Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

Genetic features of the poxtA linezolid resistance gene in human enterococci from France.

OBJECTIVES: To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. METHODS: All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. RESULTS: Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. CONCLUSIONS: The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.

Investigation of a vanA linezolid- and vancomycin-resistant Enterococcus faecium outbreak in the Southwest Indian Ocean (Reunion Island).

INTRODUCTION: Dual resistance to linezolid and glycopeptides is a milestone reached by certain extensively drug-resistant (XDR) enterococci. This paper describes the molecular and epidemiological investigations of a linezolid-resistant and vancomycin-resistant Enterococcus faecium (E. faecium) (LVREf) outbreak in the French overseas territory of Reunion Island (Indian Ocean). METHODS: All vancomycin-resistant Enterococcus (VRE) isolates detected on Reunion Island between 2015 and 2019 were included in the study. The VRE isolates were phenotypically characterised and genetically explored by whole-genome sequencing (WGS). RESULTS: Sixteen vancomycin-resistant E. faecium (VREf) isolates were retrieved between 2015 and 2019. Seven isolates obtained in 2019 were involved in the outbreak. These seven LVREf isolates from the 2019 outbreak at the University Hospital of Reunion Island (UHRI) were suspected to be related to a linezolid-susceptible VREf strain imported from India. An epidemiological link was highlighted for six of the seven outbreak cases. All the LVREf outbreak isolates were obtained from rectal swabs (colonisation) and resistant to vancomycin (MIC > 128 mg/L) and linezolid (MIC 8-32 mg/L); one isolate was also resistant to daptomycin (MIC 8 mg/L). The seven outbreak isolates were positive for the vanA and optrA genes and belonged to ST761. CONCLUSIONS: These results argue for the strict application of control and prevention measures for VRE clones at high risk of spread, particularly in areas such as Reunion Island where the risk of importation from the Indian subcontinent is high. The regional spread of optrA linezolid-resistance genes in VRE isolates is a matter of concern, due to possibility of treatment failure.