Signal Detection in Pseudomonas aeruginosa PAO1

of Biotechnology, Guru Nanak Dev University, Amritsar-143001, Punjab, India ... quantitative analysis of QS molecules by taking a low volume of cu...
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Establishment of LCMS Based Platform for Discovery of Quorum Sensing Inhibitors: Signal Detection in Pseudomonas aeruginosa PAO1 Manoj Kushwaha, Shreyans K Jain, Nisha Sharma, Vidushi Abrol, Sundeep Jaglan, and Ram A. Vishwakarma ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00875 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 6, 2018

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Establishment of LCMS Based Platform for Discovery of Quorum Sensing Inhibitors: Signal Detection in Pseudomonas aeruginosa PAO1 Manoj Kushwaha†, ‡, ∥, Shreyans K. Jainδ, ∥, Nisha Sharma†, ǁ, ∥, Vidushi Abrol,† Sundeep Jaglan*, †, ǁ, Ram A. Vishwakarma* ∆ †

Microbial Biotechnology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India ∆

Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu180001, India

δ

Natural Product Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India ‡ ǁ

Department of Biotechnology, Guru Nanak Dev University, Amritsar-143001, Punjab, India

Academy of Scientific and Innovative Research, Jammu Campus, Jammu-180001, India.

*Corresponding Author(s): Ram A. Vishwakarma, PhD Director, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India. Tel: 91-191-2584999 Fax: 91-191-2586333 Email: [email protected] Sundeep Jaglan, PhD Scientist, Microbial Biotechnology Division CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India. Tel: 91-191-2585006-13 Ext. 264 Fax: 91-191-2586333 Email: [email protected]

Contributed equally

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ABSTRACT. Targeting the main three networking systems viz. Las, RhI and PQS via natural quenchers is a new ray of hope for combating the persistent behavior of Pseudomonas aeruginosa. In the bacterial chemical vocabulary pyocyanin, N-AHLs and rhamnolipids are the main keywords, which are responsible for the social and nomadic behavior of P. aeruginosa. In the present work LC-MS based real-time qualitative and quantitative analysis of pyocyanin, green phenazine, N-AHLs, and rhamnolipids were performed on P. aeruginosa PAO1. The quantitative analysis indicates that the production of pyocyanin and NHSLs increases with time while the production of rhamnolipids discontinued after 16 Hrs. This indicates the emergence of persisters in the medium instead of planktonic cells. Rhamnolipids being acts as a surfactant enhances the motility of the bacterial cells whereas, the pyocyanin is responsible for the biofilm formation. In microtiter plate based assay an effect of capsaicin and 6-gingerol was recorded. In the presence of capsaicin and 6-gingerol, substantial fall in the production of rhamnolipids, phenazine, quinolone, and N-AHLs was observed. Most interestingly, the 6-gingerol treatment led to the drastic decrease of rhamnolipids, phenazine, quinolone and N-AHLs than capsaicin. These studies demonstrate the effectiveness of the capsaicin and 6-gingerol on Las, PQS and Rhl circuits in a bacterium in order to understand the persistent and social behavior. Here we are reporting LC-MS/MS based qualitative and quantitative analysis of QS molecules by taking a low volume of culture (up to 200 µL). This method can be used as a platform to screen the new antivirulence agents for fighting the resistant behavior of P. aeruginosa during biofilm formation. Keywords: Biofilm, Pseudomonas aeruginosa PAO1, rhamnolipids, pyocyanin, LC-MS-MRM, Quorum sensing

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Pseudomonas aeruginosa is a gram-negative opportunistic pathogenic bacterium, that primarily infects immunocompromised individuals.1 Recently World Health Organization (WHO) has announced the P. aeruginosa as one of the 12 deadliest superbugs which must be on priority to address their multidrug resistance (MDR). P. aeruginosa infections tend to be chronic as they resist innate and adaptive immune defense mechanisms as well as antibiotics. The signaling molecule 3-oxo-C12-HSL and pyocyanin (PYO) and green phenazine pigments colonize in the lungs of cystic fibrosis (CF) patients and recovered in their sputum.2-5 Phenazine and PYO are one of the most important virulence factors produced by P. aeruginosa; they penetrate the cells and generate reactive oxygen species resulting in wide spectrum damage to human cells.6, 7 Rhamnolipid acts as an antibiofilm agent by their antiadhesive and biofilm disruption abilities.8-12 In biofilm formation the production of pyocyanin and phenazine is always inversely proportional to the rhamnolipids in the culture medium.13, 14 PYO and phenazine are most commonly detected and quantified by using ultraviolet spectrophotometry, in which sample must have at least the concentration of > 100 µg/ mL.14, 15 The rhamnolipids can also be detected and quantified by UV spectrometry as reported by Koch et al.16 Rhamnolipids are poor absorber of UV light and require derivitization with p-bromoacetophenone17 to get detected in HPLC. The limitation of these methods is that the specific structural isomers of rhamnolipids cannot be distinguished in the culture medium. In recent trends the liquid chromatography-tandem mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) is widely used for metabolomics, dereplication, and identification of new metabolites in plant/ microbial extract, biosynthetic pathway elucidation, quantitative targeted/ untargeted metabolomics and pharmacokinetic studies.18-21 The LC-MS/MS is highly sensitive, it requires minute amount of sample and quantify up to femtogram / mL with excellent mass accuracy. Due to its sensitivity, specificity and accuracy the LC-MS/MS by multiple reaction monitoring (LCMRM-MS) is preferred approach in protein quantification.22 Due to their good analytical sensitivities,

