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. 2021 May 10;7(5):e07002.
doi: 10.1016/j.heliyon.2021.e07002. eCollection 2021 May.

Quorum quenching activity of Andrographis paniculata (Burm f.) Nees andrographolide compounds on metallo-β-lactamase-producing clinical isolates of Pseudomonas aeruginosa PA22 and PA247 and their effect on lasR gene expression

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Quorum quenching activity of Andrographis paniculata (Burm f.) Nees andrographolide compounds on metallo-β-lactamase-producing clinical isolates of Pseudomonas aeruginosa PA22 and PA247 and their effect on lasR gene expression

An Margarete Tan Lim et al. Heliyon. .

Abstract

Andrographis paniculata (Burm f.) Nees is a tropical plant native to Southeast Asia that has been used as an effective remedy for a wide variety of illnesses in traditional Chinese and Ayurvedic medicine. The antimicrobial activity of its crude extract had been shown to be due to its quorum quenching activity. The study determined the effect of purified extracted compounds from the leaf of A. paniculata, namely: andrographolide, 14-deoxyandrographolide, 14-deoxy-12-hydroxyandrographolide and neoandrographolide on quorum sensing-mediated virulence mechanisms in clinical isolates of metallo-β-lactamase (MβL)-producing Pseudomonas aeruginosa. Their effect on the expression of the lasR gene, which codes for LasR, a transcription activator protein of the quorum sensing system in P. aeruginosa was also determined using RT-qPCR. All the pure compounds significantly decreased the biofilm formation, protease production and swarming motility of the P. aeruginosa isolates compared to the untreated controls (p < 0.05). Results of the RT-qPCR assay showed that all compounds significantly downregulated the expression of lasR compared to the untreated control (p < 0.05), supporting the position that the lower virulence activities of the treated group were due to quorum quenching activity of the pure compounds. Multiple comparisons using Tukey's HSD analysis revealed that the means of the relative expression of lasR of the isolates treated with the different compounds were not significantly different from each other (p > 0.05), suggesting equal potencies. Results show the potential of the isolated pure compounds from A. paniculata for use as antimicrobial agents as a result of their quorum quenching activities.

Keywords: Andrographis paniculata; Andrographolides; Biofilm; Protease; Pseudomonas aeruginosa; Quorum sensing; Swarming.

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Figures

Figure 1
Figure 1
Chemical structures of the A. paniculata leaf compounds (Ragasa et al., 2016) tested in the study: (a) L5 - andrographolide, (b) L6 - 14-deoxyandrographolide, (c) L11 - 14-deoxy-12-hydroxyandrographolide, (d) L12 - neoandrographolide.
Figure 2
Figure 2
The quorum sensing inhibition activity of compounds from A. paniculata on C. violaceum shown by the inhibition of violacein pigment production in the growth zone around the well. andrographolide (L5), 14-deoxyandrographolide (L6), 14-deoxy-12-hydroxyandrographolide (L11), neoandrographolide (L12). The control well (CTRL) containing distilled water was negative for inhibition of violacein production. Unlabelled discs are not included in the study.
Figure 3
Figure 3
(A) Anti-biofilm formation assay of P. aeruginosa PA22 treated with compounds from A. paniculata. Values are the means of three independent experiments. (B) Percent inhibition of biofilm formation of P. aeruginosa PA22 treated with A. paniculata compounds: andrographolide (L5); 14-deoxyandrographolide (L6); 14-deoxy-12-hydroxyandrographolide (L11); neoandrographolide (L12); and untreated (UT).
Figure 4
Figure 4
(A) Anti-biofilm formation assay of P. aeruginosa PA247 treated with compounds from A. paniculata. Values are the means of three independent experiments. (B) Percent inhibition of biofilm formation of P. aeruginosa PA247 treated with A. paniculata compounds: andrographolide (L5); 14-deoxyandrographolide (L6); 14-deoxy-12-hydroxyandrographolide (L11); neoandrographolide (L12); and untreated (UT).
Figure 5
Figure 5
18-hour cultures of P. aeruginosa PA22 and PA247 treated with the minimum biofilm inhibitory concentrations of the andrographolide (L5), 14-deoxyandrographolide (L6), 14-deoxy-12-hydroxyandrographolide (L11), and neoandrographolide (L12), showing absence of growth inhibition.
Figure 6
Figure 6
Mean OD590 of P. aeruginosa PA22 (A) and PA247 (B) cultured in milk substrate treated and not treated with minimum biofilm inhibitory concentration (MBIC) of compounds from A. paniculata and the positive control bromofuranone in the protease assay. Asterisks (∗) indicate significant difference compared with the untreated control (p < 0.05).
Figure 7
Figure 7
Swarming motility analysis of P. aeruginosa PA22 and PA247 treated with the mean biofilm inhibitory concentrations of andrographolide (L5), 14-deoxyandrographolide (L6), 14-deoxy-12-hydroxyandrographolide (L11), and neoandrographolide (L12).
Figure 8
Figure 8
Relative fold gene expression values (FGEV) (log 2−ΔΔCt) of the 16SrRNA of P. aeruginosa PA22 (A) and PA247 (B) treated with A. paniculata compounds with respective untreated controls: andrographolide (L5); 14-deoxyandrographolide (L6); 14-deoxy-12-hydroxyandrographolide (L11); neoandrographolide (L12); and untreated (UT). There were no significant differences between all the treated and untreated groups.
Figure 9
Figure 9
Relative fold gene expression values (FGEV) (log 2−ΔΔCt) of the lasR gene of P. aeruginosa PA22 (A) and PA247 (B) treated with A. paniculata compounds and respective untreated controls: andrographolide (L5); 14-deoxyandrographolide (L6); 14-deoxy-12-hydroxyandrographolide (L11); neoandrographolide (L12); and untreated (UT). Asterisks (∗) indicate significant difference compared with the untreated control (p < 0.05).

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