Obtaining Muramic Acid from Staphylococcus aureus: A Simple Strategy for Axenic Isolation of Tannerella forsythia

Abstract

Background: The periodontal pathogen Tannerella forsythia is auxotrophic for muramic acid (MurNAc), a key component of bacterial peptidoglycan, and dependent on an external supply of MurNAc to maintain pure laboratory cultures. The focus of this study was to find a source of muramic acid and peptidoglycan fragments from a Staphylococcus aureus strain. This would facilitate the isolation of T. forsythia by incorporating peptidoglycan into conventional anaerobic media. Methods: The S. aureus strain ATCC 29213 was chosen as the source. The standardization and quantification of the method included verifying concentrations via spectrophotometry and developing a linear regression model with standard curves for muramic acid and lactic acid. The resulting lysate was used to seed Fastidious Anaerobe Agar (FAA) plates, which were inoculated with strain T. forsythia (ATCC 43037) and incubated in an anaerobic chamber for seven days. Results: The resulting lysate had an optical density ranging from 0.061 to 0.083, which corresponds to a muramic acid concentration of approximately 12 µg/mL. Pure cultures of T. forsythia could then be obtained on FAA plates supplemented with muramic acid (MurNAc) (FAA-Mur). The viability of the axenic T. forsythia culture was confirmed using muramic acid/peptidoglycan fragments of microbial origin. Conclusions: The method presented improves the growth of T. forsythia. Consequently, T. forsythia is available for further investigation into the regular performance of sensitivity tests in periodontics and the routine generation of growth curves for quantitative polymerase chain reaction (qPCR) analysis.