Elongation Factor-G (fusA) Mutations That Confer Fusidic Acid Resistance in Staphylococcus haemolyticus

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.