Staphylococcus aureus is one of the major pathogens causing community-and healthcare-acquired infections. the presence of the virulence factor panton-Valentine leukocidin (pVL) is associated with recurrent infection and clinical severity and generally regarded as a feature of community associatedmethicillin-resistant Staphylococcus aureus (MRSA). to date, the focus of pVL-positive MRSA in hospitalized patients has been on outbreaks. We aimed to investigate whether pVL-positive MRSA has penetrated the community-hospital barrier by determining the prevalence of pVL in MRSA of hospitalized patients. MRSA strains isolated from patients hospitalized > 48 h in Heidelberg University Hospital between 2015 and 2018 Isolates were analysed for the presence of PVL and subjected to spatyping. PVL-positive MRSA were then characterized by whole genome sequencing. We analysed 740 MRSA isolates in the study period and identified 6.2% (n = 46) PVL-positivity. 32.6% of PVL-positive MRSA met the criteria for nosocomial acquisition. the most frequent clones among the pVL-positive strains were ST80-t044 (21.7%, n = 10/46) and ST8-t008 (19.5%, n = 9/46). WGS identified three possible transmission clusters involving seven patients. in conclusion, we found successful epidemic pVL-positive MRSA clones entering the hospital and causing nosocomial infections. preventive measures and constant surveillance should be maintained to prevent transmissions and clonal outbreaks. Staphylococcus aureus is one of the major causes of community and hospital acquired infections. Methicillinresistance is an ongoing problem locally and globally. Although the overall prevalence of methicillin-resistant S. aureus (MRSA) in Germany is declining 1, MRSA strains harboring the pathogenic marker Panton-Valentine leukocidin (PVL) are isolated more frequently 2. The presence of PVL is relevant, as it is associated with a more severe clinical presentation, often with recurrences, deep and multiple lesions and frequent transmission to contact persons, irrespective of methicillin resistance 3-6. In addition, PVL-positive S. aureus often displays multiple resistances to commonly used antibiotics to treat S. aureus infections 3,7. PVL has been linked to community-associated (CA-) MRSA 6. Although the overall prevalence of PVL in Germany is considered low according to data acquired by Schaumburg et al. 8 , a study by Jappe et al. 9 revealed

Objectives Increasing spread of resistance could jeopardize the use of antifolates against MRSA infections. Methods We compared the prevalence of phenotypic trimethoprim/sulfamethoxazole resistance in 20 534 clinical Staphylococcus aureus isolates (19 096 MSSA and 1438 MRSA) of non-redundant patients at Heidelberg University Hospital over 8 years and performed WGS on trimethoprim/sulfamethoxazole-resistant MRSA. Results From 2012 to 2019, trimethoprim/sulfamethoxazole resistance in MSSA (674/19 096; 3.5%) ranged between 1.5% and 7.2% and in MRSA (135/1438; 9.4%) between 0.5% and 20.2%, reaching a peak in 2016 and 2018, respectively (Ptrend < 0.001). Trimethoprim/sulfamethoxazole resistance was more likely in outpatients than inpatients (P = 0.005), younger patients (P < 0.001), skin and soft tissue infections (SSTIs) (MRSA only, P = 0.05), submissions from pulmonology (MRSA only, P = 0.001), the upper respiratory tract (MSSA only, P < 0.001) and general surgery (MSSA only, P = 0.001). WGS of 76 trimethoprim/sulfamethoxazole-resistant MRSA revealed that 59% belonged to major pandemic CA-MRSA clones (ST22, ST8, ST398, ST772, ST30), 47% harboured Panton–Valentine leucocidin (PVL), 97% SCCmec IV/V, 71% dfrG and 28% dfrA. SNP-based phylogeny of trimethoprim/sulfamethoxazole-resistant MRSA core genomes favoured independent introduction over clonal expansion as the source, most prominently of dfrA+ trimethoprim/sulfamethoxazole-resistant ST22 MRSA from the Gaza Strip. Conclusions The presented results support that trimethoprim/sulfamethoxazole-resistant S. aureus, formerly associated with SSTI from outpatients and S. aureus in the (sub)tropics, is on the rise in the temperate zone, potentially due to migration. Closer monitoring of trimethoprim/sulfamethoxazole resistance in S. aureus is recommended to safeguard the effectiveness of antifolate compounds.