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

Abstract

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.

Figure 1.

Recombination-free phylogenetic tree including MLST, country, source, resistance, resistance gene and mutation data. Linezolid resistance in US isolates was attributed solely to the G2576T mutation of the 23S rRNA gene sequence. In contrast, linezolid resistance in Pakistan isolates resulted from different combinations of the acquired resistance genes optrA, poxtA and a cfr-like gene. Vancomycin resistance was observed in 90.6% (87/96) of the isolates and dalbavancin resistance was observed in 88.5% (85/96). Daptomycin resistance was observed in 3.13% (3/96) of the isolates with an additional 68.8% (66/96) classified as susceptible dose-dependent.

Figure 2.

Linezolid and tedizolid MICs and comparisons by basis of resistance mechanism. The geometric mean MIC of linezolid (a) is higher than the geometric mean MIC of tedizolid (b) at 21.83 and 2.87 mg/L, respectively. There was no difference in linezolid resistance between isolates with gene- or mutation-based resistance mechanisms (c). However, isolates that harboured poxtA and cfr-like genes had significantly lower levels of linezolid resistance than those that harboured all three linezolid resistance genes (d); statistical analysis was done using the unpaired t-test in Prism v8. Please note, y-axis values for all graphs are log₂ scaled for visual acuity.

Figure 3.

Genetic context of optrA in isolates that harbour optrA, cfr-like and poxtA genes. In isolates EF_325, EF_048, EF_111 and EF_181, optrA is downstream of the resistance gene fexA and in isolate EF_155 it is upstream of an erm(A1) resistance gene. These contexts are similar to those that optrA was in when it was first identified. However, the mobile elements surrounding optrA in our isolates differ from those previously identified. optrA’s location near mobile elements may allow it to be transferable.