Timely Cessation of Proton Pump Inhibitors in Critically Ill Patients Impacts Morbidity and Mortality: A Propensity Score-Matched Cohort Study.

OBJECTIVE: Proton pump inhibitors (PPIs) are among the drugs most commonly used in critically ill patients. Although mainly applied temporarily for stress ulcer prophylaxis, their application is frequently not terminated. Potential adverse effects of PPI treatment could impact the outcome in case of unnecessary and, therefore, avoidable long-term continuation. We tested the hypotheses that nonindicated PPI therapy continued beyond hospital discharge is associated with increased morbidity, rehospitalization rate, and mortality. DESIGN: Nationwide retrospective cohort study considering critically ill patients treated on German ICUs between January, 2017, and December, 2018 with a 2-year follow-up. SETTING: A total of 591,207 patient datasets of a German healthcare insurer were screened. PATIENTS: We identified 11,576 ICU patients who received PPI therapy for the first time during their index ICU stay without having an indication for its continuation. INTERVENTIONS: The cohort was stratified into two groups: 1) patients without further PPI therapy and 2) patients with continuation of PPI therapy beyond 8 weeks after hospital discharge. MEASUREMENTS AND MAIN RESULTS: Frequency of predescribed adverse events associated with PPI therapy, 1-year rehospitalization rate, and 2-year mortality were determined. The proportion of patients with continued PPI therapy without an objectifiable indication was 41.7% (4,825 of 11,576 patients). These patients had a 27% greater risk of pneumonia (odds ratio [OR] 1.27; 95% CI, 1.15-1.39; p < 0.001) and a 17% greater risk of cardiovascular events (OR 1.17; 95% CI, 1.08-1.26; p < 0.001). Continued PPI therapy was associated with a 34% greater risk of rehospitalization (OR 1.34; 95% CI, 1.23-1.47) and a nearly 20% greater 2-year mortality risk (hazard ratio 1.17; 95% CI, 1.08-1.27; p = 0.006). CONCLUSIONS: These data demonstrate that an unnecessary continuation of PPI therapy after hospital discharge may significantly impact morbidity and mortality. To avoid potentially harmful overuse of a PPIs, intensivists should ensure timely cessation of a temporarily indicated PPI therapy.

Anaeropeptidivorans aminofermentans gen. nov., sp. nov., a mesophilic proteolytic salt-tolerant bacterium isolated from a laboratory-scale biogas fermenter, and emended description of Clostridium colinum.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Insight into the structure, function and conjugative transfer of pLPU83a, an accessory plasmid of Rhizobium favelukesii LPU83.

Plasmids are widely distributed in rhizobia, a group of bacteria able to establish symbiotic relationships with the roots of legume plants. Two types of conjugative transfer (CT) regulation of these elements have been described in more detail. The most prevalent is through Quorum-Sensing (QS), mediated by the interaction of the TraR regulator protein and its cognate acyl-homoserine lactone (AHL) synthesized by TraI. In this study, we analyzed rhizobial plasmids classified according to their TraR regulators into four different groups. Each group has a particular genomic architecture. In one of the groups (I-C), represented by pLPU83a from Rhizobium favelukesii LPU83, CT induction requires TraR. With manual annotation, a traI was located in the plasmid distant to the traR gene. These features make pLPU83a an interesting plasmid for studying novel mechanisms of CT regulation. We mutagenized the traI gene, and found that it does not participate in CT regulation. Furthermore, we studied whether pLPU83a is subject to QS regulation by determining CT at different growth stages (cell densities). Our results showed no positive correlation between increase in culture densities and CT induction, on the contrary a slight decrease in CT was found at higher culture densities, unlike other TraR-depending plasmids. Our results show that transfer of pLPU83a is not regulated in a QS-dependent manner, and suggest that molecules not yet identified may activate its CT. Also, accumulation of a putative inhibitor cannot be disregarded.

Complete Genome Sequencing of Acinetobacter baumannii Strain K50 Discloses the Large Conjugative Plasmid pK50a Encoding Carbapenemase OXA-23 and Extended-Spectrum ß-Lactamase GES-11.

