Investigating the prevalence of CRISPR-Cas system and their association with antibiotic resistance genes and virulence factors in Enterococcus faecalis and Enterococcus faecium strains isolated from hospitalized patients

Objectives: Enterococcus faecalis and Enterococcus faecium are Gram-positive opportunistic pathogens that rank among the leading causes of nosocomial infections worldwide. This study investigates the prevalence and role of CRISPR-Cas systems in modulating antimicrobial resistance and virulence factors in clinical isolates of E. faecalis and E. faecium collected from patients in Tehran, Iran. Methods: A total of 75 clinical isolates of E. faecalis and E. faecium were collected from various hospitals in Tehran, Iran, between January and April 2023, from adult patients with urinary tract infections ( n = 55), blood infections ( n = 12), and wound infections ( n = 8). Conventional bacteriology tests and PCR were used to isolate and identify Enterococcus species. Phenotypic antibiotic and genotypic resistance were assessed. CRISPR-Cas repeat-spacer array were screened using PCR, and the relationship between CRISPR-Cas systems and antibiotic resistance and virulence genes was statistically analyzed. Phylogenetic, structural, and conservation analyses were performed to assess the degree of conservation in CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes, identify potential mutations, and evaluate their possible impact on Cas9 protein function. Results: 86.6% of the isolates harbored CRISPR-Cas repeat-spacer array, with a significantly higher prevalence in E. faecalis than in E. faecium (100% vs. 66.6%, P = 0.0001). CRISPR1-Cas, CRISPR2, and CRISPR3-Cas loci were identified in 76%, 82.6%, and 64% of isolates, respectively. Notably, the prevalence of CRISPRCas systems was significantly reduced in extensively drug-resistant (XDR) isolates (32%) compared to multidrug-resistant (MDR) isolates (68%, P = 0.0 0 01). Conservation analyses of CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes revealed conserved regions potentially linked to functional activity. Furthermore, CRISPR-Cas repeat-spacer array were correlated with specific antimicrobial resistance phenotypes and genotypes, as well as with virulence factors. Conclusions: These findings suggest that CRISPR-Cas systems may influence the resistance and virulence profiles of clinical Enterococcus isolates, potentially impacting their pathogenicity and adaptability. (c) 2025 The Authors. Published by Elsevier Ltd on behalf of International Society for Antimicrobial ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )