Estrutura populacional e cenário evolutivo do patógeno multirresistente Enterococcus faecium em instituições hospitalares do Rio de Janeiro

Abstract

Enterococcus faecium stands out as an opportunistic pathogen frequently associated with hospital-acquired infections around the world. The aim of this study was to determine the population structure among E. faecium isolates recovered in hospitals of Rio de Janeiro city, Brazil, over a period of eight consecutive years (2009 to 2016) and to evaluate the genetic events related to the evolution of hospital-adapted clonal lineages. The bacterial isolates were initially characterized according to their susceptibilites to antimicrobial agents, evaluated by the disk-diffusion method, and to the presence of genetic determinants associated with resistance to antimicrobial agents and with virulence, investigated by PCR assays. Based on the results obtained, 52 strains were subsequently selected for whole-genome sequencing (WGS) using the Illumina Hiseq platform. In addition, another 22 strains belonging to the hospital-adapted lineage ST78, previously predominant in the city hospital institutions, were included for comparative purposes. An extensive pipeline of bioinformatics tools was implemented to reconstruct phylogenetic relationships and analyze different genetic aspects of the evolution of the 74 strains studied. Phylogenetic analyses indicated the occurrence of multiple lineage changes over time. ST78 was predominant among isolates recovered from 2002 to 2008, being replaced by the emergence of ST412. This lineage was predominant until 2012, when two new lineages have emerged: ST963 in 2013, and ST896 in 2015. Genome analyses revealed different mechanisms associated with the evolution of these lineages. ST78 had its emergence and expansion associated with the enlargement of the acessory genome, with a high number of insertion sequences (ISs) and prophages. Moreover, the exclusive presence of a toxin-antitoxin system, involved in the stabilization of mobile genetic elements (MGE), corroborated these findings. On the other side, the emergence of ST412, observed in two distinct waves, was associated with several recombination events in the ancestor nodes. Additionally, a high number of SNPs, possibly related to the formation of hypermutantes due to a stop codon acquisition in a DNA repair system, and the exclusive acquisition of cell wall biogenesis genes, were associated with the expansion of this lineage. Recombination events in the ancestor nodes were detected in the ST963 and ST896 lineages. ST896 had the lowest number of acessories genes, but a frequent presence of antibiotic resistance was strongly associated with this group. The expansion of ST963 was accompanied by the exclusive acquisition of genes related to amino acid metabolism and biosynthesis of secondary metabolites. A large number of genetic novelty units (GNU) was observed in this group. Some features such as the presence of drfF gene (trimethoprim resistance), virulence determinant hyl, the ISLgar5 and the allele 44 to purK gene were also related to the lineages that replaced ST78. Overall, our results indicate that the E. faecium population investigated has undergone successive lineage replacement in a dynamic evolutionary process. The data highlighted the different genetic events that may play a role in the emergency of specialized subpopulations adapted to the hospital environment and endorse the need of continuous monitoring the infections caused by this microorganism.