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Assessment of the Quorum Sensing Inhibition Activity of a Non-Toxic Chitosan in an N-Acyl Homoserine Lactone (AHL)-Based Escherichia coli Biosensor

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Assessment of the Quorum Sensing Inhibition Activity of a Non-Toxic Chitosan in an N-Acyl Homoserine Lactone (AHL)-Based Escherichia coli Biosensor

Xiaofei Qin et al. Biomolecules.

Abstract

New approaches to deal with drug-resistant pathogenic bacteria are urgent. We studied the antibacterial effect of chitosans against an Escherichia coli quorum sensing biosensor reporter strain and selected a non-toxic chitosan to evaluate its quorum sensing (QS) inhibition activity and its effect on bacterial aggregation. To this end, chitosans of varying degree of acetylation (DA) (12 to 69%) and molecular weight (Mw) (29 to 288 kDa) were studied. Only chitosans of low DA (~12%) inhibited bacterial growth, regardless of their Mw. A chitosan with medium degree of polymerization (named MDP) DA30, with experimental DA 42% and Mw 115 kDa was selected for further QS inhibition and scanning electron microscopy (SEM) imaging studies. MDP DA30 chitosan exhibited QS inhibition activity in an inverse dose-dependent manner (≤12.5 µg/mL). SEM images revealed that this chitosan, when added at low concentration (≤30.6 µg/mL), induced substantial bacterial aggregation, whereas at high concentration (234.3 µg/mL), it did not. Aggregation explains the QS inhibition activity as the consequence of retardation of the diffusion of N-acylated homoserine lactones (AHLs).

Keywords: antibacterial activity; chitosan; quorum sensing; quorum sensing inhibition.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Experimental approach used to screen the chitosans with highest antibacterial activity, namely chitosans of low, medium or high degree of polymerization (DP) and low degree of acetylation (DA, 12%) (Step I). A non-antibacterial chitosan termed MDP DA 30 with medium degree of polymerization (MDP) and an experimental DA of 40% was selected for quorum sensing inhibition activity and agglutination assays at low (Step II) or high (Step III) concentrations. CFU: colony forming unit.
Figure 1
Figure 1
(a) Relative growth rate after re-seeding of E. coli TOP10 bacteria treated with identical charge-equivalent molar concentrations (0.389 mM) of chitosans of varying degree of acetylation and polymerization (for sample details, refer to Table 1). In the first cycle, the bacteria were incubated for 2:38 h in M9 medium at 37 °C, whereas in the second cycle, an aliquot of the bacterial culture was re-seeded in fresh M9 medium and incubated for further 2:38 h. Absorbance as measured at λ = 600 nm as a function of the chitosan concentration added to the M9 medium with E. coli. One (negative) control corresponds to bacteria cultivated in M9 medium, while the other (positive) control was kanamycin (50 µg/mL) added to the M9 medium. (b) Antibacterial effect of chitosans of varying degree of acetylation and Mw with two different amino group concentrations (0.389 and 0.778 mM) on the growth of E. coli TOP10 incubated in a 96-well-plate at 37 °C and 100 rpm for 1 h as tested in a CFU agar plate assay. The control corresponds to untreated bacteria (* p < 0.05, ** p < 0.01 and *** p < 0.001; values represent average ± standard deviation (SD), n ≥ 3; three technical replicates).
Figure 2
Figure 2
(a) Effect of varying concentrations of chitosan MDP DA30 (DA 42% and Mw 115 kDa) on the growth of E. coli TOP10 as measured by assessing the absorbance at λ = 600 nm; (b) Effect of different concentrations of chitosan on bacterial fluorescence intensity; (c) Effect of varying concentrations of chitosan on endpoint OD600 relative to the control, corresponding with the data shown in panel a; (d) Effect of varying concentrations of chitosan on fluorescence/OD600 relative to the control, corresponding with the data shown in panel b; 3OC6HSL concentration: 2.5 × 10−10 M (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; values represent average ± SD, n ≥ 3; three technical replicates).
Figure 3
Figure 3
Scanning electron microscopy images of (a) E. coli TOP10 bacteria alone; and an equivalent number of bacteria (OD600 = 0.2) mixed with varying concentrations of MDP DA30 chitosan, namely (b) 2.95 µg/mL; (c) 5.88 µg/mL; (d) 8.79 µg/mL; (e) 28.7 µg/mL; (f) 226.6 µg/mL, after incubation at 4 °C and slight shaking for 1 h, supported on track-etched polycarbonate membranes. Under these imaging conditions, bacteria appear as bright rod objects. Polycarbonate support appears as a gray background, and sharply-defined track-etched pores appear as dark circles of ~1.2 µm in diameter.

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