Methicillin-resistant Macrococcus caseolyticus strains from bovine and canine origins were found to carry a novel mecD gene conferring resistance to all classes of β-lactams including anti-MRSA cephalosporins. Association of β-lactam resistance with mecD was demonstrated by gene expression in S. aureus and deletion of the mecD-containing island in M. caseolyticus. The mecD gene was located either on an 18,134-bp M. caseolyticus resistance island (McRImecD-1) or a 16,188-bp McRImecD-2. Both islands were integrated at the 3′ end of the rpsI gene, carried the mecD operon (mecD-mecR1m-mecIm), and genes for an integrase of the tyrosine recombinase family and a putative virulence-associated protein (virE). Apart from the mecD operon, that shared 66% overall nucleotide identity with the mecB operon, McRImecD islands were unrelated to any mecB-carrying elements or staphylococcal cassette chromosome mec. Only McRImecD-1 that is delimitated at both ends by direct repeats was capable of circular excision. The recombined excision pattern suggests site-specific activity of the integrase and allowed identification of a putative core attachment site. Detection of rpsI-associated integrases in Bacillus and S. aureus reveals a potential for broad-host range dissemination of the novel methicillin resistance gene mecD.

Gram-stain-positive cocci were isolated from miscellaneous sites of the skin of healthy dogs as well as from infection sites in dogs. The closest relative by sequencing of the 16S rRNA gene was Macrococcus caseolyticus with 99.7 % sequence identity, but compared with M. caseolyticus, the novel strains shared only 90.8 to 93.5 % DNA sequence identity with cpn60, dnaJ, rpoB and sodA partial genes, respectively. The novel strains also exhibited differential phenotypic characteristics from M. caseolyticus, and the majority displayed a visible haemolysis on sheep blood agar, while M. caseolyticus did not have any haemolytic activity. They generated different matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS spectral profiles compared with the other species of the genus Macrococcus. Furthermore, strain KM 45013 shared only 53.7 % DNA-DNA relatedness with the type strain of M. caseolyticus, confirming that they do not belong to the same species. The DNA G+C content of strain KM 45013 was 36.9 mol%. The most abundant fatty acids were C14 : 0, C18 : 3ω6c (6, 9, 12) and C16 : 0 n alcohol. MK-6 was the menaquinone type of KM 45013. Cell-wall structure analysis revealed that the peptidoglycan type was A3α l-Lys-Gly2-l-Ser. Based on genotypic and chemotaxonomic characteristics, we propose to classify these strains within a novel species of the genus Macrococcus for which the name Macrococcus canis sp. nov. is proposed. The type strain is KM 45013 (=DSM 101690=CCOS 969=CCUG 68920).

This study gives some insights into the occurrence and genetic characteristics of antibiotic-resistant Macrococcus from dogs. Presence of M. canis in infection sites and resistance to antibiotics emphasized that more attention should be paid to this novel bacteria species.

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