Heterogeneidade de sistemas CRISPR em Enterococcus faecalis: perspectivas sobre a dinâmica eco-evolutiva e epidemiológica de um microrganismo generalista em ambientes hospitalares e não-hospitalares

Abstract

Enterococcus faecalis is one of the main etiological agents of healthcare-associated infections, frequently exhibiting resistance to multiple antimicrobials. Its widespread distribution across diverse environments and hosts — coupled with high genomic plasticity — facilitates the dissemination of resistance and virulence genes, thereby contributing to the global crisis of antimicrobial resistance. In this context, endogenous CRISPR-Cas systems emerge as key elements by modulating the horizontal acquisition of adaptive determinants. This study investigates the heterogeneity of CRISPR-Cas systems in E. faecalis with the aim of identifying genomic patterns underlying adaptation to distinct niches and the emergence of clinically relevant lineages. To achieve these objectives, we combined molecular biology techniques —including conventional PCR and whole-genome sequencing — with computational analyses in comparative genomics and phylogenetics. Our investigation comprised 146 isolates from a local collection (obtained from hospitalized patients, coastal waters, and wild birds) along with genomes retrieved from GenBank, representing a total diversity of 226 sequence types (STs). Our results demonstrated that the absence of CRISPR-Cas systems predominates in clinical isolates, whereas their presence is significantly higher among non-hospital-derived isolates (p < 0.0001). Clinical isolates also exhibited a greater abundance of resistance and virulence genes (p = 6.32e-10). For example, genes such as aac(6')-Iaph(2")-Ia, vanA, and cylA were more frequent in isolates lacking the system, while ant(6')-Ia, ermA, tetM, and esp were associated with its presence. Phylogenetic analysis revealed two major clades: one comprising predominantly hospital-associated STs (e.g., ST6, 103, 525, 769, 778), characterized by the absence of CRISPR-Cas systems and a high frequency of vancomycin-resistant strains; and a more diverse clade with heterogeneous CRISPR-Cas distribution, which includes ubiquitous lineages such as ST21 and highlights singular cases like ST116, where the vanA gene coexists with the CRISPR3 system. In ST116, we observed amino acid substitutions in the endonuclease domains of Cas9, alongside differential CRISPR3 array content among isolates; notably, the absence of specific spacers was associated with the presence of mobile genetic elements (MGEs), such as Tn1546. Analysis of ST21 representatives further revealed that intra-ST variation in CRISPR1 — linked to a recombination hotspot spanning the species’ pathogenicity island — drives ecological differentiation, resulting in pangenome composition differences among subclades. Our findings suggest that structural alterations in this region arise from the complex interplay between MGEs and the host, mediated by defense and counter-defense mechanisms. Finally, we identified an unusual group of E. faecalis isolates lacking CRISPR2, which form a distinct clade meeting the genomic criteria for classification as a novel subspecies. Overall, our results indicate that CRISPR-Cas heterogeneity in E. faecalis enables the discrimination of lineages at various stages of differentiation, which are indistinguishable by conventional methods. Through a reverse ecology approach, we discuss how selective pressures shape the species’ genome in a context-specific manner, preserving its diversity and ecological versatility. These insights underscore the need for continuous monitoring of the eco-evolutionary dynamics of E. faecalis by supporting both theoretical understanding and practical strategies for controlling antimicrobial resistance.