Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission

Abstract

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZD^R) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZD^R enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZD^R-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZD^R enterococci in all ecological niches and body parts to direct appropriate control strategies.

Fig. 1

Genetic environment of the optrA gene in the eight E. faecalis and E. casseliflavus isolates from healthy pigs, pig farmer, and a dog. Shown in the figure are AMR genes located in the same contigs and frames with their corresponding mobile genetic elements. The percentage of identity and scale bar legends are presented on the right side of the image. The comparison was made with a reference E. faecalis strain E349 (GenBank Accession number: KP399637) (colour figure online)

Fig. 2

Genetic representation of the optrA-carrying plasmid pURX4962. Colors and arrows indicate the represented genes and their orientation (color figure online)

Fig. 3

Schematic comparison between the environment of the cfrD gene in the E. casseliflavus isolate X4962 with E. faecalis (GenBank Accession number: CP097040) and E. faecium (GenBank Accession number: MN831413) (color figure online)

Fig. 4

Genetic environment of the poxtA gene in the E. faecium isolate X3877 in comparison with the previously described plasmid-bound poxtA gene in the E. faecium strain pGZ8 (GenBank Accession number: CP038162) (color figure online)

Fig. 5

Phylogenetic tree based on core-genome SNPs analysis of seven optrA-carrying E. faecalis isolates from healthy hosts in this study with twelve publicly available E. faecalis genomes with similar STs and linezolid resistance genes. Colors (in circles) of the AMR genes are as follows: dark purple for optrA while and light purple for fexA and cat (color figure online)