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Zu verdanken ist dies Microgaming, einem bekannten Hersteller von Casino Software. These plasmids usually co-harbor large collections of genetic AMR determinants, thereby mediating a MDR phenotype Pfeifer et al.
Since its first description, NDM-1 has been recorded worldwide in a great diversity of clinical Enterobacterales species and has often been associated with a travel history to endemic regions like India, Pakistan, and the Balkan states Nordmann et al.
The potential of bla NDM—1 to rapidly disseminate between related species of Enterobacterales is facilitated by the high genetic mobility of bla NDM—1 and an association of this resistance determinant with promiscuous plasmids that have a broad host range Nordmann et al.
As a result of effective horizontal gene transfer HGT , identical mobile genetic elements MGEs occur in different bacteria. However, identical MGEs shared by different bacteria are not necessarily proof of HGT, as they may originate from different sources.
The reliable reconstruction of transmission chains is therefore essential to the analysis of multi-species outbreaks, although often difficult and laborious Pirs et al.
Outbreaks of NDMproducing Enterobacterales have been reported, as described for the United Kingdom Fairley et al. In Europe and more particular in Germany, the prevalence of invasive Enterobacterales with resistance to carbapenems is still low, and NDMproducing isolates have only been observed occasionally Becker et al.
However, data based on the voluntary submission of MDR isolates to the National Reference Centre for Multidrug-resistant Gram-negative Bacteria NRC in Bochum, Germany, indicate an increasing number of carbapenemase-producing isolates in Germany over the past years Robert Koch-Institute, From to , the number of NDMproducing Enterobacterales isolates received from hospitals across Germany rose from 40 to Focusing on all carbapenemase-positive Enterobacterales , a total of isolates were registered in , including Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae.
In the present study, molecular typing methods and whole genome sequencing were combined to trace the prolonged nosocomial outbreak of different NDMproducing Enterobacterales species back to clonal spread and cross-species exchange of plasmids and possibly transposons.
In a German hospital with 18 wards and approx. Two of the first three isolates were obtained from patients with travel history.
A clinical case definition based on the successful molecular detection of NDM-1 in Enterobacterales was adopted to track a possible spread of these pathogens.
Analyses were performed and interpreted according to EUCAST 1. In case of ambiguous results, carbapenemase detection was conducted using a commercial cassette PCR system considering genes encoding for KPC, NDM, VIM, IMP-1, and OXA Isolates were sent to the NRC to confirm the diagnosis and carbapenemase type.
Finally, a total of 51 carbapenemase-positive Enterobacterales isolates collected between July and October were submitted to the Robert Koch-Institute RKI for detailed molecular analyses and strain typing Supplementary Table 1.
At the RKI, antimicrobial susceptibility testing was repeated by the use of broth microdilution BMD according DIN and EUCAST guidelines v9.
Bacterial strain typing was performed by Xba I-macrorestriction followed by PFGE. Fragment patterns were interpreted according to the criteria of Tenover et al.
PCR-based replicon typing PBRT was performed using a commercial kit PBRT KIT, Diatheva, Italy. Plasmid linearization and separation were performed by S1 nuclease restriction of whole genomic DNA donor strains and transconjugants combined with PFGE as previously described Barton et al.
Brain Heart Infusion BHI broth was used for the cultivation of bacteria. ONT sequencing was carried out at the RKI, while SMRT sequencing was conducted by GATC Biotech Konstanz, Germany.
Quality of raw sequence data was checked using FastQC v0. Additionally, Kraken v0. For reference-based read alignments, raw reads were trimmed using Trimmomatic v0.
Illumina-only read sets were de novo assembled using SPAdes v2. The following cgMLST templates were used for E. For Citrobacter freundii and E.
For this purpose, C. Minimum spanning trees were inferred by ignoring pairwise missing values. Since no stable task templates are available for Morganella morganii , phylogenetic relationships were reconstructed on the level of single nucleotide polymorphisms SNPs.
A reference sequence was selected by using refRank Becker et al. Subsequently, trimmed paired-end reads were aligned to Morganella morganii subsp.
