Compensatory adaptation to the loss of biological fitness associated with acquisition of fusidic acid resistance in Staphylococcus aureus

Abstract

Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in Staphylococcus aureus. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of S. aureus. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in S. aureus carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of S. aureus can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population.

FIG. 1.

Analysis of the expression of the plasmid-encoded fusA alleles in S. aureus RN4220/pSB2, S. aureus RN4220/pSB2-S416F, and RN4220/pSB2-H457Y by RT-PCR. RT-PCR was performed with the fusA-specific forward primer P9 and the reverse primer P10 with 100 pg of total RNA as the template. The PCR products obtained from S. aureus RN4220/pSB2 (lanes 1 and 2), S. aureus RN4220/pSB2-S416F (lanes 3 and 4), and S. aureus RN4220/pSB2-H457Y (lanes 5 and 6) were either directly separated by agarose gel electrophoresis (lanes 1, 3, and 5) or incubated with DraI (lanes 2 and 4) or EcoRV (lanes 2 and 6) before separation on the agarose gel. Lanes M, 123-bp DNA ladder (Invitrogen); the sizes of the marker fragments are given in base pairs.

FIG. 2.

Growth kinetics of recombinant S. aureus strains. (A) RN4220/pSB2 (wild type [WT]), RN4220/pSB2-A67T (A67T), RN4220/pSB2-P406L (P406L), RN4220/pSB2-A67T/P406L (A67T/P406L); (B) RN4220/pSB2 (WT), RN4220/pSB2-S416F (S416F), RN4220/pSB2-H457Y (H457Y), and RN4220/pSB2-S416F/H457Y (S416F/H457Y). Experiments were performed in triplicate. The results of one experiment are shown as an example.