Hordenine: A Novel Quorum Sensing Inhibitor and Antibiofilm Agent

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Hordenine, a novel quorum sensing inhibitor and anti-biofilm agent against Pseudomonas aeruginosa Jin-Wei Zhou, Huai-Zhi Luo, Huan Jiang, Ting-Kun Jian, Zi-Qian Chen, and Ai-Qun Jia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05035 • Publication Date (Web): 21 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structure of hordenine (A) and effect of hordenine on P. aeruginosa PAO1 growth (B). Growth was determined at different concentrations of hordenine (0.5, 0.75, and 1 mg mL-1) for 24 h in tube. DMSO served as the negative control. Error bars demonstrated the standard deviations of three measurements. 199x221mm (300 x 300 DPI)

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Figure 2. Relative quantification of C4-HSL and 3-oxo-C12-HSL using LC-MS/MS chromatograms. (A) MS/MS spectra of C4-HSL and 3-oxo-C12-HSL. (B) HPLC chromatograms of C4-HSL and 3-oxo-C12-HSL produced by P. aeruginosa supplemented with (c) DMSO and (d-f) hordenine (0.5, 0.75, and 1.0 mg mL-1). (a) and (b) represent the standards of C4-HSL and 3-oxo-C12-HSL. (C) Quantitative analysis of C4-HSL treated with 0.5 to 1.0 mg mL-1 of hordenine. (D) Quantitative analysis of 3-oxo-C12-HSL treated with 0.5 to 1.0 mg mL-1 of hordenine. Error bars are the standard deviations of three measurements. Statistical differences were determined by ANOVA followed by Tukey-Kramer test. **, p < 0.01 versus DMSO control. ***, p < 0.001 versus DMSO control. 177x99mm (300 x 300 DPI)


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Figure 3. Effect of hordenine coupled with netilmicin on biofilm formation. (A) Relative biofilm formation and (B) surviving planktonic cells in the culture supernatant treated with hordenine and netilmicin. (C) Fluorescence microscopy images and (D) SEM images of (a) DMSO, (b) netilmicin, (c-e) hordenine (0.5, 0.75, and 1.0 mg mL-1), (f) 0.5 mg mL-1 of hordenine with 0.4 µg mL-1 of netilmicin, (g) 0.75 mg mL-1 of hordenine with 0.4 µg mL-1 of netilmicin, and (h) 1.0 mg mL-1 of hordenine with 0.4 µg mL-1 of netilmicin treated biofilms of P. aeruginosa PAO1. Statistical differences were determined by ANOVA followed by Tukey-Kramer test. *, p < 0.05 versus corresponding control. **, p < 0.01 versus corresponding control. ***, p < 0.001 versus corresponding control. 177x99mm (300 x 300 DPI)



Figure 4. Effect of hordenine and netilmicin on preformed biofilms. (A) Quantitative analysis of biofilm biomass. (B) Quantitative analysis of surviving cells in treated biofilms. (C) Quantitative analysis of surviving cells in culture supernatant. (D) SEM images and (E) CLSM images of (a) DMSO, (b) netilmicin, (c-e) hordenine (0.5, 0.75, and 1.0 mg mL-1), (f) 0.5 mg mL-1 of hordenine with 0.8 µg mL-1 of netilmicin, (g) 0.75 mg mL-1 of hordenine with 0.8 µg mL-1 of netilmicin, and (h) 1.0 mg mL-1 of hordenine with 0.8 µg mL-1 of netilmicin treated biofilms of P. aeruginosa PAO1. Statistical differences were determined by ANOVA followed by Tukey-Kramer test. *, p < 0.05 versus corresponding control. **, p < 0.01 versus corresponding control. ***, p < 0.001 versus corresponding control. 177x99mm (300 x 300 DPI)


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Figure 5. Effect of hordenine on virulence factors secreted by P. aeruginosa PAO1. Levels of virulence factors at varying hordenine concentrations (0.5, 0.75 and 1.0 mg mL-1) were evaluated. DMSO and resveratrol (RES, 1.0 mg mL-1) were used as the negative and positive controls, respectively. (A) Protease levels. (B) Elastase levels. (C) Pyocyanin levels. (D) Rhamnolipid levels. (E) Alginate levels. (F) Pyoverdine levels. (G) and (H) represent swimming and swarming motilities, respectively, treated with (a) DMSO, (b) resveratrol (1.0 mg mL-1), and (c-e) hordenine (0.5, 0.75, and 1.0 mg mL-1). Statistical differences were determined by ANOVA followed by Tukey-Kramer test. *, p < 0.05 versus DMSO control. **, p < 0.01 versus DMSO control. ***, p < 0.001 versus DMSO control. 146x120mm (300 x 300 DPI)



Figure 6. Effect of hordenine at 1.0 mg mL-1 on gene expressions of QS-related circuits in P. aeruginosa. ***, p < 0.001 versus DMSO control. 177x124mm (300 x 300 DPI)


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Graphic for manuscript 84x47mm (300 x 300 DPI)



1

41Hordenine, a novel quorum sensing inhibitor and

2

anti-biofilm agent against Pseudomonas aeruginosa

3 4

Jin-Wei Zhou,†,‡,§, Huai-Zhi Luo†,‡,§, Huan Jiang†,‡,§, Ting-Kun Jian†,‡, Zi-Qian

5

Chen†,‡, Ai-Qun Jia*,†

6 7

†

8

Laboratory of Tropical Biological Resources of Ministry Education, Hainan

9

University, Haikou 570228, China;

State Key Laboratory of Marine Resource Utilization in South China Sea, Key

10

‡

11

Science and Technology, Nanjing 210094, China.

School of Environmental and Biological Engineering, Nanjing University of

12 13 14 15 16 17 18 19 20 21 22 1


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Abstract: The quorum sensing (QS) inhibitory activity of hordenine from sprouting

24

barley against foodborne pathogen Pseudomonas aeruginosa was evaluated for the

25

first time here. At concentrations ranging from 0.5 to 1.0 mg mL-1, hordenine

26

inhibited the levels of acyl-homoserine lactones (AHLs). The enhanced susceptibility

27

of hordenine with netilmicin on P. aeruginosa PAO1 biofilm formation as well as

28

their efficiency in disrupting preformed biofilms was also evaluated using scanning

29

electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).

