Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Sexuality, risk, and organisational misbehaviour in a secure mental healthcare facility in England.

Sexuality in secure mental healthcare has been overlooked in both clinical praxis and academic research. In the UK, there exist no formal policies to inform staff approaches to managing inpatient sexuality. The limited research that has been undertaken in this field has found that often, prohibitive approaches are favoured, which may affect how inpatients conceptualise and experience their sexuality in the long-term. The aim of this study was to identify discursive constructions of inpatient sexuality, as articulated in semi-structured group interviews with inpatients and ward staff from a secure mental healthcare facility in England. The analysis identified constructions of inpatient sexuality within two overarching and conflicting discourses: one of the normalcy and legitimacy of sexual expression in human experience; and the other of risk, wherein sexuality needed to be regulated and obstructed. Inpatients' expressions of sexuality could often only be conceptualised in terms of 'organisational misbehaviour', acts that violated the implicit norms and codes of the institution. It is suggested that recoding inpatient sexuality as misbehaviour could have implications for inpatients' long-term recovery.

The effects of provider-prescribed obesogenic drugs on post-laparoscopic sleeve gastrectomy outcomes: a retrospective cohort study.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is one of the most commonly performed bariatric procedures and has proven effective in providing weight loss. However, considerable variance has been noted in the degree of weight loss. Physician prescription practices may be negatively affecting weight loss post-LSG and, thus, contributing to the broad range of weight loss outcomes. The aim of our study was to determine whether commonly prescribed obesogenic medications negatively affect weight loss outcomes post-LSG. SUBJECTS/METHODS: This single center retrospective cohort study performed at a University hospital included 323 patients (≥18 years) within the University California, San Diego Healthcare System who underwent LSG between 2007 and 2016. We identified a list of 32 commonly prescribed medications that have weight gain as a side effect. We compared the percent excess weight loss (%EWL) of patients divided into two groups based on post-LSG exposure to obesogenic medications. A linear regression model was used to analyze %EWL at 12 months post-LSG while controlling for age, initial body mass index (BMI), and use of leptogenic medications. RESULTS: A total of 150 patients (Meds group) were prescribed obesogenic medications within the one-year post-LSG follow up period, whereas 173 patients (Control group) were not prescribed obesogenic medications. The Meds group lost significantly less weight compared to the Control group (%EWL ± SEM at 12 months 53.8 ± 2.4 n = 78, 65.0 ± 2.6, n = 84 respectively, P = 0.002). This difference could not be attributed to differences in age, gender, initial BMI, co-morbidities, or prescription of leptogenic medications between the two groups. CONCLUSIONS: The use of provider-prescribed obesogenic medications was associated with worse weight loss outcomes post-LSG. Closer scrutiny of patient medications may be necessary to help improve outcomes of weight loss treatments.

Pseudodesulfovibrio mercurii sp. nov., a mercury-methylating bacterium isolated from sediment.

The sulfate-reducing, mercury-methylating strain ND132T was isolated from the brackish anaerobic bottom sediments of Chesapeake Bay, USA. Capable of high levels of mercury (Hg) methylation, ND132T has been widely used as a model strain to study the process and to determine the genetic basis of Hg methylation. Originally called Desulfovibrio desulfuricans ND132T on the basis of an early partial 16S rRNA sequence, the strain has never been formally described. Phylogenetic and physiological traits place this strain within the genus Pseudodesulfovibrio, in the recently reclassified phylum Desulfobacterota (formerly Deltaproteobacteria). ND132T is most closely related to Pseudodesulfovibrio hydrargyri BerOc1T and Pseudodesulfovibrio indicus J2T. Analysis of average nucleotide identity (ANI) of whole-genome sequences showed roughly 88 % ANI between P. hydrargyri BerOc1T and ND132T, and 84 % similarity between ND132T and P. indicus J2T. These cut-off scores <95 %, along with a multi-gene phylogenetic analysis of members of the family Desulfovibrionacea, and differences in physiology indicate that all three strains represent separate species. The Gram-stain-negative cells are vibrio-shaped, motile and not sporulated. ND132T is a salt-tolerant mesophile with optimal growth in the laboratory at 32 °C, 2 % salinity, and pH 7.8. The DNA G+C content of the genomic DNA is 65.2 %. It is an incomplete oxidizer of short chain fatty acids, using lactate, pyruvate and fumarate with sulfate or sulfite as the terminal electron acceptors. ND132T can respire fumarate using pyruvate as an electron donor. The major fatty acids are iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0, iso-C17 : 1ω9c and anteiso-C17 : 0. We propose the classification of strain ND132T (DSM 110689, ATCC TSD-224) as the type strain Pseudodesulfovibrio mercurii sp. nov.

Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations.

Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03-14 mg kg-1 dry wt. soil) and MeHg (0.05-27 µg kg-1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB+ species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.

Vital spaces and mental health.

The impact of social and material conditions on mental health is well established but lacking in a coherent approach. We offer the concept of 'vitality' as means of describing how environments facilitate 'feelings of being alive' that cut across existing diagnostic categories. Drawing on the work of Stern, Fuchs, Worms and Duff, we argue that vitality is not solely a quality of an individual body, but rather emerges from attunements and resonances between bodies and materials. We use vitality as a lens to explore how movements within and between assembled sets of relations can facilitate or disable feelings and expressions of being alive. Building on extended discussions of both inpatient and community-based mental healthcare, we sketch out a research agenda for analysing 'vital spaces'.

Curriculum requirements and subsequent civic engagement: is there a difference between 'forced' and 'free' community service?

Despite figures showing the growth of mandatory community service programmes, there is mixed empirical evidence of their effectiveness. This paper addresses the relationship of mandated community service to one of its purported aims: subsequent volunteerism. It compares current volunteerism among four university student cohorts: those doing no service in secondary school, those volunteering with no requirement, those volunteering both before and after the introduction of a requirement, and those introduced to service through a requirement. The analysis indicates that (1) students who were introduced to service through a mandated programme exhibit current levels of engagement no greater than non-volunteers; (2) this relationship stems largely from the different service experiences of our four cohorts and relates to the fact that service satisfaction and duration, as well as background variables account for current levels of civic engagement. The findings suggest that mandatory service programmes might well be failing the very population they seek to target, particularly in weaker, less structured programmes.

Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility.

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%-56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.

Carbon Amendments Alter Microbial Community Structure and Net Mercury Methylation Potential in Sediments.

Neurotoxic methylmercury (MeHg) is produced by anaerobic Bacteria and Archaea possessing the genes hgcAB, but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution of hgcAB+ microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence of hgcAB+ organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators (Deltaproteobacteria, Firmicutes, and methanogenic Archaea) was measured with clade-specific degenerate hgcA quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria Clade-specific qPCR identified hgcA+Deltaproteobacteria and Archaea in all sites but failed to detect hgcA+Firmicutes Cellobiose shifted the communities in all samples to ∼90% non-hgcAB-containing Firmicutes (mainly Bacillus spp. and Clostridium spp.). These results suggest that either expression of hgcAB is downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment.IMPORTANCE Methylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylating Firmicutes This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.

Multifunctional Enzyme Packaging and Catalysis in the Qß Protein Nanoparticle.

The simultaneous expression in Escherichia coli cells of the Qß virus-like particle (VLP) capsid protein and protein "cargo" tagged with a positively charged Rev peptide sequence leads to the spontaneous self-assembly of VLPs with multiple copies of the cargo inside. We report the packaging of four new enzymes with potential applications in medicine and chemical manufacturing. The captured enzymes are active while inside the nanoparticle shell and are protected from environmental conditions that lead to free-enzyme destruction. We also describe genetic modifications to the packaging scheme that shed light on the self-assembly mechanism of this system and allow indirect control over the internal packaging density of cargo. The technology was extended to create, via self-assembly, VLPs that simultaneously display protein ligands on the exterior and contain enzymes within. Inverse relationships were observed between the size of both the packaged and externally displayed protein or domains and nanoparticle yield. These results provide a general method for the rapid creation of robust protein nanoparticles with desired catalytic and targeting functionalities.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum.

OBJECTIVE: To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. RESULTS: Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. CONCLUSION: A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Transcriptomic and proteomic changes from medium supplementation and strain evolution in high-yielding Clostridium thermocellum strains.