Multidrug-resistant (MDR) Acinetobacter baumannii strains appeared as serious emerging nosocomial pathogens in clinical environments and especially in intensive care units (ICUs). A. baumannii strain K50, recovered from a hospitalized patient in Kuwait, exhibited resistance to carbapenems and additionally to ciprofloxacin, chloramphenicol, sulfonamides, amikacin, and gentamicin. Genome sequencing revealed that the strain possesses two plasmids, pK50a (79.6 kb) and pK50b (9.5 kb), and a 3.75-Mb chromosome. A. baumannii K50 exhibits an average nucleotide identity (ANI) of 99.98% to the previously reported Iraqi clinical isolate AA-014, even though the latter strain lacked plasmid pK50a. Strain K50 belongs to sequence type 158 (ST158) (Pasteur scheme) and ST499 (Oxford scheme). Plasmid pK50a is a member of the Aci6 (replication group 6 [RG6]) group of Acinetobacter plasmids and carries a conjugative transfer module and two antibiotic resistance gene regions. The transposon Tn2008 carries the carbapenemase gene blaOXA-23, whereas a class 1 integron harbors the resistance genes blaGES-11, aacA4, dfrA7, qacEΔ1, and sul1, conferring resistance to all ß-lactams and reduced susceptibility to carbapenems and resistance to aminoglycosides, trimethoprim, quaternary ammonium compounds, and sulfamethoxazole, respectively. The class 1 integron is flanked by MITEs (miniature inverted-repeat transposable elements) delimiting the element at its insertion site.

Metagenome, metatranscriptome, and metaproteome approaches unraveled compositions and functional relationships of microbial communities residing in biogas plants.

The production of biogas by anaerobic digestion (AD) of agricultural residues, organic wastes, animal excrements, municipal sludge, and energy crops has a firm place in sustainable energy production and bio-economy strategies. Focusing on the microbial community involved in biomass conversion offers the opportunity to control and engineer the biogas process with the objective to optimize its efficiency. Taxonomic profiling of biogas producing communities by means of high-throughput 16S rRNA gene amplicon sequencing provided high-resolution insights into bacterial and archaeal structures of AD assemblages and their linkages to fed substrates and process parameters. Commonly, the bacterial phyla Firmicutes and Bacteroidetes appeared to dominate biogas communities in varying abundances depending on the apparent process conditions. Regarding the community of methanogenic Archaea, their diversity was mainly affected by the nature and composition of the substrates, availability of nutrients and ammonium/ammonia contents, but not by the temperature. It also appeared that a high proportion of 16S rRNA sequences can only be classified on higher taxonomic ranks indicating that many community members and their participation in AD within functional networks are still unknown. Although cultivation-based approaches to isolate microorganisms from biogas fermentation samples yielded hundreds of novel species and strains, this approach intrinsically is limited to the cultivable fraction of the community. To obtain genome sequence information of non-cultivable biogas community members, metagenome sequencing including assembly and binning strategies was highly valuable. Corresponding research has led to the compilation of hundreds of metagenome-assembled genomes (MAGs) frequently representing novel taxa whose metabolism and lifestyle could be reconstructed based on nucleotide sequence information. In contrast to metagenome analyses revealing the genetic potential of microbial communities, metatranscriptome sequencing provided insights into the metabolically active community. Taking advantage of genome sequence information, transcriptional activities were evaluated considering the microorganism's genetic background. Metaproteome studies uncovered enzyme profiles expressed by biogas community members. Enzymes involved in cellulose and hemicellulose decomposition and utilization of other complex biopolymers were identified. Future studies on biogas functional microbial networks will increasingly involve integrated multi-omics analyses evaluating metagenome, transcriptome, proteome, and metabolome datasets.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence.

BACKGROUND: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. RESULTS: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. CONCLUSIONS: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

Lifestyle-determining extrachromosomal replicon pAMV1 and its contribution to the carbon metabolism of the methylotrophic bacterium Paracoccus aminovorans JCM 7685.

Plasmids play an important role in the adaptation of bacteria to changeable environmental conditions. As the main vectors of horizontal gene transfer, they can spread genetic information among bacteria, sometimes even across taxonomic boundaries. Some plasmids carry genes involved in the utilization of particular carbon compounds, which can provide a competitive advantage to their hosts in particular ecological niches. Analysis of the multireplicon genome of the soil bacterium P. aminovorans JCM 7685 revealed the presence of an extrachromosomal replicon pAMV1 (185 kb) with a unique structure and properties. This lifestyle-determining plasmid carries genes facilitating the metabolism of many different carbon compounds including sugars, short-chain organic acids and C1 compounds. Plasmid pAMV1 contains a large methylotrophy island (MEI) that is essential not only for the utilization of particular C1 compounds but also for the central methylotrophic metabolism required for the assimilation of C1 units (serine cycle). We demonstrate that the expression of the main serine cycle genes is induced in the presence of C1 compounds by the transcriptional regulator ScyR. The extrachromosomal localization of the MEI and the distribution of related genes in Paracoccus spp. indicate that it could have been acquired by HGT by an ancestor of P. aminovorans.

Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3.

The basidiomycetes fungus Rhizoctonia solani AG3 is responsible for black scurf disease on potato and occurs in each potato growing area world-wide. In this study, the draft genome sequence of the black scurf pathogen R. solani AG3-PT isolate Ben3 is presented. The genome sequence of R. solani AG3-PT isolate Ben3 consists of 1385 scaffolds. These scaffolds amount to a size of approx. 51 Mb. Considering coverage analyses of contigs, the size of the diploid genome was estimated to correspond to 116 Mb. Gene prediction by applying AUGUSTUS (3.2.1.) resulted in 12,567 identified genes. Based on automatic annotation using GenDBE, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the R. solani AG3-PT isolate Ben3 genome. Comparative analyses including the R. solani AG3 isolate Rhs1AP, also originating from potato, revealed first insights into core genes shared by both isolates and unique determinants of each isolate.

Identification of a novel mycovirus isolated from Rhizoctonia solani (AG 2-2 IV) provides further information about genome plasticity within the order Tymovirales.

The complete genome of a novel mycovirus, named Rhizoctonia solani flexivirus 1 (RsFV-1), which infects an avirulent strain of Rhizoctonia solani AG 2-2 IV, was sequenced and analyzed. Its RNA genome consists of 10,621 nucleotides, excluding the poly-A tail, and encodes a single protein of 3477 amino acids. The identification of conserved motifs of methyltransferase, helicase and RNA-dependent RNA polymerase revealed its relatedness to members of the alphavirus-like superfamily of positive-strand RNA viruses. Phylogenetic analysis of these fused domains suggested that this virus should be assigned to the order Tymovirales. The recently described Fusarium graminearum deltaflexivirus 1 was found to be its closest relative. However, the whole genome, as well as the encoded protein of RsFV-1, is larger than that of other known members of the order Tymovirales, and unlike all other viruses belonging to this order, its methyltransferase domain is not located at the N-terminus of the replicase. Although genome diversity, as a result of recombination and gene loss, is a well-documented trait in members of the order Tymovirales, no related virus with a comparable genome alteration has been reported before. For these reasons, RsFV-1 broadens our perception about genome plasticity and diversity within the order Tymovirales.

acdc - Automated Contamination Detection and Confidence estimation for single-cell genome data.

BACKGROUND: A major obstacle in single-cell sequencing is sample contamination with foreign DNA. To guarantee clean genome assemblies and to prevent the introduction of contamination into public databases, considerable quality control efforts are put into post-sequencing analysis. Contamination screening generally relies on reference-based methods such as database alignment or marker gene search, which limits the set of detectable contaminants to organisms with closely related reference species. As genomic coverage in the tree of life is highly fragmented, there is an urgent need for a reference-free methodology for contaminant identification in sequence data. RESULTS: We present acdc, a tool specifically developed to aid the quality control process of genomic sequence data. By combining supervised and unsupervised methods, it reliably detects both known and de novo contaminants. First, 16S rRNA gene prediction and the inclusion of ultrafast exact alignment techniques allow sequence classification using existing knowledge from databases. Second, reference-free inspection is enabled by the use of state-of-the-art machine learning techniques that include fast, non-linear dimensionality reduction of oligonucleotide signatures and subsequent clustering algorithms that automatically estimate the number of clusters. The latter also enables the removal of any contaminant, yielding a clean sample. Furthermore, given the data complexity and the ill-posedness of clustering, acdc employs bootstrapping techniques to provide statistically profound confidence values. Tested on a large number of samples from diverse sequencing projects, our software is able to quickly and accurately identify contamination. Results are displayed in an interactive user interface. Acdc can be run from the web as well as a dedicated command line application, which allows easy integration into large sequencing project analysis workflows. CONCLUSIONS: Acdc can reliably detect contamination in single-cell genome data. In addition to database-driven detection, it complements existing tools by its unsupervised techniques, which allow for the detection of de novo contaminants. Our contribution has the potential to drastically reduce the amount of resources put into these processes, particularly in the context of limited availability of reference species. As single-cell genome data continues to grow rapidly, acdc adds to the toolkit of crucial quality assurance tools.

Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes.

BACKGROUND: Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. RESULTS: The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. CONCLUSIONS: The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology.

Intraspecies Transfer of the Chromosomal Acinetobacter baumannii blaNDM-1 Carbapenemase Gene.

The species Acinetobacter baumannii is one of the most important multidrug-resistant human pathogens. To determine its virulence and antibiotic resistance determinants, the genome of the nosocomial blaNDM-1-positive A. baumannii strain R2090 originating from Egypt was completely sequenced. Genome analysis revealed that strain R2090 is highly related to the community-acquired Australian A. baumannii strain D1279779. The two strains belong to sequence type 267 (ST267). Isolate R2090 harbored the chromosomally integrated transposon Tn125 carrying the carbapenemase gene blaNDM-1 that is not present in the D1279779 genome. To test the transferability of the metallo-ß-lactamase (MBL) gene region, the clinical isolate R2090 was mated with the susceptible A. baumannii recipient CIP 70.10, and the carbapenem-resistant derivative R2091 was obtained. Genome sequencing of the R2091 derivative revealed that it had received an approximately 66-kb region comprising the transposon Tn125 embedding the blaNDM-1 gene. This region had integrated into the chromosome of the recipient strain CIP 70.10. From the four known mechanisms for horizontal gene transfer (conjugation, outer membrane vesicle-mediated transfer, transformation, and transduction), conjugation could be ruled out, since strain R2090 lacks any plasmid, and a type IV secretion system is not encoded in its chromosome. However, strain R2090 possesses three putative prophages, two of which were predicted to be complete and therefore functional. Accordingly, it was supposed that the transfer of the resistance gene region from the clinical isolate R2090 to the recipient occurred by general transduction facilitated by one of the prophages present in the R2090 genome. Hence, phage-mediated transduction has to be taken into account for the dissemination of antibiotic resistance genes within the species A. baumannii.

Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L).

Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root nodules of alfalfa, grown in acid soils from Argentina and the USA. These two strains, which shared the same plasmid pattern, lipopolysaccharide profile, insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting pattern, were different from reference strains representing species of the genus Rhizobium with validly published names. On the basis of previously reported data and from new DNA-DNA hybridization results, phenotypic characterization and phylogenetic analyses, strains LPU83T and Or191 can be considered to be representatives of a novel species of the genus Rhizobium, for which the name Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is available.

Fractionation of biogas plant sludge material improves metaproteomic characterization to investigate metabolic activity of microbial communities.

With the development of high resolving mass spectrometers, metaproteomics evolved as a powerful tool to elucidate metabolic activity of microbial communities derived from full-scale biogas plants. Due to the vast complexity of these microbiomes, application of suitable fractionation methods are indispensable, but often turn out to be time and cost intense, depending on the method used for protein separation. In this study, centrifugal fractionation has been applied for fractionation of two biogas sludge samples to analyze proteins extracted from (i) crude fibers, (ii) suspended microorganisms, and (iii) secreted proteins in the supernatant using a gel-based approach followed by LC-MS/MS identification. This fast and easy method turned out to be beneficial to both the quality of SDS-PAGE and the identification of peptides and proteins compared to untreated samples. Additionally, a high functional metabolic pathway coverage was achieved by combining protein hits found exclusively in distinct fractions. Sample preparation using centrifugal fractionation influenced significantly the number and the types of proteins identified in the microbial metaproteomes. Thereby, comparing results from different proteomic or genomic studies, the impact of sample preparation should be considered. All MS data have been deposited in the ProteomeXchange with identifier PXD001508 (http://proteomecentral.proteomexchange.org/dataset/PXD001508).

Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal.

Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial isolates recovered from a BPS established for the decontamination of wastewater generated in a farmyard. Out of 1400 isolates, a collection of 75 plasmid-containing bacteria was obtained, of which 35 representative isolates comprising in total at least 50 plasmids were chosen for further characterization. Bacterial hosts were taxonomically assigned by 16S ribosomal RNA gene sequencing and phenotypically characterized according to their ability to grow in presence of different antibiotics and heavy metals. The study demonstrated that a high proportion of the isolates was tolerant to antibiotics and/or heavy metals, highlighting the on-farm BPS enrichment in such genetic traits. Several plasmids conferring such resistances in the bacterial collection were detected to be either mobilizable or selftransmissible. Occurrence of broad host range plasmids of the incompatibility groups IncP, IncQ, IncN and IncW was examined with positive results only for the first group. Presence of the IS1071 insertion sequence, frequently associated with xenobiotics degradation genes, was detected in DNA obtained from 24 of these isolates, strongly suggesting the presence of yet-hidden catabolic activities in the collection of isolates. The results showed a remarkable diversity in the plasmid mobilome of cultivable bacteria in the BPS with the presence of abundant resistance markers of different types, thus providing a suitable environment to investigate the genetic structure of the mobile genetic pool in a model on-farm biofilter for wastewater decontamination in intensive agricultural production.

