Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion

Data Availability: All relevant data are available from the Gene Expression Omnibus at the following accession number: GSE97755. Funding: This work was funded by the German Research Council (DFG) Graduation College 685, Dr. Jekyll and Mr. Hyde: A systems approach to the therapy of nosocomial infections caused by Candida albicans: a commensal organism switches to a deadly pathogen/ PTJ (FKZ: 0315409BBMBF), the Dr. Manfred Plempel-foundation, the Dr. Siegried Stettendorf-Foundation, the InfectERA Program (FunComPath; BMBF FKZ 031L0001A), the Integrated Research and Treatment Center for Sepsis Control and Care (CSCC) project CanBac (BMBF, FKZ: 01EO1002), and the German Research Council (DFG) GZ:HE7565/1-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Next-generation sequencing diagnostics of bacteremia in septic patients

Background: Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria. Methods: Here we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing. Results: We found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible. Conclusions: The complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections

Novel Transaminase and Laccase from Streptomyces spp. Using Combined Identification Approaches

Three Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (T-M = 85 degrees C) only slightly lower compared to the T-M of the most thermostable transaminases described to date (87-88 degrees C) and Sbv286-LAC being even thermoactivated at temperature gt 60 degrees C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of beta-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts

The molecular blueprint of a fungus by next-generation sequencing (NGS)

Sequencing the whole genome of an organism is invaluable for its comprehensive molecular characterization and has been drastically facilitated by the advent of high-throughput sequencing techniques. Especially in clinical microbiology the impact of sequenced strains increases as resistance and virulence markers can easily be detected. Here, we describe a combined approach for sequencing a fungal genome and transcriptome from initial nucleic acid isolation through the generation of ready-to-load DNA libraries for the Illumina platform and the final step of genome assembly with subsequent gene annotation

Enhanced performance of next-generation sequencing diagnostics compared with standard of care microbiological diagnostics in patients suffering from septic shock

Objectives: Culture-based diagnostics represent the standard of care in septic patients, but are highly insensitive and in many cases unspecific. We recently demonstrated the general feasibility of next-generation sequencing-based diagnostics using free circulating nucleic acids (cell-free DNA) in plasma samples of septic patients. Within the presented investigation, higher performance of next-generation sequencing-based diagnostics was validated by comparison to matched blood cultures. Design: A secondary analysis of a prospective, observational, single-center study. Setting: Surgical ICU of a university hospital and research laboratory. Patients: Fifty patients with septic shock, 20 uninfected patients with elective surgery as control cohort. Interventions: None. Measurements and Main Results: From 256 plasma samples of 48 septic patients at up to seven consecutive time points within the 28-day observation period, cell-free DNA was isolated and analyzed by next-generation sequencing and relevance scoring. In parallel, results from culture-based diagnostics (e.g., blood culture) were obtained. Plausibility of blood culture and next-generation sequencing results as well as adequacy of antibiotic therapy was evaluated by an independent expert panel. In contrast to blood culture with a positivity rate of 33% at sepsis onset, the positivity rate for next-generation sequencing-based pathogen identification was 72%. Over the whole study period, blood culture positivity was 11%, and next-generation sequencing positivity was 71%. Ninety-six percent of positive next-generation sequencing results for acute sepsis time points were plausible and would have led to a change to a more adequate therapy in 53% of cases as assessed by the expert evaluation. Conclusions: Our results show that next-generation sequencing-based analyses of bloodstream infections provide a valuable diagnostic platform for the identification of clinically relevant pathogens with higher sensitivity and specificity than blood culture, indicating that patients might benefit from a more appropriate therapy based on next-generation sequencing-based diagnosis

Genetic repertoires of anaerobic microbiomes driving generation of biogas

Abstract Background Biogas production is an attractive technology for a sustainable generation of renewable energy. Although the microbial community is fundamental for such production, the process control is still limited to technological and chemical parameters. Currently, most of the efforts on microbial management system (MiMaS) are focused on process-specific marker species and community dynamics, but a practical implementation is in its infancy. The high number of unknown and uncharacterized microorganisms in general is one of the reasons hindering further advancements. Results A Biogas Metagenomics Hybrid Assembly (BioMETHA) database, derived from microbiomes of biogas plants, was generated using a dedicated assembly strategy for different metagenomic datasets. Long reads from nanopore sequencing (MinION) were combined with short, more accurate second-generation sequencing reads (Illumina). The hybrid assembly resulted in 231 genomic bins each representing a taxonomic unit with an average completeness of 47%. Functional annotation identified 13,190 non-redundant genes covering roughly 207 k coding sequences. Mapping rates of metagenomics DNA derived from diverse biogas plants and laboratory reactors increased up to 73%. In addition, an EC (enzyme commission) reference sequence collection (ERSC) was generated whose genes are crucial for biogas-related processes, consisting of 235 unique EC numbers organized in 52 metabolic modules. Mapping rates of metatranscriptomic data to this ERSC revealed coverages of up to 93%. Process parameters and imbalances of laboratory reactors could be reconstructed by evaluating abundance of biogas-specific metabolic modules using metatranscriptomic data derived from various fermenter systems. Conclusion This newly established metagenomic hybrid assembly in combination with an EC reference sequence collection might help to shed light on the microbial dark matter of biogas plants by contributing to the development of a reference for biogas plant microbiome-specific gene sequences. Considering a biogas microbiome as a complex meta-organism expressing a meta-transcriptome, the approach established here could lay the foundation for a function-based microbial management system

Additional file 11: Figure S6. of Next-generation sequencing diagnostics of bacteremia in septic patients

Time course of patient S11. A 62-year-old male patient presented with a multilocular hepatocellular carcinoma with the need for a left-sided hemihepatectomy. Following the surgical procedure the patient suffered from septic shock due to severe pneumonia with Klebsiella pneumoniae as the dominant organism in blood cultures as well as tracheal secretions. Empiric antibiotic therapy was performed with imipenem, which was then switched to moxifloxacin based on the susceptibility findings. In the further course of the disease, K. pneumoniae was shown to be multidrug resistant. Although antibiotic therapy was adapted according to the findings of susceptibility testing, the pulmonary septic focus could not be removed sufficiently. In the end, the patient died from ongoing septic shock due to pneumonia with K. pneumonia 2 months after study inclusion. In addition, septic disease was shown to be accompanied by a reactivation of herpes simplex virus type 1 (HSV1) as well as cytomegalovirus (CMV) in different secretions as assessed by a PCR-based diagnostic procedure. These findings were in good agreement with next generation sequencing (NGS) of plasma. In this figure, the antibiotic treatment regime, SIQ scores for species identified via NGS, and cfDNA concentrations of the respective plasma samples are plotted over the timeline of the trial period for patient S11. Pertinent (clinical microbiology) laboratory results are marked using arrows to indicate the day the clinical specimen was obtained. Abbreviations: BC blood culture, CVC central venous catheter, TS tracheal secretion, GNST Gram-negative staphylococci, HSV1 herpes simplex virus 1, IPM imipenem, VAN vancomycin, MXF moxiflocaxin, CIP ciprofloxacin, TGC tigecycline, CAZ ceftazidime. Antibacterial antibiotics are displayed in light grey. The relative amount of bacteria found by conventional clinical microbiology is indicated with plenty (p), medium (m), or scarce (s). (For a detailed list of the anti-infective abbreviations, see Additional file 9: Table S5) (PDF 16 kb

Lonicera affinis Hook. et Arn.

原著和名: ハマニンドウ イヌニンドウ科名: スイカズラ科 = Caprifoliaceae採集地: 鹿児島県 肝属郡 佐多町 大中尾 (大隅 肝属郡 佐多町 大中尾)採集日: 1965/10/30採集者: 萩庭丈壽整理番号: JH042503国立科学博物館整理番号: TNS-VS-99250