Characterization of the CRISPR1-Cas array and its subtyping potential in Enterococcus faecalis from Malaysia

Area of Science:

• Microbiology
• Genetics
• Infectious Diseases

Background:

• *Enterococcus faecalis* is a gram-positive bacterium frequently implicated in hospital-associated infections.
• Three primary CRISPR loci (CRISPR1-cas, CRISPR2, CRISPR3-cas) are known in *E. faecalis*.

Purpose of the Study:

• To develop and test novel primers targeting the CRISPR1-cas loci in *E. faecalis*.
• To characterize CRISPR1-cas loci in Malaysian isolates and assess their utility for epidemiological surveillance.

Main Methods:

• Development of novel primers specific to CRISPR1-cas loci in *E. faecalis*.
• Testing primers on 26 *E. faecalis* isolates from Segamat, Malaysia.
• Characterization of CRISPR array sequences, including repeat and spacer identities, and comparison with CRISPR2.

Main Results:

• Half of the tested isolates carried the CRISPR1-cas9 locus.
• CRISPR1 array amplification was successful in 12 of 13 isolates harboring the *cas9* gene.
• CRISPR1-cas exhibited distinct spacer and terminal repeat (TR) sequences compared to CRISPR2, despite similar array lengths.
• Genotype analysis revealed limited correlation between CRISPR1-AS and CRISPR2-AS genotypes.
• Combined CRISPR1-cas and CRISPR2 typing demonstrated discriminatory power comparable to Multi-Locus Sequence Typing (MLST).

Conclusions:

• CRISPR1-cas typing, particularly when combined with CRISPR2, is a promising tool for short-term *E. faecalis* strain identification and epidemiological surveillance.
• This method offers a potentially lower-cost alternative to MLST for specific surveillance applications.
• The study establishes a genetic reference for *E. faecalis* research in Southeast Asia.