A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt...

Area of Science:

• Microbiology
• Molecular Biology
• Infectious Diseases

Background:

• Enterococcus faecium is a significant nosocomial pathogen.
• Vancomycin resistance in Enterococcus species poses a major public health threat.
• Accurate and rapid typing and identification methods are crucial for infection control.

Purpose of the Study:

• To develop a multiplex High Resolution Melt-PCR (HRM-PCR) method for van gene detection in Enterococcus species.
• To establish an intergenic spacer region (ISR) based HRM-PCR for Enterococcus species identification and E. faecium typing.
• To compare the discriminatory power of the developed ISR-HRM method with existing techniques like PFGE and MLST.

Main Methods:

• Development of two multiplex PCR assays for HRM-PCR: one targeting vanA, vanB, vanC, vanC23 genes, and another targeting the ISR.
• Validation using 1672 clinical Enterococcus isolates, 71 previously characterized isolates (PFGE, MLST), and ISR sequencing.
• Analysis of vanB gene sequences in GenBank and development of a numerical classification scheme for vanB genotypes.

Main Results:

• The ISR-HRM method reliably identified Enterococcus species and differentiated E. faecalis from E. faecium.
• E. faecium typing by ISR-HRM revealed 7 distinct profiles based on insertion sequence copy numbers, showing both stability and instability.
• ISR-HRM typing demonstrated comparable or superior discrimination compared to PFGE and MLST, differentiating some identical isolates.

Conclusions:

• The developed multiplex HRM-PCR and ISR-HRM methods provide a rapid, accurate, and stable approach for van gene detection and Enterococcus species identification.
• ISR-HRM typing offers high discriminatory power for E. faecium, complementing existing molecular typing techniques.
• This combined approach aids in infection control and epidemiological surveillance of vancomycin-resistant Enterococcus.