Comparative genomics of Actinomyces identifies an MRONJ-associated lineage characterized by variation in mfd

Abstract Although many cases of medication-related osteonecrosis of the jaw (MRONJ) have been reported, its pathophysiology remains unclear. Additionally, previous histological and metagenomic studies have implicated an association between MRONJ and Actinomyces ; however, verification of this association has been hindered by limited sample sizes and a lack of selective isolation media. In this study, we utilized a recently developed selective medium to isolate Actinomyces from oral specimens of 53 patients with MRONJ across multiple hospitals. We developed DNA extraction protocols suitable for Actinomyces and performed genome sequencing of these isolates using not only short-read but also the latest nanopore long-read sequencing technologies. Comparative genomic analyses, including reconstruction of a complete reference chromosome, showed that most MRONJ-derived isolates clustered within an Actinomyces lineage dominated by Actinomyces oris and characterized by a distinct variant of mfd , a gene previously implicated in bacterial evolvability. Furthermore, 3D structure prediction identified potential structural differences in Mfd among these isolates, although their functional significance and possible relevance to MRONJ remain to be determined. Thus, this study provides a basis for future genomic investigations of Actinomyces and for further exploration of its potential association with MRONJ.