Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany.

Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur. Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain.