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 ).

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.

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.

Clostridioides difficile infection is associated with differences in transcriptionally active microbial communities.

Clostridioides difficile infection (CDI) is responsible for around 300,000 hospitalizations yearly in the United States, with the associated monetary cost being billions of dollars. Gut microbiome dysbiosis is known to be important to CDI. To the best of our knowledge, metatranscriptomics (MT) has only been used to characterize gut microbiome composition and function in one prior study involving CDI patients. Therefore, we utilized MT to investigate differences in active community diversity and composition between CDI+ (n = 20) and CDI- (n = 19) samples with respect to microbial taxa and expressed genes. No significant (Kruskal-Wallis, p > 0.05) differences were detected for richness or evenness based on CDI status. However, clustering based on CDI status was significant for both active microbial taxa and expressed genes datasets (PERMANOVA, p ≤ 0.05). Furthermore, differential feature analysis revealed greater expression of the opportunistic pathogens Enterocloster bolteae and Ruminococcus gnavus in CDI+ compared to CDI- samples. When only fungal sequences were considered, the family Saccharomycetaceae expressed more genes in CDI-, while 31 other fungal taxa were identified as significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) associated with CDI+. We also detected a variety of genes and pathways that differed significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) based on CDI status. Notably, differential genes associated with biofilm formation were expressed by C. difficile. This provides evidence of another possible contributor to C. difficile's resistance to antibiotics and frequent recurrence in vivo. Furthermore, the greater number of CDI+ associated fungal taxa constitute additional evidence that the mycobiome is important to CDI pathogenesis. Future work will focus on establishing if C. difficile is actively producing biofilms during infection and if any specific fungal taxa are particularly influential in CDI.

Walnut consumption and gut microbial metabolism: Results of an exploratory analysis from a randomized, crossover, controlled-feeding study.

BACKGROUND & AIMS: The effect of walnut-related modulation of gut microbiota composition on microbiota functionality is unknown. The aim was to characterize the effect of a walnut-enriched diet (WD), compared to a fatty acid-matched diet devoid of walnuts (WFMD) and a diet where oleic acid replaces alpha-linolenic acid (ORAD), on bacterial gene expression. METHODS: A 3-period, randomized, crossover, controlled-feeding study was conducted. Participants were provided a 2-week run-in standard western diet (SWD; 50% kcal carbohydrate, 16% protein, 34% fat, 12% SFA). Following the SWD in random sequence order, participants were provided the WD, WFMD, and ORAD (48% carbohydrate; 17% protein; fat 35%; 7% SFA). The WD contained 18% of energy from walnuts (57 g/d/2100 kcal). The WFMD and ORAD were devoid of walnuts; liquid non-tropical plant oils were included in these diets. Metatranscriptomic analyses were performed as an exploratory outcome. RESULTS: The analytical sample included 35 participants (40% female) with a mean ± SD age of 43 ± 10 y and BMI of 30.3 ± 4.9 kg/m2. The ⍺-diversity of taxa actively expressing genes, assessed by observed species (p = 0.27) and Pielou's Evenness (p = 0.09), did not differ among the diets. The ⍺-diversity of actively expressed genes was greater following the WD compared to the WFMD and ORAD as assessed by the observed genes and Pielou's Evenness metrics (p < 0.05). ß-Diversity of the actively expressed genes differed following the WD compared to the WFMD (p = 0.001) and ORAD (p = 0.001); ß-diversity did not differ between the WFMD and ORAD. Active composition analyses showed increased Gordonibacter (p < 0.001) activity following the WD vs. the ORAD. Greater expression of many genes was observed following the WD compared to the WFMD and ORAD. Following the WD, greater expression of metabolism-related genes encoding glycine amidinotransferase (GATM; K00613) and arginine deiminase (K01478) was observed compared to the WFMD. Greater expression of glycine amidinotransferase (GATM; K00613) by Gordonibacter was also observed following the WD vs. the WFMD and ORAD. CONCLUSION: Our results suggest walnut intake may increase endogenous production of homoarginine through gut microbiota-mediated upregulation of GATM, which is a novel mechanism by which walnuts may lower cardiovascular disease risk. However, given the exploratory nature replication is needed. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov (NCT02210767).

Assessment of a novel continuous cleaning device using metatranscriptomics in diverse hospital environments.