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Hordenine treatment inhibited the production of QS-related extracellular virulence

31

factors of P. aeruginosa PAO1. Additionally, qRT-PCR analysis demonstrated that the

32

expressions of QS-related genes, lasI, lasR, rhlI, and rhlR, were significantly

33

suppressed. Our results indicated that hordenine can serve as a competitive inhibitor

34

for signaling molecules and act as a novel QS-based agent to defend against

35

foodborne pathogens.

36 37

KEYWORDS: hordenine, foodborne pathogen, Pseudomonas aeruginosa, quorum

38

sensing, biofilm

39 40 41 42 43 44 2



45 46

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Illness resulting from eating food contaminated with pathogens or the secreted toxins

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is a critical public health concern. In industrialized and developing countries, up to 10%

49

of the population may experience foodborne disease annually.1 One of the most

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important factors contributing to such illness is antimicrobial resistance to antibiotics.

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In addition to health consequences, food spoilage due to foodborne pathogens can

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lead to considerable economic loss to both consumers and producers.2 Microbial

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spoilage leads to excessive food loss, even with modern food preservation techniques.

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A wide range of foodborne pathogens such as Salmonella spp., Pseudomonas spp.,

55

Bacillus spp., Yersinia enterocolitica, and Campylobacter jejuni can form biofilms.3

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Biofilms have several advantages over free-living cells, including resistance to

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antibiotics. Biofilms formed on food surfaces can result in contamination of products

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and shortened shelf-life, leading to possible foodborne diseases.4

INTRODUCTION

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Food spoilage and infections due to foodborne pathogens are orchestrated processes

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regulated by quorum sensing (QS).5 The QS system is mediated by autoinducers (AIs),

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which are activated once their concentration of AIs reaches a certain threshold. The

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expressions of many genes are regulated by AIs receptors, which modulate a variety

63

of physiological activities such as bioluminescence, biofilm formation, and virulence.6

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AIs were identified as oligopeptides and acylated homoserine lactones (AHLs) in

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Gram-positive and Gram-negative bacteria, respectively.

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Pseudomonas aeruginosa, a well-documented Gram-negative and notoriously 3


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resistant bacterium, can cause a wide range of illnesses and food spoilage.7 This

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bacterium has two QS systems, that is las and rhl, which modulate the synthesis of

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AIs

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N-butanoyl-L-homoserine lactone (C4-HSL), respectively. The chemical signaling

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networks are hierarchically arranged, with rhl modulated by las. Additionally, P.

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aeruginosa also possesses a novel molecule quinolone signal (PQS), which provides

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links between las and rhl. QS signals can be adopted by P. aeruginosa directly for

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modulating the formation of biofilms and the secretion of virulence factors, such as

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exotoxins, pyocyanin, and alginate.8 As QS is vital in food spoilage and bacterial

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pathogenesis, interfering with the QS networks could be a useful strategy for

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preventing food spoilage and human infections.9

N-(3-oxododecanoyl)-L-homoserine

lactone

(3-oxo-C12-HSL)

and

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Many synthetic and natural compounds are capable of blocking QS systems in P.

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aeruginosa.10-12 However, the limited implication of these compounds in food sectors

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and mammalian cells has led to the search for novel natural QS inhibitors with broad

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applications.9 Hordenine, a dietary phenolic phytochemical from sprouting barley, has

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traditionally been used as vasoconstrictive and indirectly acting adrenergic agents,13

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however, no studies on its anti-QS activity have yet been reported. Here, hordenine

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was investigated as a novel QS inhibitor, anti-biofilm agent, and aminoglycoside

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antibiotic-accelerant against P. aeruginosa.

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MATERIALS AND METHODS

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Strains and Growth Conditions. Wild type P. aeruginosa PAO1 was obtained

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from Q. Gong (Ocean University of China). All experiments were performed in 4



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Luria-Bertani medium (LB) at 37 °C unless otherwise specified.

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Determination of Minimum Inhibitory Concentration (MIC) of Hordenine.

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Hordenine (purity > 97 %) purified from sprouting barley extract was purchased from

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Nanjing Jingzhu Biotech (Nanjing, China). Stock solutions were prepared by

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dissolving hordenine in dimethyl sulfoxide (DMSO). The Clinical and Laboratory

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Standards Institute (CLSI, 2015) was adopted for the determination of MIC of

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hordenine with an inoculum of 1-5 × 105 CFU mL-1. Hordenine (0.16-10 mg mL-1)

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was serially diluted two-fold in Müller-Hinton broth. The MIC was the lowest

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concentration of hordenine that inhibited visible growth of P. aeruginosa.

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For growth measurement, 0.1% overnight cultures of P. aeruginosa PAO1 (OD620 =

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0.5) were added to LB, and then with hordenine (0.5, 0.75, and 1.0 mg mL-1). The

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same amount of DMSO served as the negative control. After cultivation at 37 °C for

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24 h, growth was determined using a microplate reader at 620 nm (Biotek Elx800,

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USA).

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Determination of AHLs Levels. The putative anti-QS capacity of hordenine was

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assessed by quantitating C4-HSL and 3-oxo-C12-HSL levels secreted by this strain.

