Single- and Multistep Resistance Selection Studies on the Activity of Retapamulin Compared to Other Agents against Staphylococcus aureus and Streptococcus pyogenes

Abstract

Retapamulin had the lowest rate of spontaneous mutations by single-step passaging and the lowest parent and selected mutant MICs by multistep passaging among all drugs tested for all Staphylococcus aureus strains and three Streptococcus pyogenes strains which yielded resistant clones. Retapamulin has a low potential for resistance selection in S. pyogenes, with a slow and gradual propensity for resistance development in S. aureus.Pleuromutilins are a new class of antimicrobials which inhibit protein synthesis by interacting at a unique site on the 70S ribosome and demonstrate excellent in vitro activity against gram-positive and some gram-negative bacteria (2,5,9). This study used multi-and single-step passage studies to test the ability of retapamulin ( Fig. 1), compared to those of mupirocin, fusidic acid (only against Staphylococcus aureus strains), cephalexin, erythromycin, linezolid, vancomycin, and quinupristindalfopristin, to select for resistance in 12 Staphylococcus aureus and 10 Streptococcus pyogenes strains.S. aureus strains comprised three methicillin-and quinolonesusceptible, four methicillin-and quinolone-resistant, three vancomycin-intermediate, and two vancomycin-resistant (VRSA) strains. The 10 S. pyogenes strains comprised two macrolidesusceptible strains, two erm(B)-, three mef(A)-, and two erm(TR)-positive strains, and one strain with a mutation in the L4 ribosomal protein (68KGT insertion). Retapamulin and mupirocin powders were obtained from GlaxoSmithKline, Collegeville, Pa., and other drugs were obtained from their respective manufacturers. Initial MICs were determined by the CLSI agar dilution method (7).Multipassage resistance selection was done as described previously (3, 6). Daily passages were continued until a more-thanfour-fold increase in the MIC was found (minimum passage number, 14; maximum passage number, 50). If MICs of Ն32 g/ml were found, subculturing in the presence of an antibiotic ceased. Prolonged selection for the full 50 days was conducted for three random staphylococcal strains and all three streptococcal strains showing retapamulin resistance development. The stability of acquired resistance was determined by MIC determination after 10 daily passages on drug-free agar.The frequency of spontaneous single-passage mutations was determined as described previously (3, 6). Pulsed-field gel electrophoresis was used to confirm the identities of all parents and clones (3,6).Genes encoding the L3 ribosomal protein were amplified and sequenced from all parents and from clones for which an elevation in the retapamulin MIC was observed by the multipassage study. Eight S. aureus and seven S. pyogenes singlepassage mutants were randomly selected for L3 analysis. The genes encoding ribosomal proteins L4 and L22 and domains II and V of 23S rRNA were amplified and sequenced for all S.

Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

“…6 D and E). Mutations in uL4 rProtein, G69A, and T70P produce S. aureus resistance to linezolid (65), whereas Q67K, G69E, G69A, and T70P mutations produce S. aureus resistance to erythromycin (15). In addition, ΔK68 and ΔG69 are susceptible to linezolid, and K68Q mutation produces S. aureus resistance to linezolid, pleuromutilins, chloramphenicol, and tedizolid (17).…”

“…Nevertheless, the epidemiology of S. aureus is increasing (11). Resistance mutations in S. aureus are also associated with ribosomal protein (rProtein) uL3, located in proximity to the PTC, as well as with uL4 and uL22 rProteins, whose segments are exposed in the exit tunnel (12)(13)(14)(15)(16)(17)(18)(19)(20).…”

Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

“…Finally, two new L3 mutations have also been found in linezolid-resistant S. aureus strains from a cystic fibrosis patient after prolonged drug treatment, where one strain also possessed the G2576U 23S rRNA mutation (15). It is worth mentioning that mutations of L3 at some of the positions mentioned above, as well as at amino acids close by, are associated with resistance to the pleuromutilins retapamulin and tiamulin (24,42,43,66). As linezolid and the pleuromutilins binds at overlapping sites at the PTC, these findings support the relationship between L3 mutations and PTC antibiotic resistance in general.…”

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

“…† † Continuous efforts to develop pleuromutilin antibiotics yielded several semisynthetic derivatives, some of which with elevated activity over a broad spectrum of pathogens. The most advanced compound, retapamulin (SB-275833; SmithKline Beecham), has potent activity against Gram-positive pathogens (5-11) and a low propensity to select for resistance (12), has recently completed phase III clinical trials as a topical agent, showing that all strains of Staphylococcus aureus and Streptococcus pyogenes were susceptible to retapamulin with MICs Յ0.5 g/ml (13,14).…”