Optimized DNA Extraction Protocol for Staphylococcus aureus Strains Utilizing Liquid Nitrogen

BELEN GALEANO; ROBALDI STEFANIA; GORDILLO TANIA; RICARDI MARTINIANO; CASSANELLI PM; PEREDA R O; PALOMINO MARÍA MERCEDES (CO-CORRESPONDING AUTHOR); TRIBELLI PAULA (CO-CORRESPONDING AUTHOR)

REVISTA ARGENTINA DE MICROBIOLOGíA

ASOCIACION ARGENTINA MICROBIOLOGIA

Lugar: Buenos Aires; Año: 2024

0325-7541

DNA extraction is pivotal in high-throughput experiments, like whole-genome sequencing,to determine not only the complete sequence of a particular organism, but also to investigate point mutations, insertions or genomic rearrangements. To ensure adequate sensitivity of molecular biology assay, it is essential that DNA extraction is performed efficiently. with optimal reproducibility and it is desirable also at a low cost. Also, the DNA samples to be used for sequencing libraries must be pure and unfragmented. The most critical step in a DNA extraction protocol is the lysis stage that depends on the bacterial envelope characteristics. Staphylococcus aureus is a gram- positive bacterium with clinical relevance, with a thick, complex and highly cross linked peptidoglycan layer very difficult to disrupt. Different methods have been employed to extract DNA from clinical samples, including enzymatic, chemical, or thermal lysis, mechanical disruption of the cell wall using beads or sonication, or a combination of these approaches. Nucleic acids extraction from S. aureus is particularly challenging since this bacterium exhibits resistance to commonly used enzymes in enzymatic lysis, such as lysozyme and mutanolysin, arising from an elevated level of O-acetylation in peptidoglycan.The most widespread approach to disrupt S. aureus cells involves an enzymatic lysis step using lysostaphin, an enzyme from Staphylococcus simulans biovar staphylolyticus that cleaves the pentaglycine cross-bridges within the peptidoglycan of S. aureus, which is very effective, but highly expensive. Here, we present an innovative method using liquid nitrogen to disrupt S. aureus strains, the reference strain USA300 and clinical isolates. After this lysis step, genomic DNA was purified using similar procedures as those used for other Gram-positive bacteria based on phenol-chloroform DNA extraction protocol . With this method, we obtained substantial amounts of pure DNA at an average concentration of 1.042 mg/ml, which met all purity and integrity criteria to successfully tackle molecular biology assays.