Mechanisms of linezolid resistance among coagulase-negative staphylococci determined by whole-genome sequencing.

UnlabelledLinezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ≥8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate.ImportanceLinezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria

Low prevalence of Cfr-mediated linezolid resistance among methicillin-resistant Staphylococcus aureus in a Spanish hospital: case report on linezolid resistance acquired during linezolid therapy

Linezolid is an effective antimicrobial agent to treat methicillin-resistant Staphylococcus aureus (MRSA). Resistance to linezolid due to the cfr gene is described worldwide. The present study aimed to analyze the prevalence of the cfr-mediated linezolid resistance among MRSA clinical isolates in our area. A very low prevalence of cfr mediated linezolid resistance was found: only one bacteremic isolate out of 2 215 screened isolates. The only linezolid resistant isolate arose in a patient, previously colonized by MRSA, following linezolid therapy. Despite the low rate of resistance in our area, ongoing surveillance is advisable to avoid the spread of linezolid resistance

Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme.

Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown

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

Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms

An in vitro evaluation of the efficacy of tedizolid: implications for the treatment of skin and soft tissue infections

Skin and soft tissue infections (SSTI) are among the most commonly occurring infections and evidence suggests that these are increasing world-wide. The aetiology is diverse, but Staphylococcus aureus predominate and these are often resistant to antimicrobials that were previously effective. Tedizolid is a new oxazolidinone-class antibacterial indicated for the treatment of adults with SSTI caused by Gram-positive pathogens, including S. aureus. The aim of this study was to evaluate the in vitro efficacy of tedizolid in comparison to other clinically used antibacterials against antibiotic sensitive- and resistant-staphylococci, grown in planktonic cultures and as biofilms reflecting the growth of the microorganism during episodes of SSTI. Against a panel of 66 clinical staphylococci, sensitivity testing revealed that a lower concentration of tedizolid was required to inhibit the growth of staphylococci compared to linezolid, vancomycin and daptomycin; with the tedizolid MIC being 8-fold (S. aureus) or 4-fold (S. epidermidis) below that obtained for linezolid. In addition, cfr+ linezolid-resistant strains remained fully susceptible to tedizolid. Against S. aureus biofilms, 10×MIC tedizolid was superior or comparable with 10×MIC comparator agents in activity, and superior to 10×MIC linezolid against those formed by S. epidermidis (65 vs. 33% reduction, respectively). Under flow-conditions both oxazolidinones at 10×MIC statistically out-performed vancomycin in their ability to reduce the viable cell count within a S. aureus biofilm with fewer the 12% of cells surviving compared to 63% of cells. In conclusion, tedizolid offers a realistic lower-dose alternative agent to treat staphylococcal SSTI, including infections caused by multi-drug resistant strains

Mechanisms of linezolid resistance among enterococci of clinical origin in Spain—detection of optrA-and cfr(D)-carrying E. faecalis

The mechanisms of linezolid resistance among 13 E. faecalis and 6 E. faecium isolates, recovered from six Spanish hospitals during 2017–2018, were investigated. The presence of acquired linezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins was analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial agents was investigated, and the respective molecular background was elucidated by PCR-amplicon sequencing and whole genome sequencing. The transferability of the linezolid resistance genes was evaluated by filter-mating experiments. The optrA gene was detected in all 13 E. faecalis isolates; and one optrA-positive isolate also carried the recently described cfr(D) gene. Moreover, one E. faecalis isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six E. faecium isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The fexA gene was located upstream of the optrA gene in 12 of the E. faecalis isolates. Moreover, an erm(A)-like gene was located downstream of optrA in two isolates recovered from the same hospital. The optrA gene was transferable in all but one E. faecalis isolates, in all cases along with the fexA gene. The cfr(D) gene was not transferable. The presence of optrA and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among E. faecalis and E. faecium, respectively. We report the first description of the cfr(D) gene in E. faecalis. The presence of the optrA and cfr(D) genes in Spanish hospitals is a public health concern

Linezolid and Rifampicin Combination to Combat cfr-Positive Multidrug-Resistant MRSA in Murine Models of Bacteremia and Skin and Skin Structure Infection.

Linezolid resistance mediated by the cfr gene in MRSA represents a global concern. We investigated relevant phenotype differences between cfr-positive and -negative MRSA that contribute to pathogenesis, and the efficacy of linezolid-based combination therapies in murine models of bacteremia and skin and skin structure infection (SSSI). As a group, cfr-positive MRSA exhibited significantly reduced susceptibilities to the host defense peptides tPMPs, human neutrophil peptide-1 (hNP-1), and cathelicidin LL-37 (P < 0.01). In addition, increased binding to fibronectin (FN) and endothelial cells paralleled robust biofilm formation in cfr-positive vs. -negative MRSA. In vitro phenotypes of cfr-positive MRSA translated into poor outcomes of linezolid monotherapy in vivo in murine bacteremia and SSSI models. Importantly, rifampicin showed synergistic activity as a combinatorial partner with linezolid, and the EC50 of linezolid decreased 6-fold in the presence of rifampicin. Furthermore, this combination therapy displayed efficacy against cfr-positive MRSA at clinically relevant doses. Altogether, these data suggest that the use of linezolid in combination with rifampicin poses a viable therapeutic alternative for bacteremia and SSSI caused by cfr-positive multidrug resistant MRSA

