Typing of <i>mecD</i> Islands in Genetically Diverse Methicillin-Resistant <i>Macrococcus caseolyticus</i> Strains from Cattle

Abstract

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