Community shifts in a well-operating agricultural biogas plant: how process variations are handled by the microbiome.

This study provides a comprehensive, long-term microbiological study of a continuously operated, mesophilic, agricultural biogas plant fed with whole-crop silages of maize and rye, cattle manure and cattle slurry. The microbial community structure was accessed by high-throughput 16S rRNA gene amplicon sequencing. For the characterisation of the microbial dynamics, the community profiling method terminal restriction fragment length polymorphism (TRFLP) in combination with a cloning-sequencing approach as well as a LC-MS/MS approach for protein identification were applied. Our results revealed that the anaerobic digestion is a highly sensitive process: small variations in the process performance induce fluctuations in the microbial community composition and activity. In this context, it could be proven that certain microbial species were better adapted to changing process condition such as temperature (interspecies competition) and that there is a physiological compensation between different microorganisms so that the reactor efficiency was not adversely affected despite of structural and functional changes within the microbial community.

Architecture and functions of a multipartite genome of the methylotrophic bacterium Paracoccus aminophilus JCM 7686, containing primary and secondary chromids.

BACKGROUND: Paracoccus aminophilus JCM 7686 is a methylotrophic α-Proteobacterium capable of utilizing reduced one-carbon compounds as sole carbon and energy source for growth, including toxic N,N-dimethylformamide, formamide, methanol, and methylamines, which are widely used in the industry. P. aminophilus JCM 7686, as many other Paracoccus spp., possesses a genome representing a multipartite structure, in which the genomic information is split between various replicons, including chromids, essential plasmid-like replicons, with properties of both chromosomes and plasmids. In this study, whole-genome sequencing and functional genomics approaches were applied to investigate P. aminophilus genome information. RESULTS: The P. aminophilus JCM 7686 genome has a multipartite structure, composed of a single circular chromosome and eight additional replicons ranging in size between 5.6 and 438.1 kb. Functional analyses revealed that two of the replicons, pAMI5 and pAMI6, are essential for host viability, therefore they should be considered as chromids. Both replicons carry housekeeping genes, e.g. responsible for de novo NAD biosynthesis and ammonium transport. Other mobile genetic elements have also been identified, including 20 insertion sequences, 4 transposons and 10 prophage regions, one of which represents a novel, functional serine recombinase-encoding bacteriophage, ϕPam-6. Moreover, in silico analyses allowed us to predict the transcription regulatory network of the JCM 7686 strain, as well as components of the stress response, recombination, repair and methylation machineries. Finally, comparative genomic analyses revealed that P. aminophilus JCM 7686 has a relatively distant relationship to other representatives of the genus Paracoccus. CONCLUSIONS: P. aminophilus genome exploration provided insights into the overall structure and functions of the genome, with a special focus on the chromids. Based on the obtained results we propose the classification of bacterial chromids into two types: "primary" chromids, which are indispensable for host viability and "secondary" chromids, which are essential, but only under some environmental conditions and which were probably formed quite recently in the course of evolution. Detailed genome investigation and its functional analysis, makes P. aminophilus JCM 7686 a suitable reference strain for the genus Paracoccus. Moreover, this study has increased knowledge on overall genome structure and composition of members within the class Alphaproteobacteria.

IncH-type plasmid harboring bla CTX-M-15, bla DHA-1, and qnrB4 genes recovered from animal isolates.