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high-performance liquid chromatography (HPLC) in combination with mass spectrometry is a frequently employed approach for detecting PYO, phenazine and rhamnolipids and 3-oxo-C12-HSL.2-4 It is well established that the inhibition of biofilm is more beneficial than disruption,23-25 the disruption can lead to gain the acquired resistance.26, 27 Consequently, our strategy has focused to find out how the capsaicin and 6-gingerol affect the production of signaling molecules during biofilm inhibition. Since 6gingerol has been already reported to suppress the expression of QS related genes28 and have antibiofilm activity,29 similarly capsaicin showed antibacterial potential,30 inspired us to take these molecules to carry out the present work. Consequently, we assumed that the real-time qualitative analysis of bacterial crude at different time intervals could be correlated with the density of signaling molecules and pathogenesis of P. aeruginosa. This led us to hypothesize the development of LC-MS/MS (MRM) based method for quantitative measurement of these QS signaling molecules in the presence of QS modulator. This kind of quantitative or qualitative analysis protocol may lead the development of a suitable platform for drug discovery screening assay by using LC-MS. RESULTS AND DISCUSSION To establish the study, the first set of experiment was focused on chemo profiling of bacterial crude to identify the QS metabolites by HRMS and MS/MS, then compared with METLIN database. ( Supporting Information S5). In second set of experiment, study was focused on time-dependent production of identified QS signaling molecules in bacterial culture using Extracted Ion Chromatogram (EIC) in LC-MS. Finally, production and quantitative measurement of signaling molecules in the presence Capsaicin and 6-gingerol was focused. Identification and MSMS based characterization of metabolites In MSMS analysis 21 metabolites were identified in crude extract of the culture, their m/z value and mass accuracy were predicted (Table 1). The fragmentation pattern of the corresponding masses was also observed and depicted (Table 2). (Supporting Information S2 and S3 for Phenazines and

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quinolones, Supporting Information S6 and S7 for rhamnolipids and Supporting Information S9 for 3oxo-C12-HSL) Time-dependent visual inspection of Total Ion Chromatogram (TIC) By visual inspection of TIC chromatogram of crude, the major compounds of phenazine, rhamnolipids and N-acyl-homoserine lactones were detected in the extracted ion chromatogram (EIC) or direct infusion (DI) Mass Spectrometry of the crude extract of P. aeruginosa (Supporting Information S1), after we developed a method for LC separation. The EIC peak area is directly correlated with the density of signaling molecules in the culture growth medium. It clearly reveals that most abundant markers were 1-HP and PYO (Supporting Information S1). Since phenazine /quinolone, rhamnolipids and N-acylhomoserine lactones class of compounds are responsible for social and persister behavior of P. aeruginosa,31-33 also the pyocyanin binds with the extracellular DNA and helps in biofilm formation.34 The most abundant signaling molecule were isolated and selected for quantitative analysis by LCMS/MS. In multiple reaction monitoring (MRM) based quantification method, a suitable parent and daughter ion combination is required in which most abundant daughter ion was selected with the combination of the parent ion for MRM transition (Figure 1). Major marker phenazine/quinolone; 1hydroxyphenazine (1-HP), 2-nonyl-4-hydroxyquinolone (NHQ), pyocyanin (PYO), 2-nonenyl-4hydroxyquinolone (db-NHQ) and rhamnolipids (Rha-Rha-(C10-C10) Rha-Rha-(C8-C10.), Rha-Rha(C10-C12.1) and Rha-Rha-(C10-C12), were isolated using semi-preparative HPLC and used as standards. The accurate mass, observed mass and their chemical formula were recorded (Table 1). > > Peak area of metabolites was analyzed at a different time interval of their production in culture media by P. aeruginosa (Figure 2 and Supporting Information S10 and S11). The metabolite at m/z 298.2017 was identified as 3-oxo-C12-HSL, the peak intensity of 3-oxo-C12-HSL in the culture medium at 3rd h was 1.5×103 and achieved its maximum of 2×104 at 9th h. It starts decreasing after 12h and declined to a ACS Paragon Plus Environment