Introduction: Despite routine implementation of cleaning and disinfection practices in clinical healthcare settings, high-touch environmental surfaces and contaminated equipment often serve as reservoirs for the transmission of pathogens associated with healthcare-associated infections (HAIs). Methods: The current study involved the analysis of high-touch surface swabs using a metatranscriptomic sequencing workflow (CSI-Dx™) to assess the efficacy of cleanSURFACES® technology in decreasing microbial burden by limiting re-contamination. This is a non-human single center study conducted in the Emergency Department (ED) and on an inpatient Oncology Ward of Walter Reed National Military Medical Center that have followed hygienic practices during the COVID-19 pandemic environment. Results: Although there was no difference in observed microbial richness (two-tailed Wilcoxon test with Holm correction, P > 0.05), beta diversity findings identified shifts in microbial community structure between surfaces from baseline and post-intervention timepoints (Day 1, Day 7, Day 14, and Day 28). Biomarker and regression analyses identified significant reductions in annotated transcripts for various clinically relevant microorganisms' post-intervention, coagulase-negative staphylococci and Malassezia restricta, at ED and Oncology ward, respectively. Additionally, post-intervention samples predominantly consisted of Proteobacteria and to a lesser extent skin commensals and endogenous environmental microorganisms in both departments. Discussion: Findings support the value of cleanSURFACES®, when coupled with routine disinfection practices, to effectively impact on the composition of active microbial communities found on high-touch surfaces in two different patient care areas of the hospital (one outpatient and one inpatient) with unique demands and patient-centered practices.

Peanuts as a nighttime snack enrich butyrate-producing bacteria compared to an isocaloric lower-fat higher-carbohydrate snack in adults with elevated fasting glucose: A randomized crossover trial.

BACKGROUND: Tree nuts have glucoregulatory effects and influence gut microbiota composition. The effect of peanuts on the microbiota has not been investigated. OBJECTIVES: The aim was to examine the effect of 28 g/d of peanuts for 6-wks, compared to an isocaloric lower-fat higher-carbohydrate (LFHC) snack, on gut microbiota composition. A secondary aim was to identify functional and active compositional differences in a subset of participants using metatranscriptomics. METHODS: In a randomized, crossover trial, 50 adults (48% female; 42 ± 15 y; BMI 28.3 ± 5.6 kg/m2; plasma glucose 100 ± 8 mg/dL) consumed 28 g/d of dry roasted, unsalted, peanuts (164 kcal; 11% E carbohydrate, 17% E protein, 73% E fat, and 2.4 g fiber) or a LFHC snack (164 kcal; 53% E carbohydrate, 17% E protein, 33% E fat, and 3 g fiber) for 6-wk (4-wk washout period). Gut bacterial composition was measured using 16S rRNA sequencing in the whole cohort. Exploratory metatranscriptomic analyses were conducted on a random subset (n = 24) of samples from the Peanut condition. RESULTS: No between-condition differences in α- or ß- diversity were observed. Following peanut intake, Ruminococcaceae were significantly more abundant [Linear discriminant analysis score (LDA) = 2.8; P = 0.027)] compared to LFHC. Metatranscriptomics showed increased expression of the K03518 (aerobic carbon-monoxide dehydrogenase small subunit) gene following peanut intake (LDA = 2.0; P = 0.004) and Roseburia intestinalis L1-82 was identified as a contributor to the increased expression. CONCLUSION: An increased abundance of Ruminococcaceae was observed following consumption of 28 g/d of peanuts in adults with elevated fasting glucose after 6-wks. Metatranscriptomics revealed increased expression of the K03518 gene. These results suggest peanut intake enriches a known butyrate producer and the increased expression of a gene implicated in butyrate production adds further support for peanut-induced gut microbiome modulation. NCT: 03654651.

Altered gut microbiota composition with antibiotic treatment impairs functional recovery after traumatic peripheral nerve crush injury in mice: effects of probiotics with butyrate producing bacteria.

OBJECTIVE: Antibiotics (ABX) are widely used for life-threatening infections and also for routine surgical operations. Compelling evidence suggests that ABX-induced alterations of gut microbiota composition, termed dysbiosis, are linked with diverse disease states including neurological and neurodegenerative conditions. To combat the consequences of dysbiosis, probiotics (PBX) are widely used. ABX-induced dysbiosis is reported to impair neurological function after spinal cord injury. Traumatic peripheral nerve injury (TPNI) results in profound neurologic impairment and permanent disability. It is unknown whether ABX treatment-induced dysbiosis has any impact on TPNI-induced functional recovery, and if so, what role medical-grade PBX could have on TPNI recovery. RESULTS: In this study, ABX-induced dysbiosis and PBX-induced microbiota enrichment models were used to explore the potential role of gut microbiome in TPNI. Stool analysis with 16S ribosomal RNA (rRNA) gene sequencing confirmed ABX-induced dysbiosis and revealed that ABX-induced changes could be partially restored by PBX administration with an abundance of butyrate producing bacteria. Pre-injury ABX significantly impaired, but pre-injury PBX significantly improved post-TPNI functional recovery. Importantly, post-injury PBX protected against pre-injury ABX-induced functional impairment. These findings demonstrate that reestablishment of gut microbiota composition with butyrate producing PBX during ABX-induced dysbiosis could be a useful adjuvant therapy for TPNI.

