Additive Effects of N-Acetylcysteine and [R4W4] Combination Treatment on Mycobacterium avium

<i>Mycobacterium avium</i> is an opportunistic pathogen and a leading contributor to nontuberculous mycobacterial infections in immunocompromised individuals. However, treatment duration, antibiotic toxicity, and resistance present challenges in the management of mycobacterium infections, prompting the need for novel treatment. <i>N</i>-acetylcysteine (NAC) has demonstrated potent antimycobacterial activity, while antimicrobial peptides such as the cyclic [R₄W₄] have shown additive effects when combined with first-line antibiotics. This study aimed to investigate the mechanism and efficacy of NAC and [R₄W₄] combination therapy against <i>M. avium.</i> A membrane depolarization assay was used to evaluate the effects of NAC and [R₄W₄] on <i>M. avium</i> cell membrane integrity. Antimycobacterial activity was assessed by treating cultures with varying concentrations of NAC, [R₄W₄], a combination, or a sham treatment. The same regimens were applied to <i>M. avium</i>-infected THP-1-derived macrophages to assess intracellular efficacy. NAC and [R₄W₄] each disrupted the <i>M. avium</i> membrane potential, with enhanced effects in combination. The combination treatment significantly reduced <i>M. avium</i> survival in both the culture and infected macrophages compared with NAC alone and untreated controls. [R₄W₄] and NAC also demonstrated potent antibacterial activity, while the lowest MIC and the combination of [R₄W₄] and NAC displayed additive effects, indicating an improved bacterial inhibition compared to individual treatments. These findings demonstrate the additive activity of NAC and [R₄W₄] against <i>M. avium</i> in vitro and suggest that combining antioxidant compounds with antimicrobial peptides may represent a promising strategy for treating mycobacterial infections.