Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance

The increasing antimicrobial resistance (AMR) in <i>Staphylococcus pseudintermedius</i> causing skin and soft-tissue infections (SSTIs) in companion animals is a public health concern. The aim of this study was to verify if mobile genetic elements (MGEs), in particular plasmids, are related to the carriage of AMR genes among circulating and clinically relevant <i>S. pseudintermedius</i>. In total, 56 <i>S. pseudintermedius</i>, representing predominant and emerging clonal lineages associated with SSTIs in dogs and cats collected in Lisbon (Portugal), were subjected to plasmid DNA extraction and digestion with <i>Eco</i>RI and <i>Xba</i>I. Each unique restriction pattern was assigned to a plasmid profile. A subset of 17 strains was further selected for hybrid whole genome sequencing (WGS) on Oxford Nanopore MinION and Illumina MiSeq platforms. Thirty-one of the 56 <i>S. pseudintermedius</i> strains carried one or more plasmid(s), mostly of small or medium sizes, corresponding to eight plasmid profiles. Two of the identified plasmids carried AMR determinants; plasmid pSP-G3C4, isolated from ST71 strains, carried the tetracycline resistance gene <i>tet</i>(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the <i>qacG</i> biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn<i>552</i>, Tn<i>552</i>-like, Tn<i>553</i>, Tn<i>916</i>, Tn<i>5405</i>-like, Tn<i>5801</i>, Tn<i>5801</i>-like GI<i>6287</i> and pRE25-like elements. In addition, a new chromosomal cassette, carrying <i>fusC</i>, was identified in a ST1183 strain. The 12 methicillin-resistant <i>S. pseudintermedius</i> studied carried staphylococcal cassette chromosome <i>mec</i> (SCC<i>mec</i>) type III (<i>n</i> = 5), SCC<i>mec</i> type IVg (<i>n</i> = 3), SCC<i>mec</i> _NA45 (<i>n</i> = 1), ΨSCC<i>mec</i> ₅₇₃₉₅ (<i>n</i> = 1), the recently described cassettes SCC<i>mec</i> _7017-61515 (<i>n</i> = 1), or SCC<i>mec</i> type V(T)_SL/154 (<i>n</i> = 1). Most strains carried intact prophages without AMR determinants. Intact restriction-modification systems were detected in 12 out of the 17 strains and CRISPR/Cas in five strains, four of which were methicillin-susceptible. The results of this study suggest that the AMR content in <i>S. pseudintermedius</i> is mainly related to MGEs integrated into the chromosomal DNA rather than located on plasmids. These results provide important insights that may lead to a better understanding of multidrug resistance in <i>S. pseudintermedius</i> towards improved SSTIs treatment in companion animals.