Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli , although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.

Outbreaks caused by linezolid-resistant (LR) enterococci remain rare. We report the epidemiological and molecular characteristics of the multiclonal dissemination of LR enterococci in the intensive care unit (ICU) of a Greek hospital. All LR enterococcal isolates recovered from patients hospitalized in the ICU of the University Hospital of Larissa, Greece, between January 2007 and October 2008 were included. Isolates were tested by PFGE and PCR followed by sequence analysis of the entire 23S rRNA gene. Patient records were retrieved to access patterns of acquisition and outcome. Sixteen separate patients were infected and/or colonized by 22 LR enterococcal isolates (17 Enterococcus faecium and 5 Enterococcus faecalis). Linezolid MICs varied from 8 to 16 mg/L; 12 isolates showed cross-resistance to vancomycin. Genotyping revealed as many as seven and three PFGE types among E. faecium and E. faecalis isolates, respectively, indicating multiclonal spread of LR enterococci. Nine patients had received linezolid prior to the recovery of LR enterococci, while the remaining seven patients were not exposed to the drug. All isolates carried the mutation G2576T; the mutated position was heterogeneous in 12 isolates and homogeneous in 10. The multiclonal composition of LR enterococci indicates that linezolid resistance possibly occurred on several independent occasions. Its acquisition was often not related to linezolid administration; patients might have acquired their LR isolate from another patient that had received linezolid or, alternatively, resistance may have arisen by mutation that occurred independently.

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.

Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.