Novel Ribosomal Mutations in Staphylococcus aureus Strains Identified through Selection with the Oxazolidinones Linezolid and Torezolid (TR-700)

Abstract

TR-700 (torezolid), the active moiety of the novel oxazolidinone phosphate prodrug TR-701, is highly potent against gram-positive pathogens, including strains resistant to linezolid (LZD). Here we investigated the potential of Staphylococcus aureus strains ATCC 29213 (methicillin-susceptible S. aureus [MSSA]) and ATCC 33591 (methicillin-resistant S. aureus [MRSA]) to develop resistance to TR-700. The spontaneous frequencies of mutation of MSSA 29213 and MRSA 33591 resulting in reduced susceptibility to TR-700 at 2؋ the MIC were 1.1 ؋ 10 ؊10 and 1.9 ؋ 10 ؊10 , respectively. These values are ϳ16-fold lower than the corresponding LZD spontaneous mutation frequencies of both strains. Following 30 serial passages in the presence of TR-700, the MIC for MSSA 29213 remained constant (0.5 g/ml) while increasing eightfold (0.25 to 2.0 g/ml) for MRSA 33591. Serial passage of MSSA 29213 and MRSA 33591 in LZD resulted in 64-and 32-fold increases in LZD resistance (2 to 128 g/ml and 1 to 32 g/ml, respectively). Domain V 23S rRNA gene mutations (Escherichia coli numbering) found in TR-700-selected mutants included T2500A and a novel coupled T2571C/G2576T mutation, while LZD-selected mutants included G2447T, T2500A, and G2576T. We also identified mutations correlating with decreased susceptibility to TR-700 and LZD in the rplC and rplD genes, encoding the 50S ribosomal proteins L3 and L4, respectively. L3 mutations included Gly152Asp, Gly155Arg, Gly155Arg/ Met169Leu, and ⌬Phe127-His146. The only L4 mutation detected was Lys68Gln. TR-700 maintained a fourfold or greater potency advantage over LZD against all strains with ribosomal mutations. These data bring to light a variety of novel and less-characterized mutations associated with S. aureus resistance to oxazolidinones and demonstrate the low resistance potential of torezolid.Staphylococcus aureus infections pose a serious health threat worldwide. Increasing antibiotic resistance and the prevalence of methicillin (meticillin)-resistant S. aureus (MRSA) in clinical settings have created a demand for novel therapeutic agents. Linezolid (LZD) has a broad spectrum of activity against a variety of gram-positive pathogens, including MRSA, and was the first oxazolidinone antibiotic to gain FDA approval (1). LZD acts through inhibition of protein synthesis via binding to the peptidyl transferase center (PTC) of the 50S ribosomal subunit (37,65,68). Despite in vitro studies demonstrating a low resistance potential for LZD (31, 79), soon after its approval in 2000, LZD-resistant (LZD r ) MRSA (72) and LZD r , vancomycin (VAN)-resistant enterococci (22) emerged in the clinic. Although rare, resistance has most commonly occurred in patients undergoing long-term LZD therapy (10,17,22,45,72,74). Three classes of oxazolidinone resistance mechanisms have been previously characterized: mutations in the domain V region of 23S rRNA genes (69), acquisition of the ribosomal methyltransferase gene cfr (43), and mutations in the rplD gene encoding the 50S ribosomal protein L4 (76).A variety of 23S ...

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

“…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). Because the uL4 W65-Q75 loop is located in the vicinity of the phosphate of A2059, which belongs to the MLS B K-binding pocket, the aforementioned mutations may alter the W65-Q75 loop conformation, affecting A2059 conformation (Fig.…”

“…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).…”

Staphylococcal Infection, Antibiotic Resistance and Therapeutics

“…Linezolid resistance in S. aureus is uncommon though there are reports of mutations in 23S rRNA and ribosomal protein L3 and L4 encoded by rplD gene and rplC genes (Locke et al, 2009). However, the most worrisome mechanism involving acquisition of the methytransferase cfr that methylates the 23S rRNA associated with mobile genetic elements had first been identified in 16.5 kb multi-drug resistance plasmid in Staphylococcus sciuri (Kehrenberg et al, 2005).…”

Emergence of linezolid resistant Staphylococcus aureus in Bastar tribal region, India

“…7 Additional mutations in domain V of the 23S rRNA genes and substitutions in ribosomal protein L4 of the PTC are also reported in laboratory-derived lenizolid-resistant S. aureus strains. 7 The study was conducted in the microbiology laboratory of School of Life Sciences in MATS University, Raipur, Chattisgarh, India. Permission was sought to collect the pus samples from patients of Maharani Hospital, Jagdalpur, Bastar.…”