Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics

Abstract

Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites.KEYWORDS Cfr, L3 mutations, antibiotic resistance, linezolid resistance, tiamulin resistance O ver time, more and more mutations in bacterial ribosomal protein L3 (renamed uL3 in accordance with the new universal naming of ribosomal proteins [1]) have been associated with resistance to the antibiotics linezolid (LZD, an oxazolidinone) and tiamulin (TIA, a pleuromutilin). These drugs have overlapping binding sites at the peptidyl transferase center (PTC) in the ribosome (Fig. 1). Specific mutations of 23S rRNA and methylation at position 2503 also cause resistance to these antibiotics, as reviewed in references 2-4. The main part of L3 is positioned on the surface of the 50S ribosomal subunit, but a branched loop extends close to the PTC (Fig. 1), the binding site for various ribosomal antibiotics. The first L3 resistance mutation reported in bacteria was from Escherichia coli selected with TIA, and its role in resistance was verified by genetic evidence (5). Since then, L3 mutations have been associated with resistance to LZD, TIA/valnemulin, and anisomycin, as reviewed in references 2 and 6. Relationships between L3 mutations and antibiotic resistance have been reported in Brachyspira, Staphylococcus, E. coli, and Mycobacterium tuberculosis, but many of the findings lack genetic verification. Our recent study (6) demonstrated a clear antibiotic

Dissemination of cfr-mediated linezolid resistance among Staphylococcus species isolated from a teaching hospital in Beijing, China

“… 24 – 26 The combination of the cfr gene and L3 substitutions can act synergistically. 27 The mechanisms of linezolid resistance in X4535 may be the combination of cfr and fexA genes and mutations in rplC .…”

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa

“…These included insertions in PA14_42790, which encodes a predicted 50S ribosomal protein L3 N(5)-glutamine methyltransferase. The ribosomal protein L3 contains a branched loop near the PTC, and mutations in L3 are found in linezolid-resistant bacteria (75)(76)(77). L3 methylation confers resistance to PTC-targeting antibiotics in Azospirillum brasilense, although linezolid was not tested in that study (46).…”