Clostridium thermocellum is a potentially useful organism for the production of lignocellulosic biofuels because of its ability to directly deconstruct cellulose and convert it into ethanol. Previously engineered C. thermocellum strains have achieved higher yields and titers of ethanol. These strains often initially grow more poorly than the wild type. Adaptive laboratory evolution and medium supplementation have been used to improve growth, but the mechanism(s) by which growth improves remain(s) unclear. Here, we studied (1) wild-type C. thermocellum, (2) the slow-growing and high-ethanol-yielding mutant AG553, and (3) the faster-growing evolved mutant AG601, each grown with and without added formate. We used a combination of transcriptomics and proteomics to understand the physiological impact of the metabolic engineering, evolution, and medium supplementation. Medium supplementation with formate improved growth in both AG553 and AG601. Expression of C1 metabolism genes varied with formate addition, supporting the hypothesis that the primary benefit of added formate is the supply of C1 units for biosynthesis. Expression of stress response genes such as those involved in the sporulation cascade was dramatically over-represented in AG553, even after the addition of formate, suggesting that the source of the stress may be other issues such as redox imbalances. The sporulation response is absent in evolved strain AG601, suggesting that sporulation limits the growth of engineered strain AG553. A better understanding of the stress response and mechanisms of improved growth hold promise for informing rational improvement of C. thermocellum for lignocellulosic biofuel production.

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ß-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation.

Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads), and biogeochemical parameters are monitored by quantifying 53 metals, 12 organic acids, 14 anions, and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community and responded to DO. This also directly influenced the pH, and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part not only by experimental conditions but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.

Vocational Psychology: Agency, Equity, and Well-Being.

The present review organizes the vocational psychology literature published between 2007 and 2014 into three overarching themes: Promoting (a) agency in career development, (b) equity in the work force, and (c) well-being in work and educational settings. Research on career adaptability, self-efficacy beliefs, and work volition is reviewed in the agency section, with the goal of delineating variables that promote or constrain the exercise of personal agency in academic and occupational pursuits. The equity theme covers research on social class and race/ethnicity in career development; entry and retention of women and people of color in science, technology, engineering, and math (STEM) fields; and the career service needs of survivors of domestic violence and of criminal offenders. The goal was to explore how greater equity in the work force could be promoted for these groups. In the well-being section, we review research on hedonic (work, educational, and life satisfaction) and eudaimonic (career calling, meaning, engagement, and commitment) variables, with the goal of understanding how well-being might be promoted at school and at work. Future research needs related to each theme are also discussed.

Analysis of strand-specific RNA-seq data using machine learning reveals the structures of transcription units in Clostridium thermocellum.

Identification of transcription units (TUs) encoded in a bacterial genome is essential to elucidation of transcriptional regulation of the organism. To gain a detailed understanding of the dynamically composed TU structures, we have used four strand-specific RNA-seq (ssRNA-seq) datasets collected under two experimental conditions to derive the genomic TU organization of Clostridium thermocellum using a machine-learning approach. Our method accurately predicted the genomic boundaries of individual TUs based on two sets of parameters measuring the RNA-seq expression patterns across the genome: expression-level continuity and variance. A total of 2590 distinct TUs are predicted based on the four RNA-seq datasets. Among the predicted TUs, 44% have multiple genes. We assessed our prediction method on an independent set of RNA-seq data with longer reads. The evaluation confirmed the high quality of the predicted TUs. Functional enrichment analyses on a selected subset of the predicted TUs revealed interesting biology. To demonstrate the generality of the prediction method, we have also applied the method to RNA-seq data collected on Escherichia coli and achieved high prediction accuracies. The TU prediction program named SeqTU is publicly available at https://code.google.com/p/seqtu/. We expect that the predicted TUs can serve as the baseline information for studying transcriptional and post-transcriptional regulation in C. thermocellum and other bacteria.

Impact of Variations in Test Method Parameters on In Vitro Activity of Surotomycin against Clostridium difficile and Surotomycin Quality Control Limits for Broth Microdilution and Agar Dilution Susceptibility Testing.

Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 µg/ml (standard for lipopeptide testing) is recommended. Quality control ranges for Clostridium difficile (0.12 to 1 µg/ml) and Eggerthella lenta (broth, 1 to 4 µg/ml; agar, 1 to 8 µg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.

Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis.

UNLABELLED: Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE: Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.