Artemisia afra and Artemisia annua Extracts Have Bactericidal Activity against Mycobacterium tuberculosis in Physiologically Relevant Carbon Sources and Hypoxia

Abstract

Mycobacterium tuberculosis (Mtb) is a deadly pathogen and causative agent of human tuberculosis, causing ~1.5 million deaths every year. The increasing drug resistance of this pathogen necessitates novel and improved treatment strategies. A crucial aspect of the host-pathogen interaction is bacterial nutrition. In this study, Artemisia annua and Artemisia afra dichloromethane extracts were tested for bactericidal activity against Mtb strain mc²6230 under hypoxia and various infection-associated carbon sources (glycerol, glucose, and cholesterol). Both extracts showed significant bactericidal activity against Mtb, regardless of carbon source. Based on killing curves, A. afra showed the most consistent bactericidal activity against Mtb for all tested carbon sources, whereas A. annua showed the highest bactericidal activity in 7H9 minimal media with glycerol. Both extracts retained their bactericidal activity against Mtb under hypoxic conditions. Further investigations are required to determine the mechanism of action of these extracts and identify their active constituent compounds.

Keywords: Artemisia afra; Artemisia annua; Mycobacterium tuberculosis; artemisinin; carbon source; cholesterol; hypoxia.

Figure 1

Artemisia annua and Artemisia afra. Photos courtesy of James Kishlar of Atelier Temenos.

Figure 2

Comparative analysis of Mtb growth and plant extract bactericidal activity in different carbon sources. At day 0, log-phase cultures grown in the indicated carbon sources were back-diluted to OD₆₀₀ 0.1 in the same media and treated with either the indicated extract or with the extract vehicle DMSO. At the time-points indicated on the x axis, aliquots from each culture were removed, diluted, and plated on extract-free solid 7H10 media to determine the number of viable CFU. CFU counts for extract-treated cultures were compared to Day 0 and to each other by 2-way ANOVA. All samples from Day 2 onward were significantly different than Day 0 (adjusted p < 0.05). Samples grown in minimal media with individual carbon sources that were significantly different from corresponding samples grown in 7H9 are indicated with stars. * = adjusted p < 0.05; ** = adjusted p < 0.01. (A). Comparative growth/death of Mtb in the presence of 5 mg/mL A. afra extracts in all carbon sources. (B). Comparative growth/death of Mtb in the presence 9 mg/mL A. annua extracts in all carbon sources. The following are the sole carbon sources used in this experiment: 7H9 + glucose and glycerol, minimal medium (MM) + glycerol, MM + glucose, and MM + cholesterol. Data are the average from three independent experiments. Plots showing the growth of Mtb in each individual carbon source in the presence of each plant extract (n = 3) ±SD are provided in the supplementary material (Supplemental Figure S2).

Figure 3

Comparative analysis of A. afra and A. annua extract bactericidal activities against Mtb under hypoxic conditions. Quantities of 5 mg/mL A. afra extract, 9 mg/mL A. annua extract, or vehicle control were added by syringe to Mtb cultures that had previously been slowly depleted of oxygen over 14 days. At the time-points indicated on the x axis after adding extracts or vehicle, cultures were opened, diluted, and plated on extract-free 7H10 plates to determine viable CFU. N = 3 cultures per time-point; bars = ±SD. Extract-treated samples at Days 2 and 7 all had significantly lower CFU than samples at time 0 (2-way ANOVA with Dunnett’s multiple comparisons test, p < 0.0001). Samples treated with A. afra did not differ significantly from samples treated with A. annua at any time-point (p > 0.05).