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. 2025 Oct 8:16:1640322.
doi: 10.3389/fmicb.2025.1640322. eCollection 2025.

Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance

Catarina Morais  1 , Sofia Santos Costa  1 , Dennis Hanke  2   3 , Ana Santos  1 , Henrike Krüger-Haker  2   3 , Constança Pomba  4 , Andrea T Feßler  2   3 , Stefan Schwarz  2   3 , Isabel Couto  1
Affiliations

Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance

Catarina Morais et al. Front Microbiol. .

Abstract

The increasing antimicrobial resistance (AMR) in Staphylococcus pseudintermedius 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 S. pseudintermedius. In total, 56 S. pseudintermedius, 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 EcoRI and XbaI. 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 S. pseudintermedius 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 tet(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the qacG biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn552, Tn552-like, Tn553, Tn916, Tn5405-like, Tn5801, Tn5801-like GI6287 and pRE25-like elements. In addition, a new chromosomal cassette, carrying fusC, was identified in a ST1183 strain. The 12 methicillin-resistant S. pseudintermedius studied carried staphylococcal cassette chromosome mec (SCCmec) type III (n = 5), SCCmec type IVg (n = 3), SCCmec NA45 (n = 1), ΨSCCmec 57395 (n = 1), the recently described cassettes SCCmec 7017-61515 (n = 1), or SCCmec type V(T)SL/154 (n = 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 S. pseudintermedius 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 S. pseudintermedius towards improved SSTIs treatment in companion animals.

Keywords: SCCfus; Staphylococcus pseudintermedius; WGS; antimicrobial resistance; mobile genetic elements; mobilome; plasmids; transposons.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Chart displaying the presence of antimicrobial resistance (AMR) genes, AMR mutations, and mobile genetic elements across various bacterial strains. Colored squares and circles indicate the presence of specific AMR genes and mutations respectively, while triangles denote mobile genetic elements. A legend at the bottom categorizes colors and shapes by antimicrobial class, SCCmec element, and plasmid status.
FIGURE 1
Clonal lineage, antimicrobial resistance (AMR) determinants and mobile genetic elements (MGEs) of the 12 MRSP and 5 MSSP strains selected for whole genome sequencing. Squares and circles correspond to AMR genes and point mutations, respectively. Triangles correspond to MGEs. *Include variants of this element carrying different AMR genes (see Table 3).
Genetic map comparing two Staphylococcus aureus strains, RUH-32 and BIOS-V212. Features include antimicrobial resistance genes, insertion sequences, and cassette chromosome recombinase components. Identified elements are color-coded. Size scale measures 8 Kbp.
FIGURE 2
Presentation of the new SCCfus described in S. pseudintermedius carrying the fusC gene. Comparison with the SCCmec-SCCfus described in S. aureus isolate RUH-32 (MK991791). Homology is indicated through a color scale of gray: dark gray (100% homology) to light gray (87% homology). Fusidic acid resistance (fusC) and methicillin-resistance (mecA) genes are represented in yellow; cassette chromosome recombinase genes represented in brown (ccrA4, ccrB4); orfX in orange; insertion sequences in purple. Genes colored in gray represent other genes. The figure was generated using Genofig v1.1.
Genetic map of Tn5801-like GI6287 present in Staphylococcus pseudintermedius BIOS-V179 strain and comparison with the same element from another S. pseudintermedius strain. Arrows represent gene positions and orientations. Color key indicates antimicrobial resistance genes in yellow, integrase in brown; GMP synthetase in orange; insertion sequence in purple, and other coding sequences in gray. Shaded areas indicate regions of similarity between the bacterial segments and the reference. Scale bar indicates 6 kilobases.
FIGURE 3
Presentation of Tn5801-like GI6287 (BIOS-V179) carrying the tet(M) gene and comparison with strain S. pseudintermedius 2001-08-299-1 contig_3 (accession number: NZ_JTKO01000003.1, [contig 3, 177,382:198,007]). Minimum homology of 99% was detected between sequences (light gray color). Tetracycline resistance gene [tet(M)] is represented in yellow; integrase gene (int) is represented in brown; GMP synthetase gene (guaA) in orange; insertion sequence in purple. Genes colored in gray represent other genes. The figure was generated using Genofig v1.1.
“Comparison of different Tn5405-like variants with the element identified in Staphylococcus pseudintermedius ED99. Image A shows the comparison between strains carrying five AMR genes in this element. Image B represents a variant with four additional AMR genes. Genetic features are color-coded for clarity: antimicrobial resistance genes in yellow, insertion sequences in purple, integrase in orange, and other coding sequences in grey. Shaded areas indicate regions of similarity between the bacterial segments and the reference. Scale bars indicate 20 kilobases (for image A) and 8 kilobases (for image B).”
FIGURE 4
Presentation of Tn5405-like and the region until comK gene and comparison with strain S. pseudintermedius ED99 (NC_017568, [1,833,993:1,857,865]). (A) Homology of Tn5405-like from ED99 with BIOS-V141, a strain without dfrG and seven strains carrying the dfrG gene. Phages detected after the transposon are indicated in a dotted green box. (B) Representation of BIOS-V16 with the additional four antimicrobial resistance genes [aadE, spw, lsa(E) and lnu(B)]. Homology is indicated through a color scale of gray: dark gray (100% homology) to light gray (86% (A) or 89% (B) homology). Antimicrobial resistance genes are represented in yellow; integrase genes are represented in pink; insertion sequences in purple. Genes colored in gray represent other genes. The figure was generated using Genofig v1.1. The inner lines depict additional regions with homology automatically generated by Genofig.
Schematic representation of the truncated Tn4001 identified in Staphylococcus pseudintermedius and its comparison with the element described in Staphylococcus aureus. Color key indicates antimicrobial resistance genes in yellow, insertion sequences in purple, phage genes in blue, and other coding sequences in gray. Shaded areas indicate regions of similarity between the bacterial segments and the reference. Scale bar indicates 1 kilobase.
FIGURE 5
Schematic representations of the truncated Tn4001 and the region in the vicinity of aacA-aphD gene. Comparison with Tn4001 present in S. aureus plasmid pSK1 (accession number: GU565967, [23,577:28,041]). Homology of 100% was detected between sequences. Aminoglycoside resistance gene (aacA-aphD) is represented in yellow; orf132 in orange; insertion sequences in purple; phage-gene related in blue. Genes colored in gray represent other genes. The inner lines depict additional regions with homology automatically generated by Genofig.
“Genomic map comparing segments of S. aureus C2944 and E. faecium plasmid pXD4 containing antimicrobial resistance genes with the one from Staphylococcus pseudintermedius BIOS-V16. Both maps show gene structures with antimicrobial resistance genes in yellow, insertion sequences in purple, and other coding sequences in gray. Shaded areas indicate regions of similarity between the bacterial segments and the reference. Scale bar indicates 8 kilobases.”
FIGURE 6
Analysis of the carriage of erm(B), aadE, spw, lsa(E) and lnu(B) genes by BIOS-V16 strain. (A) Comparison with S. aureus strain C2944 (JQ861959.1) and (B) Enterococcus faecium strain P23 plasmid pXD4 (KF421157). Homology is indicated through a color scale of gray: dark gray (100% homology) to light gray (94% (A) or 90% (B) homology). Antimicrobial resistance genes are represented in yellow; insertion sequence in purple. Genes colored in gray represent other genes. The figure was generated using Genofig v1.1. The inner lines depict additional regions with homology automatically generated by Genofig.
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