High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide

Abstract

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

Prediction of Future Caries in 1-Year-Old Children via the Salivary Microbiome

“…In addition, the genus Streptococcus was less abundant than previous reports in 1-y-old children (Dashper et al 2019). This may be partly due to the resistance of streptococci to different lysis methods (Cho et al 2021). Since we could not analyze the abundance of S. mutans in this data set, we preliminarily analyzed culture-based mutans streptococci count in these samples.…”

A guide to DNA extraction protocols for ecologists, conservation managers, and environmental DNA researchers

“…For gram-negative bacteria, lysozyme can be combined with detergents (e.g., EDTA) to break the cell wall and membrane (Geciova et al, 2002;Islam et al, 2017). Other enzymes also include mutanolysin and lysostaphin, which are used to extract nucleic acids from susceptible bacteria (Cho et al, 2021). The enzymes commonly used to lyse yeast cells include chitinase, zymolyase, lyticase, and glucanase (Burden, 2023).…”

Chemical Trends in Sample Preparation for Nucleic Acid Amplification Testing (NAAT): A Review

“…Antimicrobial peptides (AMPs) are small, cationic, amphiphilic molecules that can permeabilize cell membranes of a broad range of microbes, and hence they are promising lytic agents. A study of the use of AMPs for the lysis of hard-to-lyse bacteria, S. typhimurium and S. aureus, systematically correlated wall structure and AMP activity [85]. AMPs including melittin, magainin analogues (MSI), bombolitin, and cecropin were utilized for lysing bacteria cells in urine samples prior to LAMP [46].…”

“…Washing buffers typically resemble the chemical composition of binding buffers and may contain chaotropic salts at reduced concentration to maintain binding, but they may also include alcohols (e.g., ethanol, isopropanol). Washing buffers used in laboratory-based NA extraction kits using silanol surfaces typically contain ethanol (50-100%) [51,85,124] or isopropanol (15-80%) [34,42,45] to remove salts and contaminants while precipitating the NAs [125]. Ethanol and isopropanol have a lower dielectric constant (ε) than water (ε of 24.6 and 18.3, respectively, vs. 78.5 [78]) [126,127], making the nucleic acids less hydrophilic by decreasing ionisation.…”