The whole sequence of plasmid pENVA carrying the extended-spectrum ß-lactamase gene blaCTX-M-15 was determined. It was identified from a series of clonally related Klebsiella pneumoniae sequence type 274 strains recovered from companion animals. This plasmid was 253,984 bp in size and harbored, in addition to blaCTX-M-15, a large array of genes encoding resistance to many antibiotic molecules, including ß-lactams (blaTEM-1, blaDHA-1), aminoglycosides (aacA2, aadA1), tetracycline (tetA), quinolones (qnrB4), trimethoprim (dfrA15), and sulfonamides (two copies of sul1). In addition, genes encoding resistance to mercury, tellurium, nickel, and quaternary compounds were identified. It also carried genes encoding DNA damage protection and mutagenesis repair and a locus for a CRISPR system, which corresponds to an immune system involved in protection against bacteriophages and plasmids. Comparative analysis of the plasmid scaffold showed that it possessed a structure similar to that of only a single plasmid, which was pNDM-MAR encoding the carbapenemase NDM-1 and identified from human K. pneumoniae isolates. Both plasmids possessed two replicons, namely, those of IncFIB-like and IncHIB-like plasmids, which were significantly different from those previously characterized. The blaCTX-M-15 gene, together with the other antibiotic resistance genes, was part of a large module likely acquired through a transposition process. We characterized here a new plasmid type carrying the blaCTX-M-15 gene identified in a K. pneumoniae isolate of animal origin. The extent to which this plasmid type may spread efficiently and possibly further enhance the dissemination of blaCTX-M-15 among animal and human isolates remains to be determined.

Towards molecular biomarkers for biogas production from lignocellulose-rich substrates.

Biogas production from lignocellulose-rich agricultural residues is gaining increasingly importance in sustainable energy production. Hydrolysis/acidogenesis (H/A) of lignocellulose as the initial rate-limiting step deserves particular optimization. A mixture of straw/hay was methanized applying two-phase digester systems with an initial H/A reactor and a one-stage system at different, meso- and thermophilic temperatures. H/A was intensified with increasing pH values and increasing temperature. H/A fermenters, however, were prone to switch to methanogenic systems at these conditions. Substrate turnover was accelerated in the bi-phasic process but did not reach the methanation efficiency of the single-stage digestion. There was no indication that two different cellulolytic inocula could establish in the given process. Bacterial communities were analyzed applying conventional amplicon clone sequencing targeting the hypervariable 16S rRNA gene region V6-V8 and by metagenome analyses applying direct DNA pyrosequencing without a PCR step. Corresponding results suggested that PCR did not introduce a bias but offered better phylogenetic resolution. Certain Clostridium IV and Prevotella members were most abundant in the H/A system operated at 38 °C, certain Clostridium III and Lachnospiraceae bacteria in the 45 °C, and certain Clostridium IV and Thermohydrogenium/Thermoanaerobacterium members in the 55 °C H/A system. Clostridium III representatives, Lachnospiraceae and Thermotogae dominated in the thermophilic single-stage system, in which also a higher portion of known syntrophic acetate oxidizers was found. Specific (RT-)qPCR systems were designed and applied for the most significant and abundant populations to assess their activity in the different digestion systems. The RT-qPCR results agreed with the DNA based community profiles obtained at the different temperatures. Up to 10(12) 16S rRNA copies mL(-1) were determined in H/A fermenters with prevalence of rRNA of a Ruminococcaceae subgroup. Besides, Thermohydrogenium/Thermoanaerobacterium rRNA prevailed at thermophilic and Prevotellaceae rRNA at mesophilic conditions. The developed (RT)-qPCR systems can be used as biomarkers to optimize biogas production from straw/hay and possibly other lignocellulosic substrates.

Rare Superior and Middle Trunk Fusion Accompanied by Altered Division Rearrangement Results in a Unique Brachial Plexus Variant: A Case Report.

During routine dissections of cadavers as part of the medical curriculum, we identified a rare unilateral variation in the brachial plexus on the right side of a female body donor. This variation consisted of four unusual changes to the regular pattering of nerve bundles and the dorsal scapular artery permeating the complex neural network. The variation included contributions of root C4 to the plexus by a root C4/C5 anastomosis, a rare fusion of the superior and middle trunks to a 'superomiddle' trunk, a preliminary, proximal branching of the suprascapular nerve off the C5 root. We further observed an accessory 'medial anterior division' branching off the fused upper and middle trunks merging with the anterior division of the inferior trunk forming the medial cord. The latter event potentially introduced nerve fibers from C5 to C7, which are absent in common patterns. We aim to relate these observations to previous categorizations and quantifications of brachial plexus patterns. We believe that the combination of different variations in this case resulted in a unique pattern. Since this observation was made in the dissection class, we further aim to raise awareness among medical students and anatomical instructors for the likelihood of variations to textbook patterns. This will hopefully foster an appreciation of uniqueness and individuality in the interaction with future patients demonstrating that proper preparation prior to surgical interventions is always a necessary prerequisite.