Background/Objectives: Staphylococcus haemolyticus is a common skin commensal that has emerged as a multidrug-resistant nosocomial pathogen, with sequence type 42 frequently implicated in clinical settings. The genetic basis of fusidic acid (FA) resistance mediated by mutations in elongation factor-G (fusA/EF-G) has not been systematically characterized in S. haemolyticus. Methods: Five representative FA-susceptible S. haemolyticus isolates were selected. In vitro FA resistance was induced by incubating each isolate on Mueller–Hinton agar containing 4 µg/mL FA at 37 °C for 48 h. Resistant colonies were recovered and fusA was sequenced by Sanger sequencing to identify mutations. Growth doubling times of EF-G mutant isolates were measured and compared with those of the parental susceptible strains. Potential fitness-compensatory changes in fusA were assessed by serial passage of selected mutants for ten successive passages and re-sequencing of fusA. Results: A total of 28 FA-resistant colonies were recovered. Sequencing identified mutations at seven nucleotide loci corresponding to ten distinct amino acid substitutions in EF-G: Q115L, L430S, E433G, G452C, H457Y, H457N, H457L, H457Q, R464L, and A655G. The mean doubling time of EF-G mutant isolates was significantly longer than that of the wild-type parental strains (mutants: 55.75 ± 7.78 min, n = 28; wild type: 40.78 ± 4.13 min, n = 5; Welch’s two-sample t test: t = −6.34, df ≈ 10.0, two-tailed p < 0.0001). Following ten serial passages, we did not detect compensatory mutations in fusA that restored the ancestral growth rate. Conclusions: FA resistance in S. haemolyticus can be rapidly induced in vitro through mutations in EF-G/fusA. Compared with previously reported EF-G mutations in other staphylococci, we identified two novel substitution sites (L430S, E433G) and previously unreported substitutions at established resistance positions (H457N, H457L, A655G). Mutations in EF-G (encoded by fusA) were associated with a measurable in vitro fitness cost; following ten serial passages, compensatory mutation was not detected in fusA. These findings support systematic surveillance of EF-G/fusA mutations in clinical S. haemolyticus isolates. Further studies should address their prevalence, stability, transmissibility, and clinical impact, particularly where FA use is increasing and patient populations are vulnerable.

Background/Objectives: Staphylococcus haemolyticus is a common commensal bacterium that has emerged as an important nosocomial pathogen. Its multi-antibiotics resistance presents substantial therapeutic challenges in healthcare settings worldwide. Despite its growing clinical relevance, most investigations into antimicrobial resistance determinants have been focused on Staphylococcus aureus or Staphylococcus epidermidis, leaving S. haemolyticus comparatively understudied. This study aimed to elucidate the genetic basis of multi-drug resistance by characterizing mobile genetic elements associated with predominant S. haemolyticus clones circulating in Taiwan. Methods: From 2010 to 2017, 140 clinical targeted isolates of S. haemolyticus were obtained from individual patients. Two representative strains, SH53 (ST3) and SH51 (ST42), were sequenced using the PacBio^TM platform. The structural organization of SCCmec cassettes and phage-associated resistance islands in the remaining 138 isolates was analyzed by polymerase chain reaction (PCR) using specifically designed primers. Results: Of the 140 isolates, 92 (65.7%) were ST42 and 48 (34.3%) were ST3. PCR analysis showed that over two-thirds harbored heavy metal resistance genes. cadD, cadX, arsC, arsB, and arsR occurred in 90.2% of ST42 isolates, with copA in 71.7%. In ST3, these five genes were present in 89.6%, and copA in 64.6%. Fusidic acid (FA) resistance was more frequent in ST42 (46.7%) than ST3 (22.9%) (p = 0.015). Only one ST42 isolate carried fusC. The remaining 52 FA-resistant isolates contained a type I aj1–leader peptide (LP)–fusB structure downstream of smpB, except for a single ST42 isolate with the type IV structure. Conclusions: MDR ST42 S. haemolyticus carrying SCCmec cassettes with heavy metal resistance genes and phage-related islands carrying type I aj1–leader peptide (LP)–fusB structures may represent emerging opportunistic pathogens in Taiwan. Continued longitudinal surveillance is warranted to track the evolution of resistance-associated mobile elements under selective antimicrobial pressure.

QacA and QacB are staphylococcal multidrug efflux proteins that share high sequence homology, differing by only six to seven amino acids. While the prevalence of these genes among clinical isolates can exceed 70% in certain regions, and qacA is well-documented to elevate chlorhexidine minimal inhibitory concentrations (MICs), the specific contribution of qacB to chlorhexidine tolerance remains incompletely defined. To investigate these differences, in silico structural analyses were performed using UCSF ChimeraX. Sequence data from NCBI were mapped onto the experimental 3.80 Å cryo-EM structure of QacA (PDB ID: 7Y58) to compare global and local conformational and electrostatic properties. Phenotypically, the pLI50 plasmid carrying either qacA or qacB was electroporated into Staphylococcus aureus strain RN4220. Chlorhexidine MICs were subsequently determined using agar dilution assays. Global in silico modeling demonstrated that the amino acid substitutions distinguishing QacB from QacA did not alter the backbone conformation, hydrogen bond counts, solvent-accessible surface area, or overall Coulombic surface potential. However, local analyses identified distinct microenvironmental perturbations attributable to the physicochemical differences of the substituted residues. Phenotypically, the introduction of qacA into S. aureus RN4220 reproducibly increased chlorhexidine MICs from 1 µg/mL to 4 µg/mL in 96% (48/50) of independent assays (adjusted OR > 999; P < 0.001). Conversely, the presence of qacB significantly increased MICs from 1 µg/mL to 2 µg/mL in 90% (45/50) of analogous assays, demonstrating a distinct but less pronounced elevation in tolerance (adjusted OR = 81.0; P = 0.0013). Both qacA and qacB contribute to increased chlorhexidine MICs in S. aureus RN4220 under the conditions tested. Given the high prevalence of these genes among clinical isolates and the widespread use of chlorhexidine in infection prevention, these findings support continued surveillance and further mechanistic studies to assess clinical and infection-control implications.