Geochemistry and Multiomics Data Differentiate Streams in Pennsylvania Based on Unconventional Oil and Gas Activity.

Unconventional oil and gas (UOG) extraction is increasing exponentially around the world, as new technological advances have provided cost-effective methods to extract hard-to-reach hydrocarbons. While UOG has increased the energy output of some countries, past research indicates potential impacts in nearby stream ecosystems as measured by geochemical and microbial markers. Here, we utilized a robust data set that combines 16S rRNA gene amplicon sequencing (DNA), metatranscriptomics (RNA), geochemistry, and trace element analyses to establish the impact of UOG activity in 21 sites in northern Pennsylvania. These data were also used to design predictive machine learning models to determine the UOG impact on streams. We identified multiple biomarkers of UOG activity and contributors of antimicrobial resistance within the order Burkholderiales. Furthermore, we identified expressed antimicrobial resistance genes, land coverage, geochemistry, and specific microbes as strong predictors of UOG status. Of the predictive models constructed (n = 30), 15 had accuracies higher than expected by chance and area under the curve values above 0.70. The supervised random forest models with the highest accuracy were constructed with 16S rRNA gene profiles, metatranscriptomics active microbial composition, metatranscriptomics active antimicrobial resistance genes, land coverage, and geochemistry (n = 23). The models identified the most important features within those data sets for classifying UOG status. These findings identified specific shifts in gene presence and expression, as well as geochemical measures, that can be used to build robust models to identify impacts of UOG development. IMPORTANCE The environmental implications of unconventional oil and gas extraction are only recently starting to be systematically recorded. Our research shows the utility of microbial communities paired with geochemical markers to build strong predictive random forest models of unconventional oil and gas activity and the identification of key biomarkers. Microbial communities, their transcribed genes, and key biomarkers can be used as sentinels of environmental changes. Slight changes in microbial function and composition can be detected before chemical markers of contamination. Potential contamination, specifically from biocides, is especially concerning due to its potential to promote antibiotic resistance in the environment. Additionally, as microbial communities facilitate the bulk of nutrient cycling in the environment, small changes may have long-term repercussions. Supervised random forest models can be used to identify changes in those communities, greatly enhance our understanding of what such impacts entail, and inform environmental management decisions.

cleanSURFACES® intervention reduces microbial activity on surfaces in a senior care facility.

As one of the top public health challenges outlined by the Centers for Disease Control (CDC), estimates report that hospital acquired infections (HAIs) claim the lives of 99,000 Americans and cost healthcare providers over $28 billion each year. In addition to underlying conditions related to age, elderly patients in long-term care facilities are at an elevated risk of acquiring HAIs. A large percentage of HAIs is attributable to contaminated surfaces and medical devices. To that end, this study utilized a metatranscriptomic sequencing workflow (CSI-Dx™) to profile active microbial communities from surfaces in the HJ Heinz Community Living Center, a long-term care facility in the Veterans Affairs Pittsburgh Health Care System. Swabs were collected from high-touch surfaces (Keyboard, Ledge, Workstation on Wheels, Worksurfaces) before (Baseline) and after cleanSURFACES® were installed at 4 timepoints (Day 1, Day 7, Day 14, and Day 30). Microbial richness was significantly reduced after cleanSURFACES® intervention (Wilcoxon test with Holm correction, p=0.000179). Beta diversity results revealed distinct clustering between Baseline and Post-intervention samples (Adonis, p<0.001). Reduction in bacterial (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis) and fungal (Malassezia restricta, Candida albicans, Candida glabrata, and Candida orthopsilosis) expression of opportunistic pathogens was observed. Additionally, a subset of taxa (Corynebacterium, Cutibacterium acnes, and Ralstonia pickettii) was present in specific Post-intervention timepoints and surface types. This study revealed decreased microbial activity, highlighting the potential for the combinatorial application of cleanSURFACES® and regular decontamination practices to reduce the prevalence of microbes causing HAIs.

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures.

Hydraulic fracturing (HF), commonly called "fracking", uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking's impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Comparative study of the effects of biocides and metal oxide nanoparticles on microbial community structure in a stream impacted by hydraulic fracturing.

