Abstract
A tedizolid-resistant isolate of MRSA was selected by serial passage. Whole genome sequencing revealed only a single nucleotide variant in rpoB. Cross-resistance to linezolid, chloramphenicol, and quinupristin-dalfopristin was observed but susceptibility to other drugs including rifampin was unchanged. Models of the RNA-polymerase-ribosomal complex revealed that the mutated residue was unlikely to interact directly with the oxazolidinone binding site. This is the first time that rpoB mutation has been associated with resistance to the PhLOPSa antimicrobials.
Tedizolid is an oxazolidinone antimicrobial with broad spectrum activity against Gram-positive bacteria including methicillin-resistant S. aureus (MRSA).1 Like other oxazolidinones tedizolid exerts its antibacterial activity by binding the 23s rRNA component of the 50s-ribosomal subunit and thus inhibiting protein synthesis. Resistance to tedizolid is uncommon but mutations in the ribosomal proteins L3, L4, and L22 (encoded by rplC, rplD, and rplV respectively), and the 23S rRNA target, which also mediate the so-called PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) resistance phenotype have been implicated.2, 3 Acquisition of the transferable rRNA methyltransferase gene, cfr, may also cause resistance to linezolid and other PhLOPSa antimicrobials but is generally believed to be insufficient to produce tedizolid resistance on its own even though it may increase the tedizolid MIC. The plasmid-carried optrA gene, which encodes an ABC transporter, has been implicated in oxazolidinone and phenicol resistance in enterococci and streptococci but has not been identified in S. aureus.4 Previous serial passage studies with tedizolid have seen limited success in selecting for tedizolid resistance and only mutations in the 23S rRNA have been recovered following tedizolid exposure.5 Genes affecting quinolone efflux such as norA and mepA, or lincosamide efflux such as mdeA have not been reported to affect oxazolidinone activity.
In this study, we observed the emergence of a mutant exhibiting a novel mechanism of PhLOPSa resistance from a well characterized MRSA strain after serial passage in escalating concentrations of tedizolid.
Using the well characterized MRSA strain, N315, we selected for tedizolid resistance by serial passage in escalating concentrations of tedizolid in Mueller Hinton II broth (MHB) starting with 0.5× the MIC. Once visible growth was observed a sample of the broth was diluted 1:1000 into fresh MHB with twice the previous concentration of tedizolid until an isolate with an MIC of ≥4 mg/mL was recovered. This MIC was selected since it is 1 log2 dilution above the breakpoint for resistance (MIC ≥ 2mg/L). After 10 passages, we recovered an isolate (N315-TDZ4) with a stable tedizolid MIC of 4mg/L or 16× the MIC of the parent strain, N315. To explore the cross resistance associated with this evolved strain we reevaluated susceptibility to a panel of other antimicrobial agents (Table 1) by broth microdilution in accordance with Clinical Laboratory Standards Institute (CLSI) guidelines6 or by gradient strip in the case of quinupristin-dalfopristin (Liofilchem®). Cross-resistance to chloramphenicol, linezolid, and quinupristin-dalfopristin was observed but susceptibility to other drugs tested was relatively unchanged (Table 1). N315 is resistant to clindamycin so lincosamide cross-resistance was unevaluable in this study and we did not have access to pleuromutilins, which are not yet a clinically important class of antimicrobials. While macrolide susceptibility is not considered part of the PhLOPSa group macrolides also target the 50s ribosome, however, erythromycin susceptibility was also unevaluable since N315 is resistant to erythromycin at baseline.
N315-TDZ4 was subjected to whole genome sequencing (WGS) using the MiSeq platform (Illumina, San Diego, CA, USA) as previously described7 to an average read depth of at least 50× per isolate and the. Sequence data from this study is freely available through the NCBI Sequence Read Archive (PRJNA578164). WGS of the N315-TDZ4 and comparison to the parent strain revealed a single nucleotide variant (SNV) in the rpoB gene (1345 A>G) corresponding to the amino acid substitution Asn449Asp. This mutation lies outside of the rifampin resistance-determining regions, which span from nucleotides 1384 – 1464 (AA 462–488) and 1543 – 1590 (AA 462–488) 8, 9, and we correspondingly did not observe any change in rifampin susceptibly (Table 1). Previous studies have reported an association between vancomycin and daptomycin susceptibility and certain rpoB mutations including Ala621Glu, and Ala477Asp, and His481Tyr.10–12 We did not observe any change in daptomycin MIC for our mutant but we did see a 1 log2-dilution increase in vancomycin MIC. To further assess the potential significance of this increase in vancomycin MIC we tested the N315-TDZ4 isolate and N315 parent strains for the heterogeneous vancomycin intermediate S. aureus (hVISA) phenotype by the gold standard population analysis profile (PAP) as previously described.13 Interestingly, we observed that PAP-AUC ratio with Mu3 of N315-TDZ4 was 0.85, up from 0.44 of the parent strain N315 (Table 1). While this increase is substantial it falls below the categorical criterion to declare this isolate an hVISA (PAP AUC ratio with Mu3 of ≥0.9).
In an effort to evaluate the potential impact of the rpoB Asn449Asp mutation on target protein function, we constructed a homology model of S. aureus rpoB using I-TASSER,14 and modeled the relative orientations of the RNA polymerase (RNAP) and the ribosome (Figure 1). This model suggests that the RNAP mutation would be unlikely to interact directly with binding sites for the oxazolidinones and other PhLOPSa drugs located in the 50S ribosomal subunit (Figure 1). This finding suggests an indirect mechanism of resistance, possibly involving one of the many factors that interact with the beta-subunit of RNAP.8
In this study, we report for the first time a novel mechanism of resistance to oxazolidinones, phenicols, and streptogramins involving mutation in rpoB, which encodes the β-subunit of RNAP. The precise molecular mechanism by which this mutation mediates this resistance phenotype is unclear but may involve transcriptional modulation by altered sigma-factor binding. Previous studies have demonstrated that the 30s-ribosomal protein S10 encoded by rpsJ links directly with the β-subunit of RNAP facilitating the tight linkage between transcription and translation in bacteria but the RNAP only interacts with the leading 30s-subunit and no known interaction between the 50s subunits and the RNAP exist. The fact that cross resistance was isolated to 50s-ribosomally active agents and not 30s active agents (doxycycline) suggests a relatively specific interaction with the 50s-ribosome and not a broader modulation of translation or protein synthesis. Based on the absence of altered susceptibility to other intracellularly active agents including moxifloxacin and doxycycline it seems unlikely that this variant facilitates multidrug efflux. For now, tedizolid resistance remains uncommon clinically and it is unknown whether this unique mutation could be selected for by other PhLOPSa drugs or would be likely to emerge after clinical exposure to tedizolid.
Acknowledgments
This study was initially presented in part at the annual ID-Week conference in San Francisco, CA 2018. This study was supported in part by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under the award numbers 1R21AI132994-01A1, 1R01AI136979-01 and by the University of Washington.