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lowest level at 48th h (Figure 2a). Similarly four metabolites m/z 621.3487 [Rha-Rha-(C8 C10.) -H]-, m/z 649.3802 [Rha-Rha-(C10-C10.) -H]-, m/z 675.3952 [Rha-Rha-(C10-C12.) -H]- and m/z 677.4105 [RhaRha-(C10-C12.1.) -H]-, were identified as dirhamnolipid and the production of these rhamnolipids were started at 3rd h and the peak intensities of Rha-Rha-(C8 C10.) were below to the detection limit. The initial peak intensity of Rha-Rha-(C10-C10.), Rha-Rha-(C10-C12.1.) and Rha-Rha-(C10-C12) was 6×102, 1×102 and 2×102 respectively. The maximum peak intensity for Rha-Rha-(C8 C10): 0.5×104, RhaRha-(C10-C10)): 2×104, Rha-Rha-(C10-C12.1): 1×104 and Rha-Rha-(C10-C12): 1×104 was observed during 12th to 24th h, and the production of rhamnolipids was completely ceased after 24th h (Figure 2b). The initial intensity (1.5×102) of pyocyanin was observed at 9th h, and it continuously increases to 1×103 (≈10 fold of initial) at 48th h. The intensity of the 1-HP at 9th h was 5.5×102 and continuously increased and reached to 7.5×104 (136 fold of initial) at 48th h (Figure 2c). By total ion chromatogram (TIC), the initial intensity of 2-nonyl-4-hydroxyquinolone and 2-nonenyl-4-hydroxyquinolone were 0.5×103 and 4×103

respectively at 9th h and the intensity observed at 48th h was 0.8×105 (160 fold of initial) and

3×105 (75 fold of initial) with continuous production. We also analysed the peak intensity of 2-heptyl-l4-hydroxyquinolone

(HHQ),

2-heptyl-4-hydroxyquinolone-N-oxide

(HQNO),

2-undecenyl-4-

hydroxyquinolone (db-UHQ), 2-nonyl-4-hydroxyquinolone N-oxide (NQNO), Rha-(C8-C10), Rha(C10-C10), Rha-(C10-C12:1), Rha-Rha(C12-C12:1) with respect to time (Supporting Information S10 and S11). The observation from TIC and EIC clearly indicated the production of 3-oxo-C12-HSL was stopped at 12th h and the production of rhamnolipids was steady at 12th to 24th h and completely ceased after 24th h whereas (Figure 2a), the production of phenazine and quinolones continuously increasing up to 48th h. This showed that the persister behavior of Pseudomonas depends on the pyocyanin and green phenazine pigments. These results prompted us to design the experiment to develop an assay for antivirulence molecules, which target the production of phenazine and quinolones class of molecules, which can be easily estimated quantitatively by LC-MS. Out of 23 characterized metabolites, we were able to isolate nine standards for the quantitative study, All these standards were subjected for MRM ACS Paragon Plus Environment