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Briefly, 0.1% overnight cultures of P. aeruginosa PAO1 were inoculated into 50 mL

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of LB in the presence or absence of hordenine and cultured at 37 °C for 48 h. The

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same amount of DMSO was added as the negative control. After cultivation, cells

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were removed by 15-min centrifugation (4 °C). The supernatant was extracted three

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times using acidified ethyl acetate (1:1, v/v). The solvent was evaporated under

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reduced pressure and residues were dissolved in methanol. LC-MS/MS was adopted 5


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for AHLs quantification.14 Briefly,

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3-oxo-C12-HSL were detected based on their MS/MS fragment ions and the retention

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time of AHLs standards. We selected the ion m/z 102 for quantification on account of

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its specificity and better signal-to-noise ratio. Peak area calculation was performed by

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the extracted ion chromatograms. Results were normalized to the DMSO control for

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relative quantification.

peaks corresponding to C4-HSL and

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Biofilm Inhibition Assay. The static biofilm inhibition assay was performed in

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96-well flat-bottom polystyrene plates (Costar 3599, Corning, USA) as described

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previously with some modifications.15 Briefly, overnight cultures of P. aeruginosa

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PAO1 (OD620 = 0.5) were diluted 1:100 into 200 µL of Trypticase Soytone broth (TSB,

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1.7% tryptone, 0.3% soy protone, 0.25% glucose, 0.5% NaCl, 0.25% KH2PO4) and

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then cultivated with hordenine at 37 °C for 24 h without agitation. After cultivation,

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planktonic cells were removed and biofilms were stained with crystal violet (0.05%)

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for 15 min. Excess crystal violet was rinsed off by distilled water and bound crystal

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violet was solubilized in 200 µL of 95% ethanol. Biofilms were quantified by reading

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the microplates at 570 nm (Biotek Elx800, USA).

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For determination of the enhanced effect of hordenine on biofilm formation with

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the addition of netilmicin (Sangon Biotech, China), 0.1% overnight cultures of P.

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aeruginosa PAO1 (OD620 = 0.5) were added to 200 µL of TSB in 96-well flat-bottom

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polystyrene plates containing netilmicin (0.4 µg mL-1) and hordenine. After

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cultivation at 37 °C for 24 h, biofilms were quantified using the method above. To

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assess planktonic cell survival, the suspension cultures (100 µL) were centrifuged at 6



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5,000 rpm at 4 °C with the pellets then resuspended in 1 mL of 0.9% NaCl and

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10-fold serially diluted. The number of surviving cells was determined by plating the

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dilutions (100 µL) at 37 °C overnight.

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Inhibition of Preformed Biofilms. P. aeruginosa PAO1 biofilms were incubated

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as described above. Once biofilm formed, the suspension cultures were removed and

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wells were washed with sterile phosphate buffer saline (PBS, pH 7.2) three times to

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remove the planktonic cells. Fresh TSB medium, supplemented with hordenine,

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netilmicin (0.8 µg mL-1), or their combination was added to the wells, with DMSO

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served as the control. The cultures were cultivated at 37 °C for 72 h. Biofilms were

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fixed with methanol for 15 min, stained with 100 µL of 0.05% crystal violet for 15

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min, washed three times to remove excess dye, and quantitated after solubilization of

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the dye with ethanol by reading the microplates at 570 nm (Biotek Elx800, USA). To

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quantify cell viability, the treated biofilms were washed with PBS three times and the

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exopolysaccharides were digested with dextranase (5 U, D8144-Sigma Aldrich, USA)

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for 30 min at 37 °C, the biofilms were then sonicated for 30 s (37 Hz, KQ-250,

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Kunshan Ultrasonic Instruments Co., Ltd., China). The number of CFU/biofilm was

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investigated by plating the resulting dilutions on LB agar at 37 °C overnight.16

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For viable colony counts of planktonic cells, the suspension cultures (100 µL) were

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centrifuged at 5,000 rpm at 4 °C with the pellet then resuspended in 1 mL of 0.9%

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NaCl. P. aeruginosa PAO1 cells were then serially diluted and the number of CFU per

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mL of cultures was calculated by plating the resulting dilutions on LB agar at 37 °C

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overnight. 7


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Microscopic Analysis. For inhibition assay, biofilms were established in 24-well

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chambered cover slides (Costar 3524, Corning, USA) and treated with hordenine,

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netilmicin, or their combination, as described above. Biofilms on the slides were

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washed with PBS, dried at 60 °C, and then stained with 0.01% acridine orange and

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observed using a fluorescence microscope (Nikon 80i, Japan). For scanning electron

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microscopy (SEM), biofilms were fixed with 2.5% glutaraldehyde and dehydrated

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with graded ethanol. Biofilms were subsequently freeze-dried, gold-coated, and

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subjected to SEM (JSM6360, JEOL, Tokyo, Japan).

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For visual observation of the preformed biofilms, samples were captured using both

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SEM and confocal laser scanning microscopy (CLSM, Zeiss LSM 700, Carl Zeiss,

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Jena). Briefly, preformed biofilms were established in 24-well chambered cover slides

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and treated with hordenine, netilmicin, or their combination as in the preformed

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biofilm inhibition assay. Biofilms on the slides were rinsed three times with PBS.

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Samples for SEM observation were prepared according to the method mentioned

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above. For CLSM imaging, acridine orange staining was performed. Briefly, samples

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were stained with acridine orange for 15 min, and then fixed with paraformaldehyde

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(4%) in the dark for 15 min. Excess dye was removed with PBS and confocal images

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of the stained biofilm cells were captured using a × 63/1.4 numerical aperture (NA)

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oil objective. Three-dimensional reconstructions were obtained adopting the IMARIS

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software package (Bitplane AG, Zürich, Switzerland).

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Effect of Hordenine on P. aeruginosa Virulence Factors. The QS inhibitor

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resveratrol (1.0 mg mL-1) served as the positive control and DMSO served as the 8



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negative control. Protease activity was measured as described previously,17 with some

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modifications. Briefly, sterile supernatant (150 µL) was mixed with 0.3% azocasein

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(250 µL, Sangon Biotech, China) in 50 mM Tris-HCl. The mixture was incubated at

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37 °C for 4 h. Trichloroacetic acid (10%) with the volume of 1.2 mL was added to

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precipitate the undigested substrate for 20 min, followed by a 10-min centrifugation.

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Subsequently, the same volume of NaOH (1M) was added to the supernatant. Protease

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activity was determined at OD440.

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Elastase activity was determined according to Ohman et al.18 In brief, sterile

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supernatant (100 µL) was mixed with elastin Congo red (ECR) buffer (900 µL, 1 mM

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CaCl2, 100 mM Tris, pH 7.5) containing 20 mg of ECR and incubated at 37 °C for 3 h.

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After 10-min centrifugation at 37 °C, the absorbance of the supernatant was

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determined at 495 nm.

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Pyocyanin was determined according to Kumar et al.19 The culture supernatant was

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extracted with chloroform (5/3, v/v). The organic phase was mixed with 1 mL of

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hydrochloric acid (0.2 M). After 10-min centrifugation at 4 °C, the organic phase was

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collected, and its absorption was measured at 520 nm.

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Rhamnolipids were assessed using the orcinol method.20 Briefly, 300 µL of culture

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supernatant was extracted twice with 600 µL of diethyl ether. The ether layer was

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evaporated at 35 °C under reduced pressure, and residuals were dissolved in 100 µL of

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deionized water. A total of 900 µL of orcinol solution (0.19% orcinol in 53% [v/v]

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H2SO4, Sigma Aldrich, USA) was mixed with 100 µL of each sample. After 30-min

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heating at 80 °C, the cooled samples were then determined at 421 nm. 9


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For assaying alginate generation, 70 µL of sterile supernatant was mixed with 600

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µL of boric acid/H2SO4 (4:1, v/v). After vortexing, 20 µL of 0.2% carbazole solution

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was added to the mixture, which was then incubated at 55 °C for 30 min. Alginate

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production was measured by reading OD530.21

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For pyoverdine assay, sterile supernatant was 10-fold diluted in Tris-HCl buffer (pH 7.4). Pyoverdine production was measured by reading OD405.

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Motility Inhibition Assays. Swimming and swarming motilities were performed as

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previously described,21 with minor modification. Briefly, 2 µL of overnight P.

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aeruginosa PAO1 cultures (OD620 = 0.5) were inoculated with hordenine at the center

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of the swimming agar (1% tryptone, 0.5% NaCl, 0.3% agar, pH 7.2) and swarming

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agar medium (1% tryptone, 0.5% NaCl, 0.5% glucose, 0.3% agar, pH 7.2),

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respectively. Resveratrol (1.0 mg mL-1) and DMSO served as the positive and

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negative control, respectively. Plates were cultivated at 37 °C overnight and migration

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was then recorded.

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Quantitative Real-Time PCR. P. aeruginosa PAO1 was grown in LB medium

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supplemented with or without hordenine (1.0 mg mL-1) at 37 °C at 180 rpm for 24 h.

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After incubation, cells were washed with sterile PBS (pH 7.2) three times and

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collected after 10-min centrifugation at 4 °C. Quantitative real-time PCR (qRT-PCR)

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was performed with the Applied Biosystems 7300 Real-time PCR System with

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primers listed in Table 1. Total RNA was extracted using an RNA extraction kit

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(Tiangen Biotech, Beijing, China). Genomic DNA was removed using the gDNA

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wiper mix and first-strand complementary DNA (cDNA) was synthesized using the 10



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HiScript® II qRT Supermix (Vazyme Biotech, Nanjing, China) according to the

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manufacturer’s recommendations. The qRT-PCR was performed using a SYBR Green

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Master Mix kit (Vazyme Biotech, Nanjing, China). The ribosomal gene rpsL was used

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as an internal control and the fold changes of the target genes were determined using

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the 2-△△Ct method, as previously described.22

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Statistical Analysis. All assays were performed at least three times and data were

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expressed as means ± standard deviation (SD). Graphs were constructed using Origin

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8.6 software (OriginLab, Northampton, MA, USA). One-way analysis of variance

229

(ANOVA) was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA)

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for comparing differences between groups, followed by the Tukey-Kramer test. A P

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value ≤ 0.05 was considered statistically significant.

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RESULTS

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Determination of MIC of Hordenine. The chemical structure of hordenine is

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presented in Figure 1A. The MIC of hordenine was evaluated by doubling dilution

235

assay with concentrations ranging from 0.625 to 10 mg mL-1. The MIC of hordenine

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was 2.5 mg mL-1. The growth profile was determined using hordenine at sub-MIC

237

concentrations for 24 h (Figure 1B). Treatment with hordenine at concentrations

238

varying from 0.5 to 1.0 mg mL-1 showed no inhibitory effect on cell growth compared

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with the control.

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Effect of Hordenine on AHLs Levels. The levels of AHLs produced by P.

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aeruginosa PAO1 were quantified to evaluate the putative anti-QS activity of

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hordenine. LC-MS/MS analysis confirmed that two major AHLs, i.e., C4-HSL and 11


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3-oxo-C12-HSL, were detected in the culture supernatants (Figure 2A). Exposure to

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hordenine (0.5, 0.75, and 1.0 mg mL-1) for 24 h caused a significant decrease in both

245

peaks and areas of C4-HSL and 3-oxo-C12-HSL (Figure 2B). Relative quantification

246

analysis demonstrated that hordenine treatment at 0.5, 0.75, and 1.0 mg mL-1 reduced

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C4-HSL by approximately 69%, 74%, and 79%, respectively compared with the

248

control (Figure 2C). Additionally, a significant decrease was also observed between

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hordenine treatment and the control for 3-oxo-C12-HSL with the inhibitory rate

250

varying from 24% to 66% (Figure 2D). These data demonstrated that hordenine

251

possesses anti-QS capacity, which might be caused by interfering with the production

252

of AHLs.

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Inhibition of Biofilm Formation. The inhibitory effect of hordenine on P.

254

aeruginosa PAO1 biofilm formation was presented in Figure 3. Treatment with

255

hordenine at concentrations of 0.75 and 1.0 mg mL-1 significantly reduced the

256

formation of biofilms by 26% and 31%, respectively (Figure 3A). After hordenine

257

treatment, planktonic cell survival was also determined. Results showed that

258

hordenine treatment (0.5 to 1.0 mg mL-1) had no effect on the viability of planktonic

259

cells compared with that of the control (Figure 3B).

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To evaluate whether hordenine increased the susceptibility of antibiotics against

261

biofilms, biofilms were treated with hordenine and netilmicin for 24 h. Results

262

indicated that hordenine significantly enhanced the effect of netilmicin on P.

