A library of human gut bacterial isolates paired with longitudinal multiomics data enables mechanistic microbiome research.

Our understanding of how the gut microbiome interacts with its human host has been restrained by limited access to longitudinal datasets to examine stability and dynamics, and by having only a few isolates to test mechanistic hypotheses. Here, we present the Broad Institute-OpenBiome Microbiome Library (BIO-ML), a comprehensive collection of 7,758 gut bacterial isolates paired with 3,632 genome sequences and longitudinal multi-omics data. We show that microbial species maintain stable population sizes within and across humans and that commonly used 'omics' survey methods are more reliable when using averages over multiple days of sampling. Variation of gut metabolites within people over time is associated with amino acid levels, and differences across people are associated with differences in bile acids. Finally, we show that genomic diversification can be used to infer eco-evolutionary dynamics and in vivo selection pressures for strains within individuals. The BIO-ML is a unique resource designed to enable hypothesis-driven microbiome research.

Health-related quality of life after general surgical intensive care.

BACKGROUND: Impaired mental and physical health are common complications after intensive care that could influence the patient's health-related quality of life (HRQoL). Earlier research has mainly focused on HRQoL in mixed surgical and medical ICU populations. This study aimed to describe and analyze factors associated with HROoL after discharge from a general surgical ICU. METHODS: A prospective cohort study was conducted in a general surgical ICU in Sweden between 2005 and 2012. Adult patients (≥18 years) with an ICU length of stay ≥96 hours were included. HRQoL was measured at 3, 6, and 12 months after discharge from the ICU using a questionnaire (SF-36). A linear mixed model was used to analyze changes over time and Wilcoxon Signed Rank Tests were used to compare the 12-months results to an age and gender matched reference population in Sweden. Linear regression analyses were performed to explore the impact on HRQoL from background variables. RESULTS: Of 447 patients eligible for the study, 276 patients (62%) answered SF-36 at least once at 3, 6 or 12 months after ICU care and were included in the study. HRQoL improved over time but was still significantly lower at 12 months compared to the reference population. Female gender, age <75 years, living single, and ICU-stay of more than 14 days were associated with lower HRQoL. CONCLUSION: General surgical ICU patients reports low HRQoL 1 year after ICU stay. The impaired HRQoL could be a long-lasting problem with major consequences for the individual, family, and society.

Pasture flock chicken cecal microbiome responses to prebiotics and plum fiber feed amendments.

When prebiotics and other fermentation substrates are delivered to animals as feed supplements, the typical goal is to improve weight gain and feed conversion. In this work, we examined pasture flock chicken cecal contents using next generation sequencing (NGS) to identify and understand the composition of the microbiome when prebiotics and fermentation substrates were supplemented. We generated 16S rRNA sequencing data for 120 separate cecal samples from groups of chickens receiving one of 3 prebiotics or fiber feed additives. The data indicated that respective feed additives enrich for specific bacterial community members and modulate the diversity of the microbiome. We applied synthetic learning in microbial ecology (SLiME) analysis to interpret 16S rRNA microbial community data and identify specific bacterial operational taxonomic units (OTU) that are predictive of the particular feed additives used in these experiments. The results suggest that feed can influence microbiome composition in a predictable way, and thus diet may have indirect effects on weight gain and feed conversion through the microbiome.

Legionellosis acquired through a dental unit: a case study.

In 2012, an elderly immunocompromised man died from legionellosis at a hospital in Uppsala, Sweden. The patient had visited a dental ward at the hospital during the incubation period. Legionella spp. at a concentration of 2000 colony-forming units/L were isolated from the cupfiller outlet providing water for oral rinsing. Isolates from the patient and the dental unit were Legionella pneumophila serogroup 1, subgroup Knoxville and ST9. Pulsed-field gel electrophoresis and whole-genome sequencing strongly suggested that the isolates were of common origin. This report presents one of few documented cases of legionellosis acquired through a dental unit.

Emergence and spread of moxifloxacin-resistant Clostridium difficile ribotype 231 in Sweden between 2006 and 2015.