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development for efficient quantification by LC-MS/MS (Figure 1), as listed in Supporting Information S13 . > Effect of 6-gingerol and Capsaicin on signaling molecule The two natural compounds 6-gingerol and Capsaicin were selected to evaluate their effect on the production of signaling molecules. To test as an antiQS agent, a 96 well plate assay was established to determine the effects of these molecules on the production of signaling molecules. 6-gingerol significantly decreased the production of rhamnolipids, a dose-dependent decrease in concentration of rhamnolipids was observed. The well having P. aeruginosa in MHB was kept as a control to compare the production of rhamnolipids with respect to the culture grown in the presence of 6-gingerol and Capsaicin. In control well the concentration of Rha-Rha-(C10-C10) was 1308.7 ± 15.9 nM and it gets reduced by 80% to 96% with 6-gingerol (2-128 µg/ mL ) and by 45% to 72% with capsaicin (2-128 µg/ mL). The control Rha-Rha-(C8-C10): 900.70 ± 16.07nM reduced by 80% to 96% with 6-gingerol (2128 µg/ mL) and 89 to 97% by capsaicin (0-128 µg/ mL). Control Rha-Rha-(C10-C12.1): 1368.04±8.60 nM reduced up to 88% to 98% and 70 to 85% by the treatment of 6-gingerol (0-128 µg/ mL ) and capsaicin (2-128 µg/ mL) respectively. Similarly, control Rha-Rha-(C10-C12): 1607.81±89.23 nM reduced by 87% to 95% with 6-gingerol and 57% to 95% reduction was noticed when treated with capsaicin (2 to 128 µg/ mL). The effect of 6-gingerol has been shown in Figure 3 and Supporting Information S21 while the effect of capsaicin is given in Supporting Information S19 and S20. The changes in the rhamnolipids concentration in the presence of 6-gingerol are in congruence as reported by Kim et al.28 The change in signaling molecule via non antibiotic molecule is also reported by Chang et al.35 PYO is one of the most important virulence factors produced by all P. aeruginosa strains, and it is a biomarker for this strain. The pyocyanin (PYO, 1-methoxy-5-methyl phenazine) and 1hydroxyphenazine are zwitterionic in nature and they can penetrate the cell membrane and generates ACS Paragon Plus Environment

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reactive oxygen species resulting in wide spectrum damage on human cells.6, 7 The production of PYO was reduced by 99% at 2 µg/ mL concentration of 6-gingerol and reduced by 94% at 2 µg/ mL of capsaicin. In the presence of 6-gingerol (2 µg/ mL), the production of db-NHQ was decreased by 93% of control concentration 12659.66 ± 515.06 nM, while in the presence of capsaicin (2 µg/ mL) it was reduced by 66% of control. The reduction of NHQ (control: 4395.32 ± 122.88 nM) was noticed by 90% with 6gingerol (at 2 µg/ mL) and by 69% with capsaicin at 32 µg/ mL. Similarly, the production of signaling molecule 1-HP (control: 28715 ±1330.7) was reduced by 95% with 2 µg/ mL of 6-gingerol and by 87% with 2 µg/ mL of capsaicin. 3-oxo-C12-HSL is produced in the log phase and it activates the transcriptional regulator LasR to initiate the expression of multiple QS target genes involved in exotoxin, exoenzyme, and secondary metabolites production, as well as the biofilm development, this represents that they respond to different stimulations and work in different ways, and thus accommodate different circumstances.36, 37 The 3-oxo-C12-HSL reduces up to 76% and 72% of control: 7132.62 ± 987.54 nM with the treatment of 6-gingerol (128 µg/ mL) and capsaicin (128 µg/ mL) (Figure 3i and Supporting Information S19, S20 and S21). In above experiments we have found 6-gingerol as more effective than capsacin while inhibiting the biofilm, it might be due to the vulnurability of 6-gingerol to make hydrogen bonding and hydrophobic interactions with the ligand molecules. Infact, 6-gingerol has been already reported to downregulate the expression of QS related genes.28 Further effect of capsaicin and 6-gingerol on P. aeruginosa was observed for the production of phenazines and quinolones. When 6-gingerol was tested at 4 µg/ mL, drastic reduction in the production of signalling molecules was observed, while capsaicin was not found to be comparable even at 32 µg/ mL (Figure 4a to 4c), P. aeruginosa grown in culture media was taken as control. As discussed phenazine and quinolone like signaling molecules are necessary for biofilm formation. The disapperance ACS Paragon Plus Environment

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of green color in the culture broth of P. aeruginosa is one of the indicator for the reduction of signalling molecules. (Supporting Information S31).We have performed the microtiter plate based biofilm assay according to Merritt et al.38 on 6 gingerol and capsaicin, the capsaicin reduce 29% of biofilm formation at 100 µg/ mL as compare to control well, the 6-gingerol reduce 44% of biofilm formation at 100 µg/ mL. (Supporting Information S30) we also performed the HPLC analysis of 96 well plate sample treated with 6-gingerol, capsaicin and ciprofloxacin and we observe that the peak at tR = 22.6 identified as db NHQ is completely suppressed in the presence of 25 and 12.5 µg/ mL 6-gingerol (Supporting Information S28)

but it detectable in 50 and 25 µg/ mL of capsaicin (Supporting

Information S27). The peak of NHQ completely disappear in the sample with minimum inhibitory concentration of ciprofloxacin (0.0312 µg/ mL) but it was detectable in 0.015 and 0.007 µg/ mL which indicates the suppression of signalling molecules by 6 gingerol. (Supporting Information S29). > > > CONCLUSION The rapid, LC-MS/MS-MRM

