Determining intracellular and extracellular activities of azithromycin against azithromycin-resistant Salmonella Typhi and their association with clinical treatment responses

Background The spread of extensively drug-resistant (XDR) Salmonella Typhi variants in South Asia has severely limited treatment options for typhoid fever, a life-threatening systemic infection affecting millions. Azithromycin has become the last effective oral drug against XDR typhoid; however, despite increasing azithromycin resistance, limited data exists concerning the correlation between in vitro susceptibility and clinical treatment efficacy. Methods We developed a THP-1 macrophage infection model to evaluate time- and concentration-dependent azithromycin activity against intracellular and extracellular S. Typhi, including azithromycin-susceptible (MIC ≤16 mg/L) and azithromycin-resistant isolates (MIC ≥ 32 mg/L). The accumulation of azithromycin in THP-1 macrophages was quantified using LC-MS/MS. Pharmacological modelling was used to estimate key parameters (E max , E min , C s , C si ), and compare azithromycin efficacy between intracellular and extracellular compartments. When available, in vitro findings were correlated with clinical treatment responses. Findings Azithromycin accumulated in THP-1 macrophages at concentrations 20–72 fold higher than in extracellular medium after 24 h. This intracellular accumulation was associated with concentration-dependent killing of S. Typhi in the intracellular assays, while only bacteriostatic effects were observed extracellularly. Importantly, pharmacological modelling data estimated that azithromycin concentrations ranging from 46.86 to 123.78 mg/L were sufficient to inhibit intracellular growth of azithromycin-susceptible isolates. In contrast, concentrations exceeding 460.63 mg/L were required to suppress intracellular growth of azithromycin-resistant isolates, substantially higher than the C max typically observed in human white blood cells (114.0–146.0 mg/L) following a 3 day course of oral azithromycin. Notably, among those with available data, three patients infected with azithromycin-resistant S. Typhi failed to respond clinically to azithromycin treatment. Interpretation We have established a THP-1 macrophage model to characterise the intracellular pharmacodynamics of azithromycin. Our findings indicate that azithromycin is likely ineffective for treating infections caused by azithromycin-resistant S. Typhi, warranting further clinical validation. Funding Wellcome International Training Fellowship.