A multilocus sequence typing method of Staphylococcus aureus DNAs in a sample from human skin.

Area of Science:

• Microbiology
• Molecular Biology
• Dermatology

Background:

• * Staphylococcus aureus (S. aureus) commonly colonizes skin and mucous membranes, especially in healthcare settings.
• * Previous research indicated higher S. aureus abundance on healed pressure injury (PI) skin compared to normal skin.
• * Traditional Multilocus Sequence Typing (MLST) is unreliable for microbiota DNA due to contamination from other bacterial species.

Purpose of the Study:

• * To develop a novel multiplex-nested PCR method for accurate S. aureus MLST directly from human skin samples.
• * To enable culture-free typing of S. aureus in clinical specimens, overcoming contamination issues.

Main Methods:

• * Designed seven pairs of conserved S. aureus-specific primers for multiplex-nested PCR.
• * Performed initial multiplex amplification followed by conventional PCR for MLST on diluted samples.
• * Validated specificity against human DNA and skin commensal bacteria.

Main Results:

• * The method specifically amplified S. aureus allele sequences in the presence of contaminating DNA.
• * Successfully determined S. aureus sequence types (STs) from DNA of skin healed from PI.
• * Identified dominant STs belonging to Clonal Complex 1 (CC1) and CC5.

Conclusions:

• * The developed multiplex-nested PCR assay provides a reliable culture-free method for S. aureus typing.
• * This approach can be applied to clinical specimens, potentially simplifying S. aureus detection and characterization without requiring bacterial isolation.