Comparative genomics of globaloptrA-carryingEnterococcus faecalisuncovers common genetic features and a chromosomal hotspot foroptrAacquisition

Abstract

27Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally 28 from multiple sources, but we still lack a comprehensive analysis of human and animal optrA-LREfs 29 strains. We investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), 30 virulence, prophages, plasmidome] and optrA-containing platforms of 28 publicly available optrA-31 positive E. faecalis genomes from different hosts in 7 countries. In the genome-level analysis, in house 32 databases with 57 virulence and 391 plasmid replication genes were tested for the first time. Our 33 analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera, 34 mainly but not exclusive from Firmicutes. The content in AMR and virulence genes was highly 35identical in contrast to the diversity of phages and plasmids observed. Epidemiologically unrelated 36 clones (ST476-like and ST21-like) obtained from human clinical and animal hosts in different 37 continents over 5 years (2012)(2013)(2014)(2015)(2016)(2017) were phylogenetically related (3-122 SNPs difference). They also 38 exhibited identical AMR and virulence profiles, highlighting a global spread of optrA-positive strains 39 with relevant adaptive traits in livestock and that they might originate from an animal reservoir. optrA 40 was located on the chromosome within a Tn6674-like element (n=9) or on medium-size plasmids (30-41 60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the 42 immediate gene vicinity of optrA was identical in chromosomal (Tn6674) and plasmid (impB-fexA-43 optrA) backbones. Tn6674 was always inserted in the same ∆radC integration site and embedded in a 44 32 kb chromosomal platform common to diverse strains from different origins (patients, healthy 45 humans, and animals) in Europe, Africa, and Asia during 2012-2018. This platform is conserved 46 among hundreds of E. faecalis genomes and we here propose a conserved chromosomal hotspot for 47 optrA integration. The finding of optrA in strains sharing identical adaptive features and genetic 48 backgrounds across different hosts and countries suggest the occurrence of common and independent 49 genetic events occurring in distant regions, and might explain the easy de novo generation of optrA-50 positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious 51 impact in the efficacy of linezolid for the treatment of Gram-positive infections. 52 53 Oxazolidinones (linezolid and tedizolid) are increasingly used for the treatment of human 55 infections caused by relevant pathogens such as methicillin-resistant Staphylococcus aureus and 56 vancomycin-resistant enterococci [1, 2]. Linezolid resistance rates remain generally low in enterococci 57 causing infections worldwide (<1%) [1, 3], however acquired linezolid resistance genes (cfr, optrA 58 and/or poxtA) are being increasingly reported in different enterococcal species and across different 59 settings [2-7]. Among t...