Comparison of the prevalence of CRISPR/Cas systems among Enterococcus faecalis strains isolated from clinical specimens of teaching and treatment hospitals of Emam Reza and pediatric of Tabriz, their association with antibiotic resistance, virulence and evaluation of CRISPR/Cas systemʹs effect on the antibacterial resistance against vancomycin and aminoglycosides

Abstract

Enterococcus faecalis is one of the important opportunistic nosocomial pathogens that can cause dangerous and life-threatening infections in humans. Several studies have demonstrated that the presence of CRISPR-cas is associated with antibiotic susceptibility and the absence of virulence factors. As well as, antibiotic resistance genes can be removed by cas9 gene. Therefore, the aim of this study was to investigate the virulence factors and antibiotic resistance and their relationship with CRISPR-Cas in E. faecalis isolates obtained from the dental-root canal and nosocomial infections and removing vanA and aac(6ʹ)-aph(2ʺ) genes by cas9 gene.

Methods: 88 isolates of E. faecalis collected from nosocomial infections and 73 isolates were collected from the dental-root canal. Virulence traits and their antibiotic resistance were investigated by phenotypic and genotypic methods. The occurrence of CRISPR loci was also determined by PCR. The pCas9 carrying the targeted gRNA of vanA and aac(6ʹ)-aph(2ʺ) were transformed into vancomycin and gentamicin-resistant isolates.

Results: Biofilm formation, gelatinase and hemolysis activity were identified in 93.8%, 29.2% and 19.2% of the isolates, respectively. The highest presence of virulence genes was related to ace and efaA and the lowest to cylA. Most isolates were resistant to tetracycline and erythromycin and the lowest resistance was to linezolid and teicoplanin. The highest presence of antibiotic resistance genes was related to aadE and tetM and the lowest presence was related to vanB, which was not found in any of the isolates. The CRISPR1-cas, CRISPR2 and CRISPR3-cas were present in 13%, 55.3% and 17.4% of the isolates, respectively, with a significant inverse correlation with esp and gelE genes. The significant inverse correlation was found between CRISPR1-cas and resistant to ciprofloxacin and rifampin, between CRISPR2 and resistant to vancomycin and fosfomycin, and between CRISPR3-cas and resistant to erythromycin, tetracycline and gentamicin. Furthermore, The significant inverse correlation was found between CRISPR1-cas and tetM, between CRISPR2, aadE and aacA-aphD, CRISPR3-cas and tetM, ermB, addE, ant(6) and aacA-aphD. Minimum inhibitory concentration (MIC) for vancomycin was significantly reduced from 256 and > 512 µg/mL to 2 µg/mL. In addition, the MIC for gentamicin was reduced from > 512 µg/mL to 1 µg/mL. As well as, in the PCR cloning method, vanA and aac(6ʹ)-aph (2ʺ) genes were not found in the individual pCas9-transformed clones.