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 (Emax, Emin, Cs, Csi), 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 Cmax 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.