Mutational resistance to linezolid and other anti-gram-positive antibiotics in staphylococci

Methicillin-resistant Staphylococcus aureus (MRSA) are endemic in UK hospitals. Treatment options for the infections caused by these multiresistant strains include vancomycin, teicoplanin and linezolid. Mutational resistance to these antibiotics, although rare in the clinic, has been detected. Resistance to linezolid requires at least one chromosomal mutation followed by subsequent internal recombination. The hypothesis tested was that resistance to linezolid or glycopeptides could emerge in hypermutable strains and that hypermutability could be co-selected with resistance to linezolid or glycopeptides. Accordingly, this work studied linezolid and teicoplanin-resistant clinical isolates and laboratory-selected linezolid-resistant S. aureus mutants for evidence of hypermutability. Mutation frequencies to a range of antibiotics were determined to evaluate the occurrence of hypermutability. Linezolid-resistant mutants were selected by serial passage in the presence of increasing concentrations of linezolid. Mutations conferring linezolid resistance were characterized by PCR-RFLP and sequencing. Pyrosequencing and hybridization were used to detect and quantify six mutations known to confer linezolid resistance. Pre-existing hypermutability increased the ability of a laboratory strain to generate linezolid resistant mutants. However, few clinical linezolid or teicoplanin-resistant isolates or mutants were found to be hypermutable, indicating that stable hypermutability is not a prerequisite for the emergence of these resistances. Likewise, no laboratory-selected linezolid-resistant mutants were hypermutable, demonstrating the lack of co-selection of linezolid resistance with hypermutability. Most linezolid-resistant laboratory-selected mutants were unstable. There was a direct correlation between the number of mutated 23 S rRNA genes and linezolid MIC in clinical and laboratory mutants, although analysis was complicated by the fact that the 23S rRNA gene copy number was variable among the laboratory mutants. Cross-resistance between linezolid and chloramphenicol was detected in some laboratory mutants. In conclusion, there was no evidence for the co-selection of hypermutability and linezolid or teicoplanin resistance, and hypermutability was not found to be a prerequisite for the emergence of linezolid resistance

Low Proportion of Linezolid and Daptomycin Resistance Among Bloodborne Vancomycin-Resistant Enterococcus faecium and Methicillin-Resistant Staphylococcus aureus Infections in Europe

Vancomycin-resistant Enterococcus faecium (VREF) and methicillin-resistant Staphylococcus aureus (MRSA) are associated with significant health burden. We investigated linezolid and daptomycin resistance among VREF and MRSA in the EU/EEA between 2014 and 2018. Descriptive statistics and multivariable logistic regression were used to analyze 6,949 VREF and 35,131 MRSA blood isolates from patients with bloodstream infection. The population-weighted mean proportion of linezolid resistance in VREF and MRSA between 2014 and 2018 was 1.6% (95% CI 1.33–2.03%) and 0.28% (95% CI 0.32–0.38%), respectively. Daptomycin resistance in MRSA isolates was similarly low [1.1% (95% CI 0.75–1.6%)]. On the European level, there was no temporal change of daptomycin and linezolid resistance in MRSA and VREF. Multivariable regression analyses showed that there was a higher likelihood of linezolid and daptomycin resistance in MRSA (aOR: 2.74, p < 0.001; aOR: 2.25, p < 0.001) and linezolid in VREF (aOR: 1.99, p < 0.001) compared to their sensitive isolates. The low proportion of linezolid and daptomycin resistance in VREF and MRSA suggests that these last-resort antibiotics remain effective and will continue to play an important role in the clinical management of these infections in Europe. However, regional and national efforts to contain antimicrobial resistance should continue to monitor the trend through strengthened surveillance that includes genomic surveillance for early warning and action.Peer Reviewe

Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan

OBJECTIVES: Linezolid is an important therapeutic option for the treatment of infections caused by VRE. Linezolid is a synthetic antimicrobial and resistance to this antimicrobial agent remains relatively rare. As a result, data on the comparative genomics of linezolid resistance determinants in Enterococcus faecium are relatively sparse. METHODS: To address this knowledge gap in E. faecium, we deployed phenotypic antibiotic susceptibility testing and Illumina WGS on hospital surface (environmental) and clinical isolates from the USA and Pakistan. RESULTS: We found complete concordance between isolate source country and mechanism of linezolid resistance, with all the US isolates possessing a 23S rRNA gene mutation and the Pakistan isolates harbouring two to three acquired antibiotic resistance genes. These resistance genes include the recently elucidated efflux-pump genes optrA and poxtA and a novel cfr-like variant. Although there was no difference in the linezolid MIC between the US and Pakistan isolates, there was a significant difference in the geometric mean of the MIC between the Pakistan isolates that had two versus three of the acquired antibiotic resistance genes. In five of the Pakistan E. faecium that possessed all three of the resistance genes, we found no difference in the local genetic context of poxtA and the cfr-like gene, but we identified different genetic contexts surrounding optrA. CONCLUSIONS: These results demonstrate that E. faecium from different geographical regions employ alternative strategies to counter selective pressure of increasing clinical linezolid use