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The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include 200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.
CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that \(Chlorella\) \(saccharophila\), \(Scenedesmus\) \(quadricauda\), and \(Micrasterias\) \(crux-melitensis\), maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant–microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.
Deep neck infections with and without mediastinal involvement: treatment and outcome in 218 patients
(2022)
Purpose
Infections of the deep neck, although becoming scarcer due to the widespread use of antibiotics, still represent a dangerous and possibly deadly disease, especially when descending into the mediastinum. Due to the different specialities involved in the treatment and the heterogenous presentation of the disease, therapeutic standard is still controversial. This study analyzes treatment and outcome in these patients based on a large retrospective review and proposes a therapeutic algorithm.
Methods
The cases of 218 adult patients treated with deep neck abscesses over a 10-year period at a tertiary university hospital were analyzed retrospectively. Clinical, radiological, microbiological and laboratory findings were compared between patients with and without mediastinal involvement.
Results
Forty-five patients (20.64%) presented with abscess formation descending into the mediastinum. Those patients had significantly (all items p < 0.0001) higher rates of surgical interventions (4.27 vs. 1.11) and tracheotomies (82% vs. 3.4%), higher markers of inflammation (CRP 26.09 vs. 10.41 mg/dl), required more CT-scans (3.58 vs. 0.85), longer hospitalization (39.78 vs 9.79 days) and more frequently needed a change in antibiotic therapy (44.44% vs. 6.40%). Multi-resistant pathogens were found in 6.67% vs. 1.16%. Overall mortality rate was low with 1.83%.
Conclusion
Despite of the high percentage of mediastinal involvement in the present patient collective, the proposed therapeutic algorithm resulted in a low mortality rate. Frequent CT-scans, regular planned surgical revisions with local drainage and lavage, as well as an early tracheotomy seem to be most beneficial regarding the outcome.
Background
Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi.
Objectives
To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms.
Methods
According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller–Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing.
Results
Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates.
Conclusions
Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations.