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

Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterisation of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differ significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterisation based on ancestral spacer (AS) and TR sequences indicate that E. faecalis with the same CRISPR1-AS genotype do not always harbour same CRISPR2-AS genotypes, and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to multilocus sequence typing (MLST), suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in the Southeast Asia region.