263

aeruginosa PAO1 biofilms in a concentration-dependent manner (Figure 3A). At

264

concentrations of 0.5, 0.75, and 1.0 mg mL-1, hordenine reduced biofilms by 12



265

approximately 35%, 52%, and 88%, respectively, with the addition of 0.4 µg mL-1 of

266

netilmicin (MIC, 1.0 µg mL-1). When used alone, netilmicin resulted in a minor

267

reduction in the biofilms (P > 0.05). Cell survival determination indicated that

268

hordenine (0.5 and 0.75 mg mL-1) coupled with netilmicin had no effect on the

269

viability of planktonic cells, though at 1.0 mg mL-1, planktonic cells were reduced by

270

10% (Figure 3B).

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In view of the promising anti-biofilm capacities of hordenine with netilmicin,

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biofilms were stained with acridine orange and observed under a fluorescence

273

microscope. Hordenine treatment showed significant biofilm reduction, as indicated

274

by the green stained cells (Figure 3Cd, e). In contrast, thickened biofilms were

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detected in the control group (Figure 3Ca). Under the combined treatment of

276

hordenine and netilmicin, the anti-biofilm capacity was significantly enhanced,

277

resulting in rare cell clusters (Figure 3Cf-h). A similar result was also obtained with

278

the SEM images, in which biofilms showed a scattered appearance in the presence of

279

hordenine (Figure 3Dd, e). After combined treatment with hordenine and netilmicin,

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the integrity of the biofilms was disrupted and cells were scattered (Figure 3Df-h).

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Effect of Hordenine on Preformed Biofilms. As shown in Figure 4A,

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approximately 23% (P < 0.05) of biofilms were eradicated after treatment with

283

hordenine at 1.0 mg mL-1. When used individually, netilmicin resulted in a minor

284

reduction in the preformed biofilms (P > 0.05) (Figure 4A). However, hordenine

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significantly enhanced the effect of netilmicin on preformed biofilms in a

286

concentration-dependent manner. Hordenine showed potent effects at 0.5, 0.75, and 13


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1.0 mg mL-1, reducing the preformed biofilms by approximately 29%, 43%, and 63%,

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respectively, with the addition of 0.8 µg mL-1 of netilmicin. Cell viability of the

289

treated biofilms further confirmed the enhanced effect mentioned above (Figure 4B).

290

Cell survival indicated that hordenine (0.5 and 0.75 mg mL-1) with netilmicin showed

291

no effect on the viability of planktonic cells in the culture supernatant, though 1.0 mg

292

mL-1 of hordenine resulted in a 10% reduction in planktonic cells (Figure 4C).

293

In addition to quantitative analysis, treated biofilms were also visualized using

294

SEM and CLSM. The SEM images showed thick biofilms in the control experiment

295

(Figure 4Da), whereas hordenine treatment at 0.75 and 1.0 mg mL-1 significantly

296

removed the microbes attached to the glass surface (Figure 4Dd, e). When hordenine

297

and netilmicin were used in combination, only small cell clusters remained attached

298

(Figure 4Df-h). The CLSM images also indicated reduced thickness in the

299

hordenine-treated biofilms (Figure 4E). After hordenine treatment (0.75 and 1.0 mg

300

mL-1), the thickness of biofilms was reduced from 20 to 16 µm. There was no

301

pronounced reduction when netilmicin was tested individually (thickness, >20 µm).

302

However, combined treatment showed major disruption to the biofilm architecture as

303

well as reduced thickness (Figure 4Ef-h).

304

Effect of Hordenine on Virulence Factors. As shown in Figure 5A, protease

305

activity was significantly suppressed by hordenine treatment (0.5 to 1.0 mg mL-1). At

306

1.0 mg mL-1, approximately 61% inhibition of protease activity was observed.

307

Resveratrol (1.0 mg mL-1), the positive control of this study and a documented QS

308

inhibitor, also showed inhibition of protease by approximately 40% (Figure 5A) 14



309

without affecting cell growth (data not shown). The inhibitory effect of hordenine on

310

elastase activity was shown in Figure 5B. The reduction in elastase was

311

concentration-dependent, with hordenine treatment causing a reduction in elastase of

312

approximately 28%, 46%, and 65%, respectively, compared with the DMSO control.

313

Resveratrol (1.0 mg mL-1) treatment resulted in a 30% inhibition of elastase activity.

314

Different concentrations of hordenine were assessed against pyocyanin production.

315

As shown in Figure 5C, a significant reduction in pyocyanin was detected after

316

treatment with hordenine. At 0.5 mg mL-1, approximately 40% reduction in pyocyanin

317

was detected, and at 1.0 mg mL-1, almost 80% inhibition was observed. When treated

318

with resveratrol (1.0 mg mL-1), a 40% reduction of pyocyanin was observed, showing

319

similar activity as hordenine at 0.5 mg mL-1. Hordenine also showed a

320

concentration-dependent reduction in the activities of rhamnolipid and alginate

321

(Figure 5D, E). At 1.0 mg mL-1, hordenine inhibited the activities of rhamnolipid and

322

alginate by 53% and 60%, respectively. Furthermore, pyoverdine production was

323

significantly decreased after hordenine treatment compared with the control (Figure

324

5F), and was reduced by 65% at a concentration of 1.0 mg mL-1.

325

Effect of Hordenine on Motilities. The swimming and swarming motilities grown

326

with hordenine were evaluated (Figure 5G, H). The mean swimming diameter in the

327

absence of hordenine was 28 mm (Figure 5Ga). Treatment with hordenine at

328

concentrations varying from 0.5 to 1.0 mg mL-1, bacterial colony were formed with

329

diameters not exceeding 13 mm (Figure 5Gb-d). Notably, the swimming diameter

330

following 1.0 mg mL-1 hordenine treatment was just 5 mm, and the green color, which 15


Page 22 of 38

Page 23 of 38


331

represents pyocyanin production, disappeared (Figure 5Gd). Additionally, in the

332

swarming motility assay, hordenine treatment significantly reduced tendril formation

333

and colony diameter (Figure 5H). An inhibitory effect of resveratrol (1.0 mg mL-1)

334

was also observed on swimming and swarming motilities (Figure 5Gb, 5Hb), which

335

showed similar effects as treated with 0.5 mg mL-1 of hordenine.

