Methanol Extract from Anogeissus leiocarpus (DC) Guill. et Perr. (Combretaceae) Stem Bark Quenches the Quorum Sensing of Pseudomonas aeruginosa PAO1

Abstract

Background: Due to its extensive arsenal of virulence factors and inherent resistance to antibiotics, Pseudomonas aeruginosa is a threat particularly in immunocompromised patients. Considering the central role of quorum sensing in the production of virulence factors, inhibition of bacterial communication mechanism constitute an opportunity to attenuate pathogenicity of bacteria resistant to available antibiotics. Our study aimed to assess the anti-quorum sensing activity of Anogeissus leiocarpus, traditionally used in Burkina Faso, for the treatment of infected burn wounds. Methods: Investigations were carried out on methanol extract from A. leiocarpus stem bark. The reporter strains Chromobacterium violaceum CV026 and P. aeruginosa PAO1 derivatives were used to evidence any interference with the bacterial quorum sensing and expression of related genes. P. aeruginosa PAO1 was used to measure the impact on pyocyanin production. Results: At a sub-inhibitory concentration (100 µg/mL), A. leiocarpus methanol extract quenched the quorum sensing mechanism of P. aeruginosa PAO1 by down-streaming the rhlR gene, with a subsequent reduction of pyocyanin production. Moreover, the antioxidant polyphenols evidenced are able to reduce the oxidative stress induced by pyocyanin. Conclusion: The antioxidant and anti-quorum sensing activities of A. leiocarpus stem bark could justify its traditional use in the treatment of infected burn wounds.

Keywords: Anogeissus leiocarpus; Chromobacterium violaceum CV026; Pseudomonas aeruginosa PAO1; pyocyanin; quorum sensing.

Figure 1

Methanol extract from A. leiocarpus reduce pyocyanin production in P. aeruginosa PAO1. (a) A. leiocarpus extract (100 µg/mL) significantly reduced (p > 0.05) pyocyanin production within 18 h compared to DMSO used as control; (b) A. leiocarpus extract (100 µg/mL) did not exhibit a significant effect on P. aeruginosa PAO1 kinetic growth within 18 h. Dimethyl sulfoxide (DMSO) was used as negative control. P. aeruginosa PAO1 was grown (37 °C, 175 rpm agitation) in the LB broth. Mean values ± SD of triplicate independent experiments are shown.

Figure 2

Anti-quorum sensing (QS) activity of methanol extract from A. leiocarpus. (a) A. leiocarpus extract (100 µg/mL) significantly reduces (p > 0.05) violacein production by C. violaceum CV026 within 48 h (b) A. leiocarpus extract (100 µg/mL) did not exhibit a significant effect of on C. violaceum CV026 kinetic growth within 48 h. Dimethyl sulfoxide (DMSO) was used as a negative control. C. violaceum CV026 was grown (30 °C, 175 rpm agitation) in LB broth supplemented with HHL (10 µM in DMSO). Mean values of triplicate independent experiments and SD are shown. Data with different letters in superscript are significantly different (p > 0.05).

Figure 3

Effect of A. leiocarpus extract on the transcription level of lasRI and rhlRI QS genes. Data are expressed as mean ± SD of 3 independent essays. Dimethyl sulfoxide (DMSO) was used as negative control. Salicylic acid was used as positive control. *** Significantly different compared with DMSO treatment (p < 0.05). ns: not significantly different compared to DMSO treatment. Gene expression was measured as the β-galactosidase activity of the lacZ gene fusions and expressed in Miller units. Expression of the aceA gene is used as a quorum-sensing independent control. P. aeruginosa PAO1 strains were grown.