, where they were masked by a more abundant serovar(s) in a sample. Serovars Enteritidis and Typhimurium, especially, were masked in 71 and 78% of samples where they were detected, respectively. Whole-genome sequencing-based phylogeny demonstrated that strains within the same serovar collected throughout the watershed were also very diverse. The Susquehanna River basin is the largest system where Salmonella prevalence and serovar diversity have been temporally and spatially investigated, and this study reveals an extraordinary level of inter- and intraserovar diversity.IMPORTANCESalmonella is a leading cause of bacterial foodborne illness in the United States, and outbreaks linked to fresh produce are increasing. Understanding Salmonella ecology in freshwater is of importance, especially where irrigation practices or recreational use occur. As the third largest river in the United States east of the Mississippi, the Susquehanna River is the largest freshwater contributor to the Chesapeake Bay, and it is the largest river system where Salmonella diversity has been studied. Rainfall and subsequent high river discharge rates were the greatest indicators of Salmonella presence in the Susquehanna and its tributaries. Several Salmonella serovars were identified, including eight commonly associated with foodborne illness. Many clinically important serovars were present at a low frequency within individual samples and so could not be detected by conventional culture methods. The technologies employed here reveal an average of three serovars in a 10-liter sample of water and up to 10 serovars in a single sample.

Bone Marrow-Derived Cells Restore Functional Integrity of the Gut Epithelial and Vascular Barriers in a Model of Diabetes and ACE2 Deficiency.

RATIONALE: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. OBJECTIVE: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. METHODS AND RESULTS: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2-/y, Akita (type 1 diabetes mellitus), and ACE2-/y-Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2-/y-Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2-/y-Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. CONCLUSIONS: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2-/y-Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

Walnuts and Vegetable Oils Containing Oleic Acid Differentially Affect the Gut Microbiota and Associations with Cardiovascular Risk Factors: Follow-up of a Randomized, Controlled, Feeding Trial in Adults at Risk for Cardiovascular Disease.

BACKGROUND: It is unclear whether the favorable effects of walnuts on the gut microbiota are attributable to the fatty acids, including α-linolenic acid (ALA), and/or the bioactive compounds and fiber. OBJECTIVE: This study examined between-diet gut bacterial differences in individuals at increased cardiovascular risk following diets that replace SFAs with walnuts or vegetable oils. METHODS: Forty-two adults at cardiovascular risk were included in a randomized, crossover, controlled-feeding trial that provided a 2-wk standard Western diet (SWD) run-in and three 6-wk isocaloric study diets: a diet containing whole walnuts (WD; 57-99 g/d walnuts; 2.7% ALA), a fatty acid-matched diet devoid of walnuts (walnut fatty acid-matched diet; WFMD; 2.6% ALA), and a diet replacing ALA with oleic acid without walnuts (oleic acid replaces ALA diet; ORAD; 0.4% ALA). Fecal samples were collected following the run-in and study diets to assess gut microbiota with 16S rRNA sequencing and Qiime2 for amplicon sequence variant picking. RESULTS: Subjects had elevated BMI (30 ± 1 kg/m2), blood pressure (121 ± 2/77 ± 1 mmHg), and LDL cholesterol (120 ± 5 mg/dL). Following the WD, Roseburia [relative abundance (RA) = 4.2%, linear discriminant analysis (LDA) = 4], Eubacterium eligensgroup (RA = 1.4%, LDA = 4), LachnospiraceaeUCG001 (RA = 1.2%, LDA = 3.2), Lachnospiraceae UCG004 (RA = 1.0%, LDA = 3), and Leuconostocaceae (RA = 0.03%, LDA = 2.8) were most abundant relative to taxa in the SWD (P ≤ 0.05 for all). The WD was also enriched in Gordonibacter relative to the WFMD. Roseburia (3.6%, LDA = 4) and Eubacterium eligensgroup (RA = 1.5%, LDA = 3.4) were abundant following the WFMD, and Clostridialesvadin BB60group (RA = 0.3%, LDA = 2) and gutmetagenome (RA = 0.2%, LDA = 2) were most abundant following the ORAD relative to the SWD (P ≤ 0.05 for all). Lachnospiraceae were inversely correlated with blood pressure and lipid/lipoprotein measurements following the WD. CONCLUSIONS: The results indicate similar enrichment of Roseburia following the WD and WFMD, which could be explained by the fatty acid composition. Gordonibacter enrichment and the inverse association between Lachnospiraceae and cardiovascular risk factors following the WD suggest that the gut microbiota may contribute to the health benefits of walnut consumption in adults at cardiovascular risk. This trial was registered at clinicaltrials.gov as NCT02210767.

Enteric Virome and Carcinogenesis in the Gut.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in both the USA and the world. Recent research has demonstrated the involvement of the gut microbiota in CRC development and progression. Microbial biomarkers of disease have focused primarily on the bacterial component of the microbiome; however, the viral portion of the microbiome, consisting of both bacteriophages and eukaryotic viruses, together known as the virome, has been lesser studied. Here we review the recent advancements in high-throughput sequencing (HTS) technologies and bioinformatics, which have enabled scientists to better understand how viruses might influence the development of colorectal cancer. We discuss the contemporary findings revealing modulations in the virome and their correlation with CRC development and progression. While a variety of challenges still face viral HTS detection in clinical specimens, we consider herein numerous next steps for future basic and clinical research. Clinicians need to move away from a single infectious agent model for disease etiology by grasping new, more encompassing etiological paradigms, in which communities of various microbial components interact with each other and the host. The reporting and indexing of patient health information, socioeconomic data, and other relevant metadata will enable identification of predictive variables and covariates of viral presence and CRC development. Altogether, the virome has a more profound role in carcinogenesis and cancer progression than once thought, and viruses, specific for either human cells or bacteria, are clinically relevant in understanding CRC pathology, patient prognosis, and treatment development.

The role of fungi in C. difficile infection: An underappreciated transkingdom interaction.

Novel culture independent technologies have further elucidated the composition of the human mycobiome, though the role of fungi in human health and disease remains largely unknown. Recent studies have suggested conflicting roles for fungi in the gastrointestinal tract, underscoring the complexity of the interactions between the mycobiome, its bacterial counterpart, and the host. One key example is the observation that fungal taxa are overrepresented in patients with Clostridioides difficile infection (CDI), suggesting a role for fungi in this disease. Recent studies in murine models have demonstrated the ability of the commensal fungus Candida albicans to alter the course of CDI, supporting the notion that fungi play a role in this infection. This review summarizes current data on fungi and CDI, and shows that views of the dysbiotic state that is central to the pathogenesis of CDI are incomplete without consideration of the mycobiome.

Surface Water Microbial Community Response to the Biocide 2,2-Dibromo-3-Nitrilopropionamide, Used in Unconventional Oil and Gas Extraction.

Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA.IMPORTANCE Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.

HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants.

High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq.

Salmonella enterica is represented by >2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample. Poultry is a major reservoir of Salmonella spp., including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of Salmonella spp. in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003% of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species.IMPORTANCESalmonella enterica is the leading bacterial cause of foodborne illness in the United States and is represented by over 2,600 distinct serovars. Some of these serovars are pathogenic in humans, while others are not. Current surveillance for this pathogen is limited by the detection of only the most abundant serovars, due to the culture-based approaches that are used. Thus, pathogenic serovars that are present in a minority remain undetected. By exploiting serovar-specific differences in the CRISPR arrays of Salmonella spp., we have developed a high-throughput sequencing tool to be able to identify multiple serovars in a single sample and tested this in multiple poultry samples. This novel approach allows differences in the dynamics of individual Salmonella serovars to be measured and can have a significant impact on understanding the ecology of this pathogen with respect to zoonotic risk and public health.

Impacts of Glutaraldehyde on Microbial Community Structure and Degradation Potential in Streams Impacted by Hydraulic Fracturing.

The environmental impacts of hydraulic fracturing, particularly those of surface spills in aquatic ecosystems, are not fully understood. The goals of this study were to (1) understand the effect of previous exposure to hydraulic fracturing fluids on aquatic microbial community structure and (2) examine the impacts exposure has on biodegradation potential of the biocide glutaraldehyde. Microcosms were constructed from hydraulic fracturing-impacted and nonhydraulic fracturing-impacted streamwater within the Marcellus shale region in Pennsylvania. Microcosms were amended with glutaraldehyde and incubated aerobically for 56 days. Microbial community adaptation to glutaraldehyde was monitored using 16S rRNA gene amplicon sequencing and quantification by qPCR. Abiotic and biotic glutaraldehyde degradation was measured using ultra-performance liquid chromatography--high resolution mass spectrometry and total organic carbon. It was found that nonhydraulic fracturing-impacted microcosms biodegraded glutaraldehyde faster than the hydraulic fracturing-impacted microcosms, showing a decrease in degradation potential after exposure to hydraulic fracturing activity. Hydraulic fracturing-impacted microcosms showed higher richness after glutaraldehyde exposure compared to unimpacted streams, indicating an increased tolerance to glutaraldehyde in hydraulic fracturing impacted streams. Beta diversity and differential abundance analysis of sequence count data showed different bacterial enrichment for hydraulic fracturing-impacted and nonhydraulic fracturing-impacted microcosms after glutaraldehyde addition. These findings demonstrated a lasting effect on microbial community structure and glutaraldehyde degradation potential in streams impacted by hydraulic fracturing operations.

Natural Attenuation of Nonionic Surfactants Used in Hydraulic Fracturing Fluids: Degradation Rates, Pathways, and Mechanisms.

Hydraulic fracturing fluids are injected into shales to extend fracture networks that enhance oil and natural gas production from unconventional reservoirs. Here we evaluated the biodegradability of three widely used nonionic polyglycol ether surfactants (alkyl ethoxylates (AEOs), nonylphenol ethoxylates (NPEOs), and polypropylene glycols (PPGs)) that function as weatherizers, emulsifiers, wetting agents, and corrosion inhibitors in injected fluids. Under anaerobic conditions, we observed complete removal of AEOs and NPEOs from solution within 3 weeks regardless of whether surfactants were part of a chemical mixture or amended as individual additives. Microbial enzymatic chain shortening was responsible for a shift in ethoxymer molecular weight distributions and the accumulation of the metabolite acetate. PPGs bioattenuated the slowest, producing sizable concentrations of acetone, an isomer of propionaldehyde. Surfactant chain shortening was coupled to an increased abundance of the diol dehydratase gene cluster (pduCDE) in Firmicutes metagenomes predicted from the 16S rRNA gene. The pduCDE enzymes are responsible for cleaving ethoxylate chain units into aldehydes before their fermentation into alcohols and carboxylic acids. These data provide new mechanistic insight into the environmental fate of hydraulic fracturing surfactants after accidental release through chain shortening and biotransformation, emphasizing the importance of compound structure disclosure for predicting biodegradation products.

Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy.

UNLABELLED: Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. IMPORTANCE: In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment.

Diets high in resistant starch increase plasma levels of trimethylamine-N-oxide, a gut microbiome metabolite associated with CVD risk.

Production of trimethylamine-N-oxide (TMAO), a biomarker of CVD risk, is dependent on intestinal microbiota, but little is known of dietary conditions promoting changes in gut microbial communities. Resistant starches (RS) alter the human microbiota. We sought to determine whether diets varying in RS and carbohydrate (CHO) content affect plasma TMAO levels. We also assessed postprandial glucose and insulin responses and plasma lipid changes to diets high and low in RS. In a cross-over trial, fifty-two men and women consumed a 2-week baseline diet (41 percentage of energy (%E) CHO, 40 % fat, 19 % protein), followed by 2-week high- and low-RS diets separated by 2-week washouts. RS diets were assigned at random within the context of higher (51-53 %E) v. lower CHO (39-40 %E) intake. Measurements were obtained in the fasting state and, for glucose and insulin, during a meal test matching the composition of the assigned diet. With lower CHO intake, plasma TMAO, carnitine, betaine and γ-butyrobetaine concentrations were higher after the high- v. low-RS diet (P<0·01 each). These metabolites were not differentially affected by high v. low RS when CHO intake was high. Although the high-RS meal reduced postprandial insulin and glucose responses when CHO intake was low (P<0·01 each), RS did not affect fasting lipids, lipoproteins, glucose or insulin irrespective of dietary CHO content. In conclusion, a lower-CHO diet high in RS was associated with higher plasma TMAO levels. These findings, together with the absence of change in fasting lipids, suggest that short-term high-RS diets do not improve markers of cardiometabolic health.

FOAM (Functional Ontology Assignments for Metagenomes): a Hidden Markov Model (HMM) database with environmental focus.

A new functional gene database, FOAM (Functional Ontology Assignments for Metagenomes), was developed to screen environmental metagenomic sequence datasets. FOAM provides a new functional ontology dedicated to classify gene functions relevant to environmental microorganisms based on Hidden Markov Models (HMMs). Sets of aligned protein sequences (i.e. 'profiles') were tailored to a large group of target KEGG Orthologs (KOs) from which HMMs were trained. The alignments were checked and curated to make them specific to the targeted KO. Within this process, sequence profiles were enriched with the most abundant sequences available to maximize the yield of accurate classifier models. An associated functional ontology was built to describe the functional groups and hierarchy. FOAM allows the user to select the target search space before HMM-based comparison steps and to easily organize the results into different functional categories and subcategories. FOAM is publicly available at http://portal.nersc.gov/project/m1317/FOAM/.

Diversity and dynamics of dominant and rare bacterial taxa in replicate sequencing batch reactors operated under different solids retention time.

In this study, 16S rRNA gene pyrosequencing was applied in order to provide a better insight on the diversity and dynamics of total, dominant, and rare bacterial taxa in replicate lab-scale sequencing batch reactors (SBRs) operated at different solids retention time (SRT). Rank-abundance curves showed few dominant operational taxonomic units (OTUs) and a long tail of rare OTUs in all reactors. Results revealed that there was no detectable effect of SRT (2 vs. 10 days) on Shannon diversity index and OTU richness of both dominant and rare taxa. Nonmetric multidimensional scaling analysis showed that the total, dominant, and rare bacterial taxa were highly dynamic during the entire period of stable reactor performance. Also, the rare taxa were more dynamic than the dominant taxa despite expected low invasion rates because of the use of sterile synthetic media.