An aggregation of moxifloxacin-resistant Clostridium difficile ribotype 231 (RT231) isolates was first identified in the county of Stockholm in 2008, and by the end of 2015 isolates of RT231 had spread to 13 of 21 Swedish counties. We investigated the epidemiology of C. difficile RT231 in Sweden between 2006 and 2015 using whole genome sequencing (WGS) and evaluated whether its emergence could be associated with extended moxifloxacin use. We performed WGS and phylogenetic analysis of 51 C. difficile RT231 strains isolated in Sweden over a 10-year period. We also calculated the county-specific prescription rates for moxifloxacin between 2005 and 2015. Using WGS and detailed single nucleotide polymorphism analysis, we demonstrated three divergent sublineages of moxifloxacin-resistant C. difficile RT231 in Sweden from 2008 to 2015. A set of closely related RT231 was identified in hospitals located in the counties of Stockholm and Uppsala in 2008. Another set of RT231 isolates was found in four different counties in the Uppsala-Örebro Health Care Region. A gradual drop in moxifloxacin use in the county of Stockholm coincided with a reduction of RT231 in the area. However, RT231 continued to be frequent in surrounding counties including Uppsala, a county that also had the highest moxifloxacin prescription rates. We demonstrated frequent transmission of C. difficile RT231 within and between counties, indicating the importance of careful monitoring of hospitalized individuals infected with moxifloxacin-resistant C. difficile as well as the need for a strict moxifloxacin prescription policy.

Mobile genes in the human microbiome are structured from global to individual scales.

Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.

Association between fluid balance and mortality in patients with septic shock: a post hoc analysis of the TRISS trial.

BACKGROUND: Several studies have shown an association between a positive fluid balance and increased mortality in patients with septic shock. This may have led to a more restrictive use of intravenous fluids. The association between fluid accumulation and mortality in the setting of a more restrictive use of intravenous fluids, however, is uncertain. We therefore aimed to investigate the association between a cumulative fluid balance 3 days after randomization and 90-day mortality in a recent Nordic multicentre cohort of patients with septic shock. METHODS: A post hoc analysis of patients from the Transfusion Requirements in Septic Shock (TRISS) trial treated for 3 days or more in the ICU after randomization. The patients were categorized into four groups depending on their weight-adjusted cumulative fluid balance after 3 days. We performed multivariable Cox regression analysis, adjusting for important prognostics (study site, age, chronic cardiovascular and chronic lung disease, haematologic malignancy, chronic dialysis, source of infection, baseline SOFA score and plasma lactate). RESULTS: The median cumulative fluid balance of the 841 included patients was 2480 ml (IQR 47-5045). The median time from ICU admission to inclusion in the trial was 22 h. The overall 90-day mortality was 52%. There was no statistically significant association between fluid balance 3 days from inclusion and 90-day mortality after the adjustment for the prognostics (P = 0.37). CONCLUSION: In our cohort of patients with septic shock and a comparably low cumulative fluid balance, there was no association between fluid balance and mortality. However, the study design and the limited power preclude strong conclusions. There is an urgent need for high-quality trials assessing the benefit and harm of different fluid volume strategies in patients with septic shock.

Recurrent Clostridium difficile infection associates with distinct bile acid and microbiome profiles.

BACKGROUND: The healthy microbiome protects against the development of Clostridium difficile infection (CDI), which typically develops following antibiotics. The microbiome metabolises primary to secondary bile acids, a process if disrupted by antibiotics, may be critical for the initiation of CDI. AIM: To assess the levels of primary and secondary bile acids associated with CDI and associated microbial changes. METHODS: Stool and serum were collected from patients with (i) first CDI (fCDI), (ii) recurrent CDI (rCDI) and (iii) healthy controls. 16S rRNA sequencing and bile salt metabolomics were performed. Random forest regression models were constructed to predict disease status. PICRUSt analyses were used to test for associations between predicted bacterial bile salt hydrolase (BSH) gene abundances and bile acid levels. RESULTS: Sixty patients (20 fCDI, 19 rCDI and 21 controls) were enrolled. Secondary bile acids in stool were significantly elevated in controls compared to rCDI and fCDI (P < 0.0001 and P = 0.0007 respectively). Primary bile acids in stool were significantly elevated in rCDI compared to controls (P < 0.0001) and in rCDI compared to fCDI (P = 0.02). Using random forest regression, we distinguished rCDI and fCDI patients 84.2% of the time using bile acid ratios. Stool deoxycholate to glycoursodeoxycholate ratio was the single best predictor. PICRUSt analyses found significant differences in predicted abundances of bacterial BSH genes in stool samples across the groups. CONCLUSIONS: Primary and secondary bile acid composition in stool was different in those with rCDI, fCDI and controls. The ratio of stool deoxycholate to glycoursodeoxycholate was the single best predictor of disease state and may be a potential biomarker for recurrence.

