Bactericidal and Fungicidal Activity of N-Chlorotaurine Is Enhanced in Cystic Fibrosis Sputum Medium

Abstract

Lung infections with multiresistant pathogens are a major problem among patients suffering from cystic fibrosis (CF). N-Chlorotaurine (NCT), a microbicidal active chlorine compound with no development of resistance, is well tolerated upon inhalation. The aim of this study was to investigate the in vitro bactericidal and fungicidal activity of NCT in artificial sputum medium (ASM), which mimics the composition of CF mucus. The medium was inoculated with bacteria (Staphylococcus aureus, including some methicillin-resistant S. aureus [MRSA] strains, Pseudomonas aeruginosa, and Escherichia coli) or spores of fungi (Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Scedosporium apiospermum, Scedosporium boydii, Lomentospora prolificans, Scedosporium aurantiacum, Scedosporium minutisporum, Exophiala dermatitidis, and Geotrichum sp.), to final concentrations of 10⁷ to 10⁸ CFU/ml. NCT was added at 37°C, and time-kill assays were performed. At a concentration of 1% (10 mg/ml, 55 mM) NCT, bacteria and spores were killed within 10 min and 15 min, respectively, to the detection limit of 10² CFU/ml (reduction of 5 to 6 log₁₀ units). Reductions of 2 log₁₀ units were still achieved with 0.1% (bacteria) and 0.3% (fungi) NCT, largely within 10 to 30 min. Measurements by means of iodometric titration showed oxidizing activity for 1, 30, 60, and >60 min at concentrations of 0.1%, 0.3%, 0.5%, and 1.0% NCT, respectively, which matches the killing test results. NCT demonstrated broad-spectrum microbicidal activity in the milieu of CF mucus at concentrations ideal for clinical use. The microbicidal activity of NCT in ASM was even stronger than that in buffer solution; this was particularly pronounced for fungi. This finding can be explained largely by the formation, through transhalogenation, of monochloramine, which rapidly penetrates pathogens.

Keywords: N-chlorotaurine; anti-infective; antimicrobial; antiseptic; cystic fibrosis; lung.

FIG 1

Killing of S. aureus ATCC 25923 (A), P. aeruginosa ATCC 27853 (B), E. coli ATCC 11229 (C), and MRSA ATCC 43300 (D) by 0.1% (▲), 0.3% (■), and 1.0% (●) NCT in ASM at 37°C. Controls were in phosphate buffer (◆) and ASM (▼) without NCT (dashed lines). The data are presented as means ± SDs (n = 3). The detection limit was 2 log₁₀ units. **, P < 0.01; *, P < 0.05, versus ASM control, by one-way ANOVA. a, P < 0.01, 1.0% versus 0.3% NCT; b, P < 0.01, 0.3% versus 0.1% NCT, by one-way ANOVA.

FIG 2

Killing of C. albicans ATCC 90028 (A), A. fumigatus ATCC 204305 (B), A. terreus ATCC 3633 (C), and S. apiospermum IHEM 21170 (D) by 0.3% (■), 0.5% (▼), and 1.0% (●) NCT in ASM at 37°C. Controls were in phosphate buffer (◆) and ASM (▼) without NCT (dashed lines). The data are presented as means ± SDs (n = 3). The detection limit was 2 log₁₀ units. **, P < 0.01, versus ASM control, by one-way ANOVA. a, P < 0.01, 1.0% versus 0.5% NCT; b, P < 0.01, 0.5% versus 0.3% NCT, by one-way ANOVA.

FIG 3

Oxidative capacity (COX) of 1.0% (●), 0.5% (▼), 0.3% (■), and 0.1% (▲) NCT in ASM, measured by iodometric titration. The data are presented as means ± SDs (n = 3). P values were <0.01 for all curves.

FIG 4

Comparison of the fungicidal activities of 1% NCT against conidia of S. apiospermum IHEM 21170, Scedosporium boydii, and Lomentospora prolificans IHEM 21176 in ASM (▼) and phosphate buffer (■) at 37°C and pH of 7.0 ± 0.1. Respective controls were without NCT (dashed lines). Values are summarized from three independent experiments for each strain as means ± SDs (n = 9). P values were <0.01 for 1% NCT in ASM versus 1% NCT in phosphate for all time points except zero. *, P < 0.05; **, P < 0.01, versus control without NCT.