336

Effect of Hordenine on QS-Related Gene Expression. qRT-PCR assay was

337

performed to investigate the efficiency of hordenine on changes in the expressions of

338

four QS-related genes, that is, lasI, lasR, rhlI, and rhlR, in P. aeruginosa PAO1. The

339

most significant change was found for lasR, which was down-regulated in P.

340

aeruginosa PAO1 by approximately 60% after exposure to 1.0 mg mL-1 of hordenine

341

(Figure 6). Similarly, exposure to hordenine also caused a significant reduction in the

342

expressions of lasI, rhlI, and rhlR (Figure 6).

343

344

Hordenine

345

properties23, inhibition of monoamine oxidase B24 and stimulation of gastrin release.25

346

However, hordenine as a QS inhibitor has not yet been reported. In this study,

347

hordenine was evaluated for its potential to block QS-controlled phenotypes and

348

biofilm formation in foodborne pathogen P. aeruginosa. The anti-QS potential of

349

hordenine was initially screened by quantifying AHLs levels. Hordenine showed a

350

concentration-dependent reduction in AHLs production, thus indicating potent

351

anti-QS capacity. Hordenine also effectively reduced biofilm formation, virulence

352

factors, and QS-related gene expression of P. aeruginosa PAO1.

DISCUSSION possesses diverse

pharmaceutical effects,

16


including antibacterial


353

Biofilm formation by foodborne pathogens is one of the most notable aspects of

354

their pathogenicity.9 As QS plays a vital role in biofilm formation, interfering with the

355

QS system might be a preferable and convenient way to block biofilm formation. As

356

AHLs can bind to receptor lasR or rhlR and subsequently activate the expressions of

357

QS-related genes responsible for virulence production and biofilm formation, we first

358

evaluated the effect of hordenine on AHLs production. Results showed that AHLs

359

demonstrated a reduction of more than 65% when treated with 1.0 mg mL-1 of

360

hordenine. We next employed qRT-PCR to evaluate the expressions of QS-related

361

genes in P. aeruginosa PAO1. The expressions of lasI, lasR, rhlI, and rhlR were

362

reduced by about 50% compared with that of the control. However, when treated with

363

hordenine at 1.0 mg mL-1, biofilm formation and preformed biofilms were only

364

reduced by ~30% and ~23%, respectively. The results from the biofilm assay did not

365

well-correlated with the AHLs and QS-related gene expression results. The above data

366

indicated that biofilm formation is a sophisticated process and is not regulated by QS

367

alone. At present, the relationship between biofilm formation and QS has not been

368

fully clarified.26 Studies have shown that P. aeruginosa cells lacking Las-QS can also

369

form biofilms.27 Polysaccharides are essential for the formation of biofilms, and are

370

major components of biofilms in P. aeruginosa. The biofilm matrix polysaccharides

371

are encoded by two loci, pel and psl. LasR- and RhlR-QS can affect the pel operon

372

indirectly, with pel also regulated by another transcriptional regulator.28 The psl

373

operon is mediated transcriptionally by RpoS and post- transcriptionally by RsmA.29

374

However, RsmA has been proven to negatively regulate lasI and rhlI translation.30 17


Page 24 of 38

Page 25 of 38


375

These results suggest that QS is a vital regulatory mechanism for biofilm formation,

376

but not the only one.

377

Given the vital role of QS in P. aeruginosa pathogenesis, the use of hordenine

378

might restore or enhance the effectiveness of antibiotics against P. aeruginosa. We

379

therefore evaluated hordenine and antibiotic netilmicin against P. aeruginosa biofilms

380

to determine whether hordenine could function as a potential antibiotic accelerant.

381

Our results demonstrated that hordenine had the remarkable capacity to enhance the

382

susceptibility of P. aeruginosa PAO1 biofilms to netilmicin. Netilmicin, an

383

aminoglycoside antibiotic, is notable for its capacity to treat infections, particularly

384

pulmonary infections in cystic fibrosis patients.31 Recently, P. aeruginosa has shown

385

increased resistance to aminoglycoside antibiotics.32 Our results indicated that

386

hordenine can enhance the effect of netilmicin against both biofilm formation and

387

preformed biofilms. The prime mechanism of this combination might be the

388

permeability of the bacteria to antibiotic entrance. Biofilms are multicellular

389

three-dimensional structures held together by an extracellular matrix. Alginate is a

390

constituent of the matrix and displays a vital role in maintaining biofilm structure and

391

preventing the entrance of antibiotics into bacterial cells. The suppressed production

392

of alginate and loosed architecture and morphology of biofilms by hordenine, as

393

observed through SEM and CLSM, ultimately resulted in the reduced antibiotic

394

resistance of bacteria. The enhanced interaction between hordenine and netilmicin

395

could enhance the efficacy and lifespan of antibiotics, reduce antibiotic dosages, and

396

attenuate the emergence of resistant organisms. 18



397

Extracellular virulence factors such as protease, pyocyanin, elastase, rhamnolipid

398

and pyoverdine are important indicators of QS operon in P. aeruginosa. Therefore,

399

reduced levels of these factors are necessary to inhibit QS. Protease and elastase are

400

essential for colonizing host tissues and are regulated by the las system.33 Pyocyanin

401

and pyoverdine are important virulence factors for infection and biofilm formation

402

and can chelate the bound iron from transferrin.17 Rhamnolipid, an important

403

surfactant, is regulated by the rhl system and plays a vital role in surface motility and

404

biofilm initiation.34 Motility (swimming and swarming) is associated with biofilm

405

formation, virulence factor expression, and colonization, and plays a fundamental role

406

in the pathogenesis of P. aeruginosa.35 Data from the present study showed that these

407

factors were all reduced when treated with hordenine and hordenine was much more

408

effective than the QS inhibitor resveratrol in inhibiting virulence factors of P.

