Objectives: To identify and characterize a novel tetracycline resistance gene on a multiresistance plasmid from Staphylococcus aureus SA01 of chicken origin. Methods: MICs were determined by broth microdilution according to CLSI recommendations. The whole genome sequence of S. aureus SA01 was determined via Illumina HiSeq and Oxford Nanopore platforms followed by a hybrid assembly. The new tet gene was cloned and expressed in S. aureus. The functionality of the corresponding protein as an efflux pump was tested by efflux pump inhibition assays. Results: A novel tetracycline resistance gene, tet(63), was identified on a plasmid in S. aureus SA01. The cloned tet(63) gene was functionally expressed in S. aureus and shown to confer resistance to tetracycline and doxycycline, and a slightly elevated MIC of minocycline. The tet(63) gene encodes a 459 amino acid efflux protein of the major facilitator superfamily that consists of 14 predicted transmembrane helices. The results of efflux pump inhibitor assays confirmed the function of Tet(63) as an efflux protein. The deduced amino acid sequence of the Tet(63) protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). The plasmid pSA01-tet, on which tet(63) was located, had a size of 25664 bp and also carried the resistance genes aadD, aacA-aphD and erm(C). Conclusions: A novel tetracycline resistance gene, tet(63), was identified in S. aureus. Its location on a multiresistance plasmid might support the co-selection of tet(63) under the selective pressure imposed by the use of macrolides, lincosamides and aminoglycosides.

(1) Background: methicillin‑resistant Staphylococcus aureus (MRSA) is a zoonotic pathogen, and its hemolysins serve as key virulence factors. Functional membrane microdomains (FMMs) are protein-enriching platforms and regulate diverse physiological functions by recruiting and assembling various proteins. However, whether and how FMMs regulate the hemolytic ability of MRSA remains unclear. This study aimed to investigate FMM-mediated regulation of MRSA hemolysins and the underlying mechanisms. (2) Methods: Homologous recombination was employed to generate FMM-disrupted (N315ΔfloA) and complemented (N315ΔfloA::floA) strains from the MRSA N315 wild-type strain (N315 WT). The three strains were compared with respect to hemolytic activity, transcript levels of key virulence and regulatory genes, and in vivo virulence. (3) Results: Disruption of FMMs significantly enhanced hemolytic activity compared with N315 WT and complemented strains. Meanwhile, FMM disruption repressed the two-component system genes (vraS and vraR), while activating the agr operon (agrB, agrD, agrC and agrA) and its effector molecule RNAIII, leading to upregulation of hemolysin genes (hla, hlb, hld). In vivo, N315ΔfloA infection markedly increased mortality in G. mellonella larvae and BALB/c mice, with significantly elevated pro-inflammatory factors (TNF-α, IL-6, and IL-1β) in mouse plasma. All these phenotypes were effectively reversed in N315ΔfloA::floA. (4) Conclusions: Disruption of FMMs potentiates both hemolytic activity and overall virulence in MRSA, with the potential underlying mechanism involving the VraS/R-Agr regulatory axis that drives transcriptional upregulation of hemolysin-encoding genes.