Phenotypic Response of Actinomyces israelii to Antibiotic Exposure

Abstract

Introduction and Objective: Actinomyces israelii is an early colonizer of the dental biofilm and a recognized causative agent of actinomycosis that has been implicated as a conditional pathogen in chronic periodontal inflammation. Clinical studies have demonstrated that A. israelii is more frequently isolated from diseased periodontal pockets than from healthy sites, yet its antimicrobial susceptibility profile and adaptive responses to commonly prescribed periodontal antibiotics remain poorly characterized. This study investigated the phenotypic, transcriptional, and biofilm-level responses of A. israelii to amoxicillin and metronidazole through a three-phase experimental design. Methods: In Phase 1, minimum inhibitory concentrations (MICs) of amoxicillin and metronidazole were determined for four Actinomyces species (A. gerencseriae, A. israelii, A. naeslundii, and A. oris) using the E-test method. In Phase 2, transcriptomic profiling was performed on planktonic A. israelii cultures exposed to amoxicillin (0.04 µg/mL) and metronidazole (13 µg/mL) for 2 hours, with differential gene expression analysis conducted using DESeq2. In Phase 3, a 40-species multispecies biofilm model grown on hydroxyapatite-coated pegs (MBEC Assay® system) was exposed to both antibiotics and assessed by scanning electron microscopy (SEM) and the triphenyltetrazolium chloride (TTC) metabolic assay at 3- and 7-day timepoints. Results: All Actinomyces species demonstrated marked susceptibility to amoxicillin (MICs ≤0.058 µg/mL) and intrinsic resistance to metronidazole (MICs ≥37 µg/mL), with A. israelii showing MIC .016 and >256 µg/mL, respectively. Transcriptomic analysis revealed antibiotic-specific gene expression programs: amoxicillin selectively upregulated resistance genes aph(3')-Ia (log₂FC = 4.70) and tet(C) (log₂FC = 2.81), while metronidazole predominantly upregulated virulence and stress response genes including uvrA, recA, and dnaK. Biofilm metabolic activity was transiently reduced at day 3 across treatment groups but recovered by day 7, with amoxicillin-treated biofilms exceeding control levels — suggesting compensatory metabolic responses within the multispecies community. SEM imaging confirmed that amoxicillin markedly disrupted biofilm architecture at day 7, whereas metronidazole had minimal structural impact consistent with the intrinsic resistance profile. Conclusions: A. israelii mounts distinct, antibiotic-specific adaptive programs in response to amoxicillin and metronidazole. Multispecies biofilm resilience is not predicted by planktonic susceptibility testing, highlighting the need for biofilm-based assessments to inform more targeted periodontal antimicrobial strategies.