409

aeruginosa PAO1. These results correlated well with the QS-related gene expressions,

410

confirming the transcription level inhibition of virulence after hordenine treatment.

411

There are two main mechanisms for QS inhibition, signal mimicry and signal

412

degradation, which lead to the inhibition of downstream virulence and biofilm

413

genes.36 The prominent reduction of AHLs after hordenine treatment indicated the

414

possibility of QS suppression by the autoinducer degradation mechanism. These

415

results are similar to those reported in previous research.37 Therefore, hordenine might

416

act as an efficient and promising agent for combating foodborne pathogens.

417

418

Corresponding Author

AUTHOR INFORMATION

19


Page 26 of 38

Page 27 of 38


419

*

420

84303216.

421

Author Contributions

422

§

423

Notes

424

The authors declare no competing financial interests.

425

426

This work was supported by grants from the National Key Research and Development

427

Program of China (2017YFD0201401), Natural Science Foundation of Jiangsu

428

Province, China (BK20170859), Science and Technology Development Program of

429

Modern Agriculture, Nanjing (201608052), Six talent peaks project in Jiangsu

430

Province, Fundamental Research Funds for the Central Universities (30916011307),

431

and National Natural Science Foundation of China (41766006, 31170131).

A.-Q.J.: E-mail: [email protected]. Tel: +86 25 84303216. Fax: +86 25

J.W.Z., H.Z.L. and H.J. contributed equally to this work.

ACKNOWLEDGEMENTS

432 433 434 435 436 437 438 439 440 20



441 442 443 444 445 446 447 448

449

(1) Sandri, I.; Zacaria, J.; Fracaro, F.; Delamare, A.; Echeverrigaray, S. Antimicrobial

450

activity of the essential oils of Brazilian species of the genus Cunila against

451

foodborne pathogens and spoiling bacteria. Food Chem. 2007, 103, 823-828.

452

(2) Monente, C.; Bravo, J.; Vitas, A. I.; Arbillaga, L.; De Peña, M. P.; Cid, C. Coffee

453

and spent coffee extracts protect against cell mutagens and inhibit growth of

454

food-borne pathogen microorganisms. J. Funct. Foods 2015, 12, 365-374.

455

(3) Bridier, A.; Sanchez-Vizuete, P.; Guilbaud, M.; Piard, J.-C.; Naïtali, M.; Briandet,

456

R. Biofilm-associated persistence of food-borne pathogens. Food microbiol. 2015, 45,

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167-178.

458

(4) Abdallah, M.; Khelissa, O.; Ibrahim, A.; Benoliel, C.; Heliot, L.; Dhulster, P.;

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Chihib, N.-E. Impact of growth temperature and surface type on the resistance of

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Pseudomonas aeruginosa and Staphylococcus aureus biofilms to disinfectants. Int. J.

461

Food Microbiol. 2015, 214, 38-47.

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(5) Rahman, M. R.; Lou, Z.; Yu, F.; Wang, P.; Wang, H. Anti-quorum sensing and

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Terminalia chebula Retz. increase the susceptibility of Pseudomonas aeruginosa to

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williamsii), and hordenine. J. Pharm. Pharmacol. 1970, 22, 544-545.

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hordenine by monoamine oxidase and its action on vasa deferentia of the rat. J.

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Pharm. Pharmacol. 1989, 41, 421-3.

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(25) Yokoo, Y.; Kohda, H.; Kusumoto, A.; Naoki, H.; Matsumoto, N.; Amachi, T.;

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(27) Davies, D. G.; Parsek, M. R.; Pearson, J. P.; Iglewski, B. H.; Costerton, J. T.; 24



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Greenberg, E. The involvement of cell-to-cell signals in the development of a

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(29) Irie, Y.; Starkey, M.; Edwards, A. N.; Wozniak, D. J.; Romeo, T.; Parsek, M. R.

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Pseudomonas

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transcriptionally by RpoS and post-transcriptionally by RsmA. Mol. Microbiol. 2010,

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78, 158-172.

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(30) Pessi, G.; Williams, F.; Hindle, Z.; Heurlier, K.; Holden, M. T. G.; Camara, M.;

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Haas, D.; Williams, P. The global posttranscriptional regulator RsmA modulates

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production of virulence determinants and N-acylhomoserine lactones in Pseudomonas

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aeruginosa. J. Bacteriol. 2002, 184, 335-335.

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(31) Smyth, A. R.; Campbell, E. L. Prescribing practices for intravenous

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aminoglycosides in UK Cystic Fibrosis clinics: a questionnaire survey. J. Cyst. Fibros.

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2015, 14, 160-160.

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(32) Prickett, M. H.; Hauser, A. R.; McColley, S. A.; Cullina, J.; Potter, E.; Powers,

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C.; Jain, M. Aminoglycoside resistance of Pseudomonas aeruginosa in cystic fibrosis

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results from convergent evolution in the mexZ gene. Thorax 2017, 72, 40-47.

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(33) Stehling, E. G.; Silveira, W. D.; Leite, D. S. Study of biological characteristics

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of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and

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from patients with extra-pulmonary infections. Braz. J. Infect. Dis. 2008, 12, 86-8.

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(34) O'May, C.; Tufenkji, N. The swarming motility of Pseudomonas aeruginosa is

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blocked by cranberry proanthocyanidins and other tannin-containing materials. Appl.

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Environ. Microbiol. 2011, 77, 3061-7.

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(35) Luo, J.; Kong, J. L.; Dong, B. Y.; Huang, H.; Wang, K.; Wu, L. H.; Hou, C. C.;

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Liang, Y.; Li, B.; Chen, Y. Q. Baicalein attenuates the quorum sensing-controlled

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virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response

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in P. aeruginosa-infected macrophages by downregulating the MAPK and NFkappaB

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signal-transduction pathways. Drug Des., Dev. Ther. 2016, 10, 183-203.