based technique is sensitive and specific screening method for

discovering anti-QS molecules. Our method provides an important starting point for the design of effective antivirulence strategies for future studies of the complex QS circuitry in P. aeruginosa as well as a platform to discover new antibiotic compounds. Finally in this study, we have screened capsaicin and 6-gingerol on non-native AHLs for their ability to modulate production of important virulence factors i.e. pyocyanin, rhamnolipids and 3-oxo-C12-HSL in P. aeruginosa PAO1. The 6-gingerol was found to be effective QS modulator which suppresses the signaling molecules. The time-dependent LCMS study of culture and the density of signaling molecule at different time interval clearly correlate with the social and nomadic behavior of P. aeruginosa. We have observed the inhibitory effect of capsaicin ACS Paragon Plus Environment

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and 6-gingerol on the density of pyocyanin, 3-oxo-C12-HSL, and rhamnolipids and found 6-gingerol as more effective than capsaicin. The molecular mechanism via 6-gingerol acts on P. aeruginosa has been already reported,28 similarly it would be interesting to find out such mechanisms for capsacin in future work. The present method is fast, accurate and reliable with high specificity which can be implicated to manage the clinical applications. Furrthermore, the quantitative and qualitative analysis via LC-MS/MS may lead the development of a suitable platform for screening assay in drug discovery program. METHODS The culture media which was procured from Himedia Laboratories, India, rest all of the reagents and chemicals viz.LC-MS grade acetonitrile, acetic acid, ethyl acetate, methanol, water, formic acid and Lactones (3-oxo-C12-HSL) were of Sigma-Aldrich. The capsaicin was purchased from Sigma-Aldrich, whereas, the 6-gingerol was isolated from the rhizomes of Zingiber officinale Roscoe. Tandem mass spectroscopy was studied on Agilent UHD Q-TOF 6540 equipped with Agilent 1290 HPLC infinity series and 6410B QqQ system equipped with Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA). Bacterial culture and metabolite extraction. In this study, the bacteria P. aeruginosa strain PAO1 was used. The working culture of the bacteria was maintained on Luria Bertani Agar plates. The Erlenmeyer flask (100 mL) containing Luria Bertani broth (25 mL) was seeded with loopful of bacterial culture to get the fresh inoculum. For real-time based analysis of QS molecules, a set of 18 Erlenmeyer flasks (100 mL) each containing 25 mL Luria Bertani broth was inoculated with 1 mL of a bacterial suspension having a count of 5 × 105 colony forming units (CFU) / milliliters. The flasks were incubated at 37 °C in dark under constant shaking of 200 rpm for 48 h. The sample was retrieved (500 µL) from flasks at an interval of 3 h and extracted twice with equal volume of ethyl acetate. The organic layer was air-dried and subjected to LC-MS analysis. The experiment was performed in triplicate.

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Isolation of Pure 6-gingerol. For extraction of 6 gingerol, 500 grams of fresh ginger was purchased from the market. The raw ginger was grinded to make a paste and extracted twice with dichloromethane. The organic extract was subjected to a rotary evaporator to obtain the dry crude extract. 200 mg of crude extract was taken and dissolved in 1 mL of ethanol, subjected to semipreparative separation. The Column 10 × 250 mm equipped with waters HPLC model purospher with eluent mixture 1:1 acetonitrile /water with the flow of 3 mL/ min. (Supporting Information S17 and S18). Bioassay. The anti-quorum sensing effect of the 6 gingerol was performed by following the microdilution based method as per the CLSI antimicrobial susceptibility testing standards.39 MuellerHinton Agar (MHA) and Mueller-Hinton Broth (MHB) was prepared as per the manufacturer’s guidelines (HiMedia Laboratories, India). The pre-culture of the bacterial strain PAO1 was prepared in MHB by fresh inoculums of the culture incubated at 37 °C for 18-24 h with 100 rpm shaking to obtain growth of approximately 5 to 6 log CFU/ mL (evaluated and adjusted photometrically at 600 nm). The bacterial suspension was further diluted with MHB to obtain a final inoculum of 5 × 105 CFU/ mL. The assays were performed in transparent 96 well microtiter plates. Isolation and characterization of rhamnolipids, pyocyanin, and phenazines. Pseudomonas aeruginosa was grown in 1L Erlenmeyer flask containing 400 mL LB broth to obtain a culture volume of 2.5 liters. After 24 h the broth was centrifuged at 13000g, the supernatant was recovered while the cell mass was discarded. The supernatant was extracted twice with equal volume of chloroform and methanol mixture (1:1), concentrated in vacuo at low temperature. The extract was subