Impact of process temperature and organic loading rate on cellulolytic / hydrolytic biofilm microbiomes during biomethanation of ryegrass silage revealed by genome-centered metagenomics and metatranscriptomics.

BACKGROUND: Anaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage two-phase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To follow the adaptive response of the biomass-attached cellulolytic/hydrolytic biofilms at increasing ammonium/ammonia contents, genome-centered metagenomics and transcriptional profiling based on metagenome assembled genomes (MAGs) were conducted. RESULTS: In total, 78 bacterial and archaeal MAGs representing the most abundant members of the communities, and featuring defined quality criteria were selected and characterized in detail. Determination of MAG abundances under the tested conditions by mapping of the obtained metagenome sequence reads to the MAGs revealed that MAG abundance profiles were mainly shaped by the temperature but also by the OLR. However, the OLR effect was more pronounced for the mesophilic systems as compared to the thermophilic ones. In contrast, metatranscriptome mapping to MAGs subsequently normalized to MAG abundances showed that under thermophilic conditions, MAGs respond to increased OLRs by shifting their transcriptional activities mainly without adjusting their proliferation rates. This is a clear difference compared to the behavior of the microbiome under mesophilic conditions. Here, the response to increased OLRs involved adjusting of proliferation rates and corresponding transcriptional activities. The analysis led to the identification of MAGs positively responding to increased OLRs. The most outstanding MAGs in this regard, obviously well adapted to higher OLRs and/or associated conditions, were assigned to the order Clostridiales (Acetivibrio sp.) for the mesophilic biofilm and the orders Bacteroidales (Prevotella sp. and an unknown species), Lachnospirales (Herbinix sp. and Kineothrix sp.) and Clostridiales (Clostridium sp.) for the thermophilic biofilm. Genome-based metabolic reconstruction and transcriptional profiling revealed that positively responding MAGs mainly are involved in hydrolysis of grass silage, acidogenesis and / or acetogenesis. CONCLUSIONS: An integrated -omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs. Genome-based knowledge on the metabolic potential and transcriptional activity of responsive microbiome members will contribute to the development of improved microbiological AD management strategies for biomethanation of renewable biomass.

Screening of microbial communities associated with endive lettuce during postharvest processing on industrial scale.

In this study, the composition of the microbial community on endive lettuce (Cichorium endivia) was evaluated during different postharvest processing steps. Microbial community structure was characterized by culture-dependent and culture-independent methods. Endive lettuce was sampled exemplarily at four different stages of processing (raw material, cut endive lettuce, washed endive lettuce, and spin-dried (ready to pack) endive lettuce) and analysed by plate count analysis using non-selective and selective agar plates with subsequent identification of bacteria colonies by matrix-assisted laser desorption/ionization time-of light mass spectrometry (MALDI-TOF MS). Additionally, terminal-restriction fragment length polymorphism (TRFLP) analysis and 16S rRNA gene nucleotide sequence analysis were conducted. The results revealed structural differences in the lettuce microbiomes during the different processing steps. The most predominant bacteria on endive lettuce were detected by almost all methods. Bacterial species belonging to the families Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, and Moraxellaceae were detected in most of the examined samples including some unexpected potentially human pathogenic bacteria, especially those with the potential to build resistance to antibiotics (e.g., Stenotrophomonas maltophilia (0.9 % in cut sample, 0.4 % in spin-dried sample), Acinetobacter sp. (0.6 % in raw material, 0.9 % in cut sample, 0.9 % in washed sample, 0.4 % in spin-dried sample), Morganella morganii (0.2 % in cut sample, 3 % in washed sample)) revealing the potential health risk for consumers. However, more seldom occurring bacterial species were detected in varying range by the different methods. In conclusion, the applied methods allow the determination of the microbiome's structure and its dynamic changes during postharvest processing in detail. Such a combined approach enables the implementation of tailored control strategies including hygienic design, innovative decontamination techniques, and appropriate storage conditions for improved product safety.

DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison.

Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.

Impact of surface structure and feed gas composition on Bacillus subtilis endospore inactivation during direct plasma treatment.

