Propriedades fenotípicas e moleculares associadas aos aspectos evolutivos de Enterococcus faecium: análise comparativa de estratégias de um patógeno oportunista

Abstract

Enterococcus faecium stands out as a bacterial species that have been often associated with hospital-acquired infections around the world. The aim of this study was to compare the characteristics of two E. faecium strains SS-1274 (type strain) and CL-6729 (clinical isolate from nosocomial urinary tract infection). The bacterial strains were characterized by antimicrobial susceptibilities, presence of genetic determinants of virulence and resistance and molecular typing methods as PFGE, MLVA, and MLST. It was also evaluated the biofilm formation and architecture by confocal laser scanning microscopy (CLSM). The response of macrophages J.774 to biofilms and planktonic growth was identified by enzyme-linked immunosorbent assay quantification of cytokines TNF-α, IFN-γ, IL-6 and IL-10, determination of nitric oxide concentration and CLSM. Proteomic profiles of the bacterial cells from biofilms and planktonic growth were carried out by ESI-Q-TOF mass spectrometry methodology from prior results achieved by SDS-PAGE. It was also investigated the whole genome sequencing (WGS) of CL-6729 using Illumina Hiseq platform and genes annotation was performed on PATRIC 3.2.75. Our results showed that CL-6729 is multi-drug resistant and harbored the genes vanA and esp. This isolated was molecular typed as MT-12 and ST-78, therefore belonging to the clonal complex 17 (CC17). This isolate also showed constitutive resistance to vancomycin, an unusual characteristic for the species. Analysis of Tn1546 revealed the presence of IS19 inserted into the gene vanS. According to the data obtained from qPCR and partial sequencing of the genes involved with vancomycin resistance, the presence of IS19 seemed to be influencing directly or indirectly on the changes of the resistance mechanisms expression as was identified from both biofilms and planktonic growing. Biofilms of E. faecium showed protein-rich and DNA-rich biomass after 24 h and 72 h of formation, respectively. Interaction assays demonstrated a negative modulation of nitrite production by macrophages J.774. On the other hand, the data suggest ainduction of cytokines production, TNF-α, IFN-, IL-6 e IL-10, by biofilms and planktonic cells from both E. faecium strains. In addition, CLSM has shown that macrophages were able to penetrate the biofilms structure. The results relating to mass spectrometry suggest that biofilm cells of E. faecium had seemed to be metabolicaly active, according to the identification of a significant number of proteins related to metabolism and cell division. WGS revealed a 2.9 Mb size, with 3,125 predicted genes, among which about 2,000 genes are associated with the carbohydrates and nucleic acids metabolisms, and cell division. Genes associated with antimicrobial resistance, virulence, insertion sequences, phage and plasmids were annotated. Our data point to a dynamic and evolutionary process, indicating significant changes between samples of the same species. These data highlights the continuous evolution process of this species that changes the concept from an ordinary opportunistic pathogen with low virulence potential to a severely problem to human health. We believe that our results reaffirm the need of continuous monitoring the infections caused by these microorganisms, considering their skills of adaptation and persistence.