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(36) Ni, N. T.; Li, M. Y.; Wang, J. F.; Wang, B. H. Inhibitors and antagonists of

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properties by competitively binding to quorum sensing receptors. Biofouling 2017,

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1-16.

563 564 565 566 567 568 569 570 571 572

26



573 574 575 576 577 578 579 580

Figure captions:

581

Figure 1. Chemical structure of hordenine (A) and effect of hordenine on P.

582

aeruginosa PAO1 growth (B). Growth was determined at different concentrations of

583

hordenine (0.5, 0.75, and 1 mg mL-1) for 24 h in a tube. DMSO served as the negative

584

control. Error bars demonstrated the standard deviations of three measurements.

585 586

Figure 2. Relative quantification of C4-HSL and 3-oxo-C12-HSL using LC-MS/MS

587

chromatograms. (A) MS/MS spectra of C4-HSL and 3-oxo-C12-HSL. (B) HPLC

588

chromatograms of C4-HSL and 3-oxo-C12-HSL produced by P. aeruginosa

589

supplemented with (c) DMSO and (d-f) hordenine (0.5, 0.75, and 1.0 mg mL-1). (a)

590

and (b) represent the standards of C4-HSL and 3-oxo-C12-HSL. (C) Quantitative

591

analysis of C4-HSL treated with 0.5 to 1.0 mg mL-1 of hordenine. (D) Quantitative

592

analysis of 3-oxo-C12-HSL treated with 0.5 to 1.0 mg mL-1 of hordenine. Error bars

593

are the standard deviations of three measurements. Statistical differences were

594

determined by ANOVA followed by Tukey-Kramer test. **, p < 0.01 versus DMSO 27


Page 34 of 38

Page 35 of 38


595

control. ***, p < 0.001 versus DMSO control.

596 597

Figure 3. Effect of hordenine coupled with netilmicin on biofilm formation. (A)

598

Relative biofilm formation and (B) surviving planktonic cells in the culture

599

supernatant treated with hordenine and netilmicin. (C) Fluorescence microscopy

600

images and (D) SEM images of P. aeruginosa PAO1 biofilms treated with (a) DMSO,

601

(b) netilmicin, (c-e) hordenine (0.5, 0.75, and 1.0 mg mL-1), (f) 0.5 mg mL-1 of

602

hordenine with 0.4 µg mL-1 of netilmicin, (g) 0.75 mg mL-1 of hordenine with 0.4 µg

603

mL-1 of netilmicin, and (h) 1.0 mg mL-1 of hordenine with 0.4 µg mL-1 of netilmicin.

604

Statistical differences were determined by ANOVA followed by Tukey-Kramer test. *,

605

p < 0.05 versus corresponding control. **, p < 0.01 versus corresponding control. ***,

606

p < 0.001 versus corresponding control.

607 608

Figure 4. Effect of hordenine and netilmicin on preformed biofilms. (A) Quantitative

609

analysis of biofilm biomass. (B) Quantitative analysis of surviving cells in treated

610

biofilms. (C) Quantitative analysis of surviving cells in culture supernatant. (D) SEM

611

images and (E) CLSM images of P. aeruginosa PAO1 biofilms treated with (a)

612

DMSO, (b) netilmicin, (c-e) hordenine (0.5, 0.75, and 1.0 mg mL-1), (f) 0.5 mg mL-1

613

of hordenine with 0.8 µg mL-1 of netilmicin, (g) 0.75 mg mL-1 of hordenine with 0.8

614

µg mL-1 of netilmicin, and (h) 1.0 mg mL-1 of hordenine with 0.8 µg mL-1 of

615

netilmicin. Statistical differences were determined by ANOVA followed by

616

Tukey-Kramer test. *, p < 0.05 versus corresponding control. **, p < 0.01 versus 28



617

corresponding control. ***, p < 0.001 versus corresponding control.

618 619

Figure 5. Effect of hordenine on virulence factors secreted by P. aeruginosa PAO1.

620

Levels of virulence factors at varying hordenine concentrations (0.5, 0.75 and 1.0 mg

621

mL-1) were evaluated. DMSO and resveratrol (RES, 1.0 mg mL-1) were used as the

622

negative and positive controls, respectively. (A) Protease levels. (B) Elastase levels.

623

(C) Pyocyanin levels. (D) Rhamnolipid levels. (E) Alginate levels. (F) Pyoverdine

624

levels. (G) and (H) represent swimming and swarming motilities, respectively, treated

625

with (a) DMSO, (b) resveratrol (1.0 mg mL-1), and (c-e) hordenine (0.5, 0.75, and 1.0

626

mg mL-1). Statistical differences were determined by ANOVA followed by

627

Tukey-Kramer test. *, p < 0.05 versus DMSO control. **, p < 0.01 versus DMSO

628

control. ***, p < 0.001 versus DMSO control.

629 630

Figure 6. Effect of hordenine at 1.0 mg mL-1 on the gene expressions of QS-related

631

circuits in P. aeruginosa. ***, p < 0.001 versus DMSO control.

632 633 634 635 636 637 638 29


Page 36 of 38

Page 37 of 38


639 640 641 642 643 644 645 646

Table 1. PCR primers for qRT-PCR. Primer Gene

Amplicon size Sequence (5’-3’)

direction

(bp)

Forward

GGCTGGGACGTTAGTGTCAT

Reverse

AAAACCTGGGCTTCAGGAGT

Forward

ACGCTCAAGTGGAAAATTGG

Reverse

TCGTAGTCCTGGCTGTCCTT

Forward

AAGGACGTCTTCGCCTACCT

Reverse

GCAGGCTGGACCAGAATATC

Forward

CATCCGATGCTGATGTCCAACC

Reverse

ATGATGGCGATTTCCCCGGAAC

Forward

GCAACTATCAACCAGCTGGTG

Reverse

GCTGTGCTCTTGCAGGTTGTG

lasI

104

lasR

111

rhlI

130

rhlR

101

rpsL

231

647 648 649 650 651 30



652 653 654 655 656 657

31


Page 38 of 38

Hordenine: A Novel Quorum Sensing Inhibitor and Antibiofilm Agent

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