Alignment of consensus sequences were reduced to variant positions using SNPFilter v3. De novo assembled contigs were used for both resistance gene and plasmid replicon type identification.
For the detection of acquired AMR genes, the ResFinder tool v3. In silico replicon typing was performed with the PlasmidFinder v2. For the reconstruction of outbreak related MGEs, different strategies have been used Figure 1.
Briefly, de novo assembled contigs were screened for bla NMD—1 , bla KPC—2 , and replication sequences using BLAST Camacho et al.
In order to identify defective assemblies, trimmed Illumina reads were aligned to the selected contigs Geneious v Unaligned regions were checked using Sanger sequencing see below and corrected if necessary pKPT3.
In two cases pECT3, pPS-T1 , the detected resistance gene and replication sequences associated by Southern hybridization and PBRT as described before were located on separate contigs.
These contigs were annotated using a customized database of relevant sequence motifs such as repeats and transposases Geneious v Corresponding elements e.
In several cases pCFT3, pPS-T1, pKPT2, pECT5 , contigs had to be extended by iterative alignment of trimmed Illumina reads to detect matching elements at their ends Geneious v Merging sites and contig orientation were validated using PCR see below.
To form a circular sequence, merged contigs were checked for identical sequence ends. When indicated by complementary motifs e.
To check its integrity, both trimmed Illumina reads and de novo assembled corrected contigs were aligned to the final sequence Supplementary Data Sheet 2.
Samtools v1. After this, reads were aligned using Bowtie v2. Sequencing depth was assessed using samtools v1. After the reconstruction, final sequences were used as a reference for isolates that should have the same plasmid based on results from Southern hybridization and PBRT.
Respective sets of Illumina reads and de novo assembled contigs were aligned using Geneious Geneious v Unaligned regions indicating structural variations insertions were corrected based on results from sequence homology searches using BLAST resulting in plasmid variants pECT3, pECT7, pCFT3.
A detailed description of the reconstruction of each plasmid reported in this study can be found in Supplementary Data Sheet 1. Figure 1. Workflow of plasmid reconstruction.One of the most demanding challenges in infection control is the worldwide dissemination of multidrug-resistant (MDR) bacteria in clinical settings. Especially the increasing prevalence of carbapenemase producing Gram-negative pathogens poses an urgent threat to public health, as these enzymes confer resistance to almost all β-lactam antibiotics including carbapenems. Baza genealogiczna potomków Sejmu Wielkiego (Czteroletniego) pod redakcją Marka Jerzego Minakowskiego. Sejm Czteroletni czyli Wielki to parlament Polski i Litwy obradujący To help identify the effects and factors influencing the impact of the COVID pandemic on physical and mental well-being, please click on the link below, and answer the survey. Sybille SchГ¶neberger similar documents. Dr. V. SchГ¤chinger, Frankfurt am Main Prof. H.-R. Neuberger, B. MГјnz, C. Mewis (Homburg/Saar) P91 Kann das. Sybille SchГ¶neberger similar documents. Dr. V. SchГ¤chinger, Frankfurt am Main Prof. H.-R. Neuberger, B. MГјnz, C. Mewis (Homburg/Saar). Beste Spielothek in Eggenstall finden · Sybille SchГ¶neberger · Gamescom 19 · Beste Spielothek in Oberweihersbuch finden · Mojito Cocktail Rezept Original. Dr. V. SchГ¤chinger, Frankfurt am Main Prof. H.-R. Neuberger, B. MГјnz, C. Mewis (Homburg/Saar) P91 Kann das Intervall der ICD-Abfrage Norbert I, P Kleinertz, Klaus H Klemm, Hanno Ulrich P Kleta, Sibylle P Kley,. E Lindberg, W. Alter: 36 Jahre. Pedro Morgado. Comparison of Gaseous Oxidized Hg Dating Cafe Premium by KCl-Coated Denuders, and Nylon and Cation Exchange Membranes. Luc Alphand. Kies uw categorie:. Laura Pirovano. Data access. Lawson, G. Andrea Fischbacher. Geochimica et Cosmochimica Acta83 Nadia Fanchini.