EndoNUclease Heteroduplex cleavage typing a new technique for rapid typing of bacterial isolates in the context of nosocomial outbreaks: proof of concept with Bacillus cereus.

INTRODUCTION: Bacillus cereus is a Gram-positive, spore-forming bacterium implicated in foodborne illnesses and severe nosocomial infections, particularly in immunocompromised patients. In France, B. cereus has been associated with neonatal infections, including septicemia and central nervous system infections, with a significant mortality rate. The bacterium's spores are highly resistant to disinfectants, contributing to hospital-acquired infections through environmental contamination. Current typing methods, such as Multi-Locus Sequence Typing (MLST) are time-consuming and not routinely performed in clinical settings, limiting real-time outbreak management.

METHODS: This study evaluates the EndoNUclease Heteroduplex Cleavage Typing (ENUHCT) as a rapid alternative to MLST for B. cereus strain typing. ENUHCT identifies single nucleotide polymorphisms by cleaving mismatched DNA heteroduplexes, enabling rapid discrimination between strains. 14 B. cereus strains (clinical and environmental) from two hospitals were analyzed using both MLST and ENUHCT. ENUHCT results were congruent with MLST, successfully distinguishing identical and closely related strains. ENUHCT provided results within hours, compared to days for MLST, and required no prior sequencing.

RESULTS: ENUHCT demonstrated high discriminatory power, with strains sharing identical STs showing no cleavage, while strains with differing STs exhibited distinct cleavage patterns. Phylogenetic analysis based on ENUHCT data closely matched MLST-derived trees, highlighting its potential for real-time outbreak tracking. However, ENUHCT's accuracy decreased with high Single Nucleotide Polymorphisms (SNP) counts (≥4), as peak detection became less reliable. Despite this limitation, ENUHCT proved effective for initial strain screening, reducing the need for full genome sequencing in unrelated strains.

CONCLUSION: In conclusion, ENUHCT offers a rapid, cost-effective alternative to MLST for B. cereus strain typing, particularly in outbreak settings. Its ability to provide real-time results supports timely infection control measures, though further validation is needed for broader application. ENUHCT's adaptability to other pathogens with established MLST schemes underscores its potential as a first line typing tool in clinical microbiology.