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Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven blaOXA-48 gene-carrying Klebsiella pneumoniae isolates, which were isolated between 2013 and 2014 at the University Medical Center in Göttingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively, and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All blaOXA-48 genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in K. pneumoniae in Germany.

Young children are frequently colonized with Clostridioides (C.) difficile. Depending on their resistance patterns, antibiotic treatment can facilitate gastrointestinal spreading in colonized individuals, potentially leading to transmission to others. C. difficile was isolated from stool samples from infants born in two hospitals in Göttingen and Darmstadt, Germany. All isolates were subjected to phenotypic antimicrobial resistance testing, PCR-based screening for toxin genes and mass spectrometry-based exclusion of ribotypes 027 and 176. Within an initial cohort of 324 neonates with a longitudinal survey of C. difficile, 137 strains were isolated from 48 individuals. Antimicrobial resistance was recorded against metronidazole in one (0.7%), erythromycin in 16 (11.7%) and moxifloxacin in 2 (1.5%) of the strains, whereas no resistance was observed against vancomycin (0.0%) or rifampicin (0.0%). Newly observed resistance against erythromycin in children with detection of previously completely sensitive isolates was reported for C. difficile isolates from 2 out of 48 children. In 20 children (42%), non-toxigenic strains were detected, and from 27 children (56%), toxigenic strains were isolated, while both toxigenic and non-toxigenic strains were recorded for 1 child (2%). Ribotypes 027 or 176 were not observed. In conclusion, the German C. difficile strains isolated from the children showed mild to moderate resistance with predominance of macrolide resistance, a substance class which is frequently applied in children. The observed switches to the dominance of macrolide-resistant isolates suggests likely selection of resistant C. difficile strains already in children.

Clostridioides difficile, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for C. difficile. A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the C. difficile proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the C. difficile proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based C. difficile clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool. Open-Access-Publikationsfonds 2019 peerReviewed

Motility assay of C. difficile isolates. RT 012 strains (630; DSM 27639), RT 027 strains (R20291; DSM 27640; DSM 27638) and non-motile RT 078 (DSM 29747) as a reference were analyzed on 0.3% BHI-agar. Following 1 to 2 days of post inoculation, the diffusion radius was monitored in semi-solid hungate tubes (A) and on semi-solid agar plates (B), respectively. Note, all strains produced gas. (DOCX 11038 kb)

Genome comparison of C. difficile isolates DSM 27638 and DSM 27640. The genome share a complete highly conserved chromosome reflected by the main diagonal. The repeats are depicted by the blue dots evenly distributed within the graph. The comparison has been calculated with Mummer 3 using default parameters. (DOCX 603 kb)

Sporulation assay of DSM 27638, DSM 27639 and DSM 27640 on ChromID plates after 5 days incubation on BHIS. All three isolates show a comparable count of spores that germinated on the plate. (DOCX 275 kb)

Oligonucleotides. (DOCX 18 kb)

Distribution of tlps in poultry and cattle. (DOCX 13 kb)

DNA gyrase subunit A alignment. The resistance phenotype is encoded by the amino acid substitution at the position 82 of GyrA. (DOCX 33 kb)

Mauve whole genome alignments of ten linearized C. difficile strains from four different PCR ribotypes. In this alignment process locally collinear blocks (LCBs) were generated. Boxes with identical colors represent LCB, indicating homologous DNA regions shared between the chromosomes without sequence rearrangement. Lines collate aligned segments between genomes. The vertical bars denote the conservation level, and upward and downward orientations relative to the genome line indicates collinear and inverted regions, respectively. The only non syntenic region of the genomes is indicated by the red box. Sequences outside colored blocks do not have homologues in the other genome. (DOCX 419 kb)

Growth curves in BHIS of DSM 27638, DSM 27639 and DSM 27640. The isolates were grown in brain heart infusion medium containing 0.5% (w/v) yeast extract und 0.03% (w/v) L-cysteine. All isolates have the same growth rate under laboratory conditions as shown as the means of three replicates with standard deviation. (DOCX 55 kb)

Genes assigned to resistance. (DOCX 16 kb)