Clostridium difficile associated risk of death score (CARDS): a novel severity score to predict mortality among hospitalised patients with C. difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) is a public health threat and associated with significant mortality. However, there is a paucity of objectively derived CDI severity scoring systems to predict mortality. AIM: To develop a novel CDI risk score to predict mortality entitled: Clostridium difficile associated risk of death score (CARDS). METHODS: We obtained data from the United States 2011 Nationwide Inpatient Sample (NIS) database. All CDI-associated hospitalisations were identified using discharge codes (ICD-9-CM, 008.45). Multivariate logistic regression was utilised to identify independent predictors of mortality. Clostridium difficile associated risk of death score was calculated by assigning a numeric weight to each parameter based on their odds ratio in the final logistic model. Predictive properties of model discrimination were assessed using the c-statistic and validated in an independent sample using the 2010 NIS database. RESULTS: We identified 77 776 hospitalisations, yielding an estimate of 374 747 cases with an associated diagnosis of CDI in the US, 8% of whom died in the hospital. The eight severity score predictors were identified on multivariate analysis: age, cardiopulmonary disease, malignancy, diabetes, inflammatory bowel disease, acute renal failure, liver disease and ICU admission, with weights ranging from -1 (for diabetes) to 5 (for ICU admission). The overall risk score in the cohort ranged from 0 to 18. Mortality increased significantly as CARDS increased. CDI-associated mortality was 1.2% with a CARDS of 0 compared to 100% with CARDS of 18. The model performed equally well in our validation cohort. CONCLUSION: Clostridium difficile associated risk of death score is a promising simple severity score to predict mortality among those hospitalised with C. difficile infection.

Ancestral Reconstruction of a Pre-LUCA Aminoacyl-tRNA Synthetase Ancestor Supports the Late Addition of Trp to the Genetic Code.

The genetic code was likely complete in its current form by the time of the last universal common ancestor (LUCA). Several scenarios have been proposed for explaining the code's pre-LUCA emergence and expansion, and the relative order of the appearance of amino acids used in translation. One co-evolutionary model of genetic code expansion proposes that at least some amino acids were added to the code by the ancient divergence of aminoacyl-tRNA synthetase (aaRS) families. Of all the amino acids used within the genetic code, Trp is most frequently claimed as a relatively recent addition. We observe that, since TrpRS and TyrRS are paralogous protein families retaining significant sequence similarity, the inferred sequence composition of their ancestor can be used to evaluate this co-evolutionary model of genetic code expansion. We show that ancestral sequence reconstructions of the pre-LUCA paralog ancestor of TyrRS and TrpRS have several sites containing Tyr, yet a complete absence of sites containing Trp. This is consistent with the paralog ancestor being specific for the utilization of Tyr, with Trp being a subsequent addition to the genetic code facilitated by a process of aaRS divergence and neofunctionalization. Only after this divergence could Trp be specifically encoded and incorporated into proteins, including the TyrRS and TrpRS descendant lineages themselves. This early absence of Trp is observed under both homogeneous and non-homogeneous models of ancestral sequence reconstruction. Simulations support that this observed absence of Trp is unlikely to be due to chance or model bias. These results support that the final stages of genetic code evolution occurred well within the "protein world," and that the presence-absence of Trp within conserved sites of ancient protein domains is a likely measure of their relative antiquity, permitting the relative timing of extremely early events within protein evolution before LUCA.

Patterns and persistence of antibiotic resistance in faecal indicator bacteria from freshwater recreational beaches.

