Macrococcus caseolyticus belongs to the normal bacterial flora of dairy cows and does not usually cause disease. However, methicillin-resistant M. caseolyticus strains were isolated from bovine mastitis milk. These bacteria had acquired a chromosomal island (McRI _mecD -1 or McRI _mecD -2) carrying the methicillin resistance gene mecD To gain insight into the distribution of McRI _mecD types in M. caseolyticus from cattle, 33 mecD-containing strains from Switzerland were characterized using molecular techniques, including multilocus sequence typing, antibiotic resistance gene identification, and PCR-based McRI _mecD typing. In addition, the same genetic features were analyzed in 27 mecD-containing M. caseolyticus strains isolated from bovine bulk milk in England/Wales using publicly available whole-genome sequences. The 60 strains belonged to 24 different sequence types (STs), with strains belonging to ST5, ST6, ST21, and ST26 observed in both Switzerland and England/Wales. McRI _mecD -1 was found in different STs from Switzerland (n = 19) and England/Wales (n = 4). McRI _mecD -2 was only found in 7 strains from Switzerland, all of which belonged to ST6. A novel island, McRI _mecD -3, which contains a complete mecD operon (mecD-mecR1_m-mecI_m [where the subscript m indicates Macrococcus]) combined with the left part of McRI _mecD -2 and the right part of McRI _mecD -1, was found in heterogeneous STs from both collections (Switzerland, n = 7; England/Wales, n = 21). Two strains from England/Wales carried a truncated McRI _mecD -3. Phylogenetic analyses revealed no clustering of strains according to geographical origin or carriage of McRI _mecD -1 and McRI _mecD -3. Circular excisions were also detected for McRI _mecD -1 and McRI _mecD -3 by PCR. The analyses indicate that these islands are mobile and may spread by horizontal gene transfer between genetically diverse M. caseolyticus strains.IMPORTANCE Since its first description in 2017, the methicillin resistance gene mecD has been detected in M. caseolyticus strains from different cattle sources and countries. Our study provides new insights into the molecular diversity of mecD-carrying M. caseolyticus strains by using two approaches to characterize mecD elements: (i) multiplex PCR for molecular typing of McRI _mecD and (ii) read mapping against reference sequences to identify McRI _mecD types in silico In combination with multilocus sequence typing, this approach can be used for molecular characterization and surveillance of M. caseolyticus carrying mecD.

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