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Background: Escherichia coli carrying clinically important antimicrobial resistances [i.e., against extended-spectrum-beta-lactamases (ESBL)] are of high concern for human health and are increasingly detected worldwide. Worryingly, they are often identified as multidrug-resistant (MDR) isolates, frequently including resistances against quinolones/fluoroquinolones., Results: Here, the occurrence and genetic basis of the fluoroquinolone resistance enhancing determinant qnrB in ESBL-/non-ESBL-producing E. coli was investigated. Overall, 33 qnrB-carrying isolates out of the annual German antimicrobial resistance (AMR) monitoring on commensal E. coli (incl. ESBL-/AmpC-producing E. coli) recovered from food and livestock between 2013 and 2018 were analysed in detail. Whole-genome sequencing, bioinformatics analyses and transferability evaluation was conducted to characterise the prevailing qnrB-associated plasmids. Furthermore, predominant qnrB-carrying plasmid-types were subjected to in silico genome reconstruction analysis. In general, the qnrB-carrying E. coli were found to be highly heterogenic in their multilocus sequence types (STs) and their phenotypic resistance profiles. Most of them appeared to be MDR and exhibited resistances against up to ten antimicrobials of different classes. With respect to qnrB-carrying plasmids, we found qnrB19 located on small Col440I plasmids to be most widespread among ESBL-producing E. coli from German livestock and food. This Col440I plasmid-type was found to be highly conserved by exhibiting qnrB19, a pspF operon and different genes of unassigned function. Furthermore, we detected plasmids of the incompatibility groups IncN and IncH as carriers of qnrB. All qnrB-carrying plasmids also exhibited virulence factors and various insertion sequences (IS). The majority of the qnrB-carrying plasmids were determined to be self-transmissible, indicating their possible contribution to the spread of resistances against (fluoro)quinolones and other antimicrobials., Conclusion: In this study, a diversity of different plasmid types carrying qnrB alone or in combination with other resistance determinants (i.e., beta-lactamase genes) were found. The spread of these plasmids, especially those carrying antimicrobial resistance genes against highest priority critically important antimicrobial agents, is highly unfavourable and can pose a threat for public health. Therefore, the dissemination pathways and evolution of these plasmids need to be further monitored., (© 2022. The Author(s).)

Background: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods., Results: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach., Conclusions: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis., (© 2021. The Author(s).)

Background: Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest., Results: Strains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection., Conclusions: We investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

Background: Salmonella enterica subsp. enterica serotype Enteritidis is known as an important and pathogenic clonal group which continues to cause worldwide sporadic cases and outbreaks in humans. Here a new multiple-locus variable-number tandem repeat analysis (MLVA) method is reported for highly-discriminative subtyping of Salmonella Enteritidis. Emphasis was given on the most predominant phage types PT4 and PT8. The method comprises multiplex PCR specifically amplifying repeated sequences from nine different loci followed by an automatic fragment size analysis using a multicolor capillary electrophoresis instrument. A total of 240 human, animal, food and environmental isolates of S. Enteritidis including 23 definite phage types were used for development and validation. Furthermore, the MLVA types were compared to the phage types of several isolates from two recent outbreaks to determine the concordance between both methods and to estimate their in vivo stability. The in vitro stability of the two MLVA types specifically for PT4 and PT8 strains were determined by multiple freeze-thaw cycles., Results: Seventy-nine different MLVA types were identified in 240 S. Enteritidis strains. The Simpson's diversity index for the MLVA method was 0.919 and Nei diversity values for the nine VNTR loci ranged from 0.07 to 0.65. Twenty-four MLVA types could be assigned to 62 PT4 strains and 21 types to 81 PT8 strains. All outbreak isolates had an indistinguishable outbreak specific MLVA type. The in vitro stability experiments showed no changes of the MLVA type compared to the original isolate., Conclusion: This MLVA method is useful to discriminate S. Enteritidis strains even within a single phage type. It is easy in use, fast, and cheap compared to other high-resolution molecular methods and therefore an important tool for surveillance and outbreak studies for S. Enteritidis.

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.