AIMS: This study was conducted to determine antibiotic susceptibility patterns among the faecal indicator bacteria (FIB), Escherichia coli and enterococci, and to determine the potential for freshwater beaches to serve as reservoirs of resistance genes where transfer of resistant phenotypes takes place or de novo resistance may evolve. METHODS AND RESULTS: One hundred and forty-seven E. coli and 150 enterococci collected from sand and water at recreational beaches along Lake Huron, Michigan, USA were screened against commonly used antibiotics. Resistance was apparent in both E. coli (19% resistant) and enterococci (65% resistant). Antibiotic-resistant E. coli were capable of growing in beach sand microcosms and were able to transfer a plasmid-encoded kanamycin-resistance gene in sand microcosms. Furthermore, resistant phenotypes were stable in the sand environment even in the absence of the corresponding antibiotic. CONCLUSIONS: Antibiotic-resistant FIB were prevalent and persistent in the beach habitat. SIGNIFICANCE AND IMPACT OF THE STUDY: Active populations of FIB at beaches express antibiotic resistance phenotypes and have the ability to transfer antibiotic resistance. These human-associated bacteria may be intermediaries in the movement of resistance between environmental and clinical reservoirs.

Modifying the gastrointestinal ecology in alternatively raised poultry and the potential for molecular and metabolomic assessment.

Consumer demand for nonconventional poultry products continues to increase in the United States. In pasture flock and organic poultry production, probiotics and prebiotic feed additives have potential advantages because they are thought to promote intestinal health and may offer a replacement for current intervention strategies that are not considered acceptable for these production systems. Prebiotics have been demonstrated to produce effects on the gastrointestinal tract including modulation of microflora by promoting selective increases in beneficial bacteria concomitant with decreases in undesirable bacteria. In-depth assessment of microbial community changes during host growth and development as well as the establishment of beneficial microbial species by adding biologicals such as probiotics and prebiotics is important to achieve predictable and consistent improvements in chicken health and productivity. To analyze microflora shifts and metabolites produced by bacteria in the gut as well as host responses to biological additives, sophisticated molecular techniques are now available and are becoming more widely used. Polymerase chain reaction assays, denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis offer approaches for detecting microbial shifts in the gut. Likewise, the employment of microarrays and molecular analysis of gut tissues can reveal insight into gut physiological and responses to dietary and other changes. Recent application of 16S rDNA sequencing and analysis utilizing basic local alignment search tool (BLAST) and FASTA databases on poultry gut samples have the potential to provide a much more in-depth assessment of the gut microbiome. Utilizing ultra pressure liquid chromatography-mass spectroscopy profiling, metabolomic assessment of gut contents will also allow for parallel comparisons of changes in the gut contents with microbiome and physiological responses. Combining all these technologies will provide a plenary understanding of poultry gut health in alternative production systems.

Temporal transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into stationary phase during electron donor depletion.

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >/= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-gel electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.

Seasonal and spatial variability in Lake Michigan sediment small-subunit rRNA concentrations.

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4 degrees C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.

Distribution and abundance of Gram-positive bacteria in the environment: development of a group-specific probe.

We developed a 16S rRNA-targeted oligonucleotide probe (S-P-GPos-1200-a-A-13) for the Gram-positive bacteria, confirmed its specificity by database searches and hybridization studies, and investigated the effects of humic acids on membrane hybridizations with this probe. S-P-GPos-1200-a-A-13 was used to estimate the abundance of Gram-positive populations in the bovine rumen and Lake Michigan sediments. This probe should be useful for studies of the environmental distribution of Gram-positive bacteria and the detection of uncultured, phylogenetically Gram-positive bacteria with variable or negative Gram staining reactions, and could serve for Gram staining in some diagnostic settings.

Mechanisms of protein folding.

The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.

The presence of humic substances and DNA in RNA extracts affects hybridization results.

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.

Critical role of beta-hairpin formation in protein G folding.

Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the approximately 60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed beta-hairpins. We demonstrate that, as in the case of protein L, one of the two beta-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second beta-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain-main chain hydrogen bond in the second beta-turn slows the folding rate approximately 20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact beta-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated beta-hairpins.