This study investigated the inactivation efficiency of cold atmospheric pressure plasma treatment on Bacillus subtilis endospores dependent on the used feed gas composition and on the surface, the endospores were attached on. Glass petri-dishes, glass beads, and peppercorns were inoculated with the same endospore density and treated with a radio frequency plasma jet. Generated reactive species were detected using optical emission spectroscopy. A quantitative polymerase chain reaction (qPCR) based ratio detection system was established to monitor the DNA damage during the plasma treatment. Argon + 0.135% vol. oxygen + 0.2% vol. nitrogen as feed gas emitted the highest amounts of UV-C photons and considerable amount of reactive oxygen and nitrogen species. Plasma generated with argon + 0.135% vol. oxygen was characterized by the highest emission of reactive oxygen species (ROS), whereas the UV-C emission was negligible. The use of pure argon showed a negligible emission of UV photons and atomic oxygen, however, the emission of vacuum (V)UV photons was assumed. Similar maximum inactivation results were achieved for the three feed gas compositions. The surface structure had a significant impact on the inactivation efficiency of the plasma treatment. The maximum inactivation achieved was between 2.4 and 2.8 log10 on glass petri-dishes and 3.9 to 4.6 log10 on glass beads. The treatment of peppercorns resulted in an inactivation lower than 1.0 log10. qPCR results showed a significant DNA damage for all gas compositions. Pure argon showed the highest results for the DNA damage ratio values, followed by argon + 0.135% vol. oxygen + 0.2% vol. nitrogen. In case of argon + 0.135% vol. oxygen the inactivation seems to be dominated by the action of ROS. These findings indicate the significant role of VUV and UV photons in the inactivation process of B. subtilis endospores.

Detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms.

BACKGROUND: In recent years biogas plants in Germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. In particular, biogas plants are discussed to contribute to the spreading of Escherichia coli infections in humans or chronic botulism in cattle caused by Clostridium botulinum. Metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at different temperatures and conditions were analyzed for the presence of putative pathogenic bacteria and virulence determinants by various bioinformatic approaches. RESULTS: All datasets featured a low abundance of reads that were taxonomically assigned to the genus Escherichia or further selected genera comprising pathogenic species. Higher numbers of reads were taxonomically assigned to the genus Clostridium. However, only very few sequences were predicted to originate from pathogenic clostridial species. Moreover, mapping of metagenome reads to complete genome sequences of selected pathogenic bacteria revealed that not the pathogenic species itself, but only species that are more or less related to pathogenic ones are present in the fermentation samples analyzed. Likewise, known virulence determinants could hardly be detected. Only a marginal number of reads showed similarity to sequences described in the Microbial Virulence Database MvirDB such as those encoding protein toxins, virulence proteins or antibiotic resistance determinants. CONCLUSIONS: Findings of this first study of metagenomic sequence reads of biogas producing microbial communities suggest that the risk of dissemination of pathogenic bacteria by application of digestates from biogas fermentations as fertilizers is low, because obtained results do not indicate the presence of putative pathogenic microorganisms in the samples analyzed.

Temperature increases from 55 to 75 °C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure.

Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75 °C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65 °C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70 °C led to slight improvements in the reactor performance; these did not persist at 75 °C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55 °C, altered by the temperature increase in the LBR. At an LBR temperature of 55 °C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65 °C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas.

Impact of the postthrombotic syndrome on quality of life after primary upper extremity deep venous thrombosis.

BACKGROUND: To determine the impact of the postthrombotic syndrome (PTS) on quality of life after primary upper extremity deep venous thrombosis (UEDVT). PATIENTS AND METHODS: Twenty-five patients with a history of primary UEDVT, treated with anticoagulation alone, and twenty healthy controls were retrospectively identified and prospectively assessed for health-related quality of life (SF-36 and VEINES-QOL-questionnaire) and upper extremity functional impairment (DASH-score). Presence of PTS was classified according to the modified Villalta-score. Comparisons between patients and controls and between patients with and without PTS were performed using Fisher`s exact test (categorical variables) and Mann-Whitney-U-test (continuous variables). RESULTS: According to the modified Villalta-score, 32 % of the patients suffered from mild to moderate PTS. None of the patients developed severe PTS. Compared to healthy control subjects, patients with a history of primary UEDVT reported on considerably worse health-related quality of life and significantly stronger upper extremity functional impairment. Within the cohort of patients with UEDVT, subjects with PTS had a significantly reduced quality of life and a more severe functional limitation. CONCLUSIONS: Quality of life and functional performance are impaired in patients with a history of conservatively treated primary UEDVT. Impairment is most pronounced in patients with mild to moderate PTS occurring in every third patient.

Involvement of the femoropopliteal arteries in giant cell arteritis: clinical and color duplex sonography.

OBJECTIVE: To determine the extent and clinical significance of giant cell arteritis (GCA) of the femoropopliteal arteries. METHODS: This was a retrospective clinical color duplex sonography (CDS) study; 60 of 112 consecutive patients with the diagnosis of GCA underwent complete clinical examination of the lower extremities including the vasculature, systolic ankle pressure measurement, and CDS scans of the femoropopliteal arteries within 1 year after diagnosis of GCA. Circumferential, hypoechogenic, homogenous wall thickening was regarded as a hallmark of femoropopliteal GCA. RESULTS: GCA of femoropopliteal arteries was present in 32 (53.3%) of 60 patients. In general, femoropopliteal GCA developed bilaterally (100%) and 14 patients (23.3%) had significant lower extremity artery obstructions secondary to vasculitis, all leading to symptomatic lower extremity ischemia, with development of critical leg ischemia in 4 patients. Compared with subjects without lower extremity vasculitis, patients with femoropopliteal involvement had a significant time delay until diagnosis (mean 23.9 vs 11.1 weeks; p = 0.03) and a higher frequency of concomitant vasculitis of the arm arteries (74.2% vs 42.9%; p = 0.02). CONCLUSION: Femoropopliteal artery involvement appears to be a clinically relevant manifestation of GCA, frequently leading to symptomatic lower extremity ischemia. CDS of the femoropopliteal arteries is a noninvasive diagnostic tool for detection of lower extremity vasculitis in GCA.

Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing.

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.

Plantar vein thrombosis due to busy night duty on intensive care unit.

A 32-year-old woman with severe foot pain came to our emergency department after a busy night duty in hospital followed by an extended sleep period. Physical examination revealed a discrete swelling of the medial aspect of the right foot and a painful plantar arch during digital examination. Magnetic resonance imaging (MRI) with intravenous gadolinium showed filling defects in the lateral plantar vein. Doppler sonography displayed noncompressible structures in the plantar veins without flow signals, suggesting a plantar vein thrombosis. Therapy was initiated with low-molecular-weight heparin in combination with customized elastic bandages for the lower leg. Follow-up sonography 6 weeks later showed complete patency of the plantar veins. To our knowledge, we present the first case of isolated plantar vein thrombosis independent of trauma, surgery, or malignant disease, most probably caused by a busy night duty on the intensive care unit (ICU) followed by a prolonged sleeping period.

Successful antibiotic treatment of severe staphylococcal infection of a long stent graft in the superficial femoral artery with graft preservation in the long term.

INTRODUCTION: Bacterial infection of endovascular stent grafts is a serious condition, regularly leading to graft replacement by open bypass surgery. CASE REPORT: We describe the case of a staphylococcal infection of a 150-mm covered stent graft (Gore Viabahn), placed in the superficial femoral artery. Stent graft infection was successfully treated by oral administration of penicillinase-resistant flucloxacillin and the lipopeptide daptomycin with complete graft preservation, not requiring surgical treatment. During 1-year follow-up, the graft infection did not reappear. However, the patient developed restenosis at the proximal margin of the stent with recurrence of mild claudication, so far treated conservatively. CONCLUSION: With the increased use of covered stent grafts in the peripheral vasculature, the frequency of graft infection will increase. We demonstrate that with newly developed antibiotics, it is possible to treat this severe complication conservatively, with complete graft preservation and without the need for bypass surgery in selected cases.

Large external iliac vein aneurysm in a patient with a post-traumatic femoral arteriovenous fistula.

We report about a young patient with a large aneurysm of the left external iliac vein associated with a traumatic arteriovenous fistula between the left superficial femoral artery and the femoral vein after a stab wound 20 years ago. The patient presented with swelling of the left leg, which developed during the past years and worsened after saphenectomy 12 months before hospital admission. The chronically hyperperfused common iliac artery proximal to the arteriovenous fistula was compressing the common iliac vein. The venous outflow obstruction and subsequent venous hypertension render a possible explanation for the formation of the iliac vein aneurysm. Surgical repair of the venous aneurysm by interposition grafting and closure of the arteriovenous fistula was successful. A postoperative computed tomography scan showed a 50% size reduction of the feeding artery, underlining the ability of the arterial system to normalize arterial diameter in response to flow reduction, even after a high flow situation had existed for probably >20 years.