Alexandra Simbeck

Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.

Background: A variety of rapid molecular PCR tests has been developed and commercialised that interrogate the junction site between the staphylococcal core genome, and the mobile genetic element (SCC mec ) which harbours the gene responsible for methicillin-/beta-lactam-resistance. Aim: The purpose of the present study was to investigate why a clinical MRSA isolate yielded false negative test results with widely used, commercial orfX /SCC mec junction site PCR tests. Methods: A collection of isolates that belonged to the same epidemic strain as the index isolate were investigated with commercial MRSA assays and all isolates were sequenced in order to explain this observation. Results: It was found that isolates of the epidemic “European CC1-MRSA-IV” clone, which likely originated in South-Eastern Europe and subsequently spread to Western Europe, generally exhibit this behaviour. The failure of the assays was attributable to a characteristic large insertion in the orfX /SCC mec integration site presumably targeted by such tests. In contrast to MW2 (GenBank BA000033.2 , a CC1 “USA400” strain which also harbours SCC mec IVa), the European CC1 clone harbours an insertion of ca . 5,350 nucleotides adjacent to orfX . This sequence starts with a novel SCC terminal sequence alternate to dcs and encodes six different hypothetical proteins (E7MHX1, ydiL 2, C5QAP8, A8YYX4, npd- SCC, H4AYD7; nucleotide positions 280,690–286,024 of GenBank RBVO000005.1). An SCC mec element with the same insertion was previously found in a Staphylococcus epidermidis isolate (GenBank MH188467.1 ) suggesting transfer between staphylococcal species. Conclusion: In order to ensure the reliability of molecular MRSA tests, it is vital to monitor the emergence of new SCC mec junction sites, not only in Staphylococcus aureus but also in coagulase-negative staphylococci.