Our study goal was to investigate the impact of biocides and nanoparticles (NPs) on the microbial diversity in a hydraulic fracturing impacted stream. Biocides and NPs are known for their antimicrobial properties and controlling microbial growth. Previous work has shown that biocides can alter the microbial community composition of stream water and may select for biocide-resistant bacteria. Additional studies have shown that nanoparticles can also alter microbial community composition. However, previous work has often focused on the response to a single compound. Here we provide a more thorough analysis of the microbial community response to three different biocides and three different nanoparticles. A microcosm-based study was undertaken that exposed stream microbial communities to either biocides or NPs. Our results showed a decrease in bacterial abundance with different types of nanoparticles, but an increase in microbial abundance in biocide-amended treatments. The microbial community composition (MCC) was distinct from the controls in all biocide and NP treatments, which resulted in differentially enriched taxa in the treatments compared to the controls. Our results indicate that NPs slightly altered the MCC compared to the biocide-treated microcosms. After 14 days, the MCC in the nanoparticle-treated conditions was similar to the MCC in the control. Conversely, the MCC in the biocide-treated microcosms was distinct from the controls at day 14 and distinct from all conditions at day 0. This finding may point to the use of NPs as an alternative to biocides in some settings.

Temporal Transcriptomics of Gut Escherichia coli in Caenorhabditis elegans Models of Aging.

Host-bacterial interactions over the course of aging are understudied due to complexities of the human microbiome and challenges of collecting samples that span a lifetime. To investigate the role of host-microbial interactions in aging, we performed transcriptomics using wild-type Caenorhabditis elegans (N2) and three long-lived mutants (daf-2, eat-2, and asm-3) fed Escherichia coli OP50 and sampled at days 5, 7.5, and 10 of adulthood. We found host age is a better predictor of the E. coli expression profiles than host genotype. Specifically, host age was associated with clustering (permutational multivariate analysis of variance [PERMANOVA], P = 0.001) and variation (Adonis, P = 0.001, R2 = 11.5%) among E. coli expression profiles, whereas host genotype was not (PERMANOVA, P > 0.05; Adonis, P > 0.05, R2 = 5.9%). Differential analysis of the E. coli transcriptome yielded 22 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 100 KEGG genes enriched when samples were grouped by time point [LDA, linear discriminant analysis; log(LDA), ≥2; P ≤ 0.05], including several involved in biofilm formation. Coexpression analysis of host and bacterial genes yielded six modules of C. elegans genes that were coexpressed with one bacterial regulator gene over time. The three most significant bacterial regulators included genes relating to biofilm formation, lipopolysaccharide production, and thiamine biosynthesis. Age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, building on a growing literature of host-microbial interactions. IMPORTANCE Previous research has reported effects of the microbiome on health span and life span of Caenorhabditis elegans, including interactions with evolutionarily conserved pathways in humans. We build on this literature by reporting the gene expression of Escherichia coli OP50 in wild-type (N2) and three long-lived mutants of C. elegans. The manuscript represents the first study, to our knowledge, to perform temporal host-microbial transcriptomics in the model organism C. elegans. Understanding changes to the microbial transcriptome over time is an important step toward elucidating host-microbial interactions and their potential relationship to aging. We found that age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, which contributes to our growing knowledge about host-microbial interactions.

Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania.

Unconventional oil and gas (UOG) extraction, also known as hydraulic fracturing, is becoming more prevalent with the increasing use and demand for natural gas; however, the full extent of its environmental impacts is still unknown. Here we measured physicochemical properties and bacterial community composition of sediment samples taken from twenty-eight streams within the Marcellus shale formation in northeastern Pennsylvania differentially impacted by hydraulic fracturing activities. Fourteen of the streams were classified as UOG+, and thirteen were classified as UOG- based on the presence of UOG extraction in their respective watersheds. One stream was located in a watershed that previously had UOG extraction activities but was recently abandoned. We utilized high-throughput sequencing of the 16S rRNA gene to infer differences in sediment aquatic bacterial community structure between UOG+ and UOG- streams, as well as correlate bacterial community structure to physicochemical water parameters. Although overall alpha and beta diversity differences were not observed, there were a plethora of significantly enriched operational taxonomic units (OTUs) within UOG+ and UOG- samples. Our biomarker analysis revealed many of the bacterial taxa enriched in UOG+ streams can live in saline conditions, such as Rubrobacteraceae. In addition, several bacterial taxa capable of hydrocarbon degradation were also enriched in UOG+ samples, including Oceanospirillaceae. Methanotrophic taxa, such as Methylococcales, were significantly enriched as well. Several taxa that were identified as enriched in these samples were enriched in samples taken from different streams in 2014; moreover, partial least squares discriminant analysis (PLS-DA) revealed clustering between streams from the different studies based on the presence of hydraulic fracturing along the second axis. This study revealed significant differences between bacterial assemblages within stream sediments of UOG+ and UOG- streams and identified several potential biomarkers for evaluating and monitoring the response of autochthonous bacterial communities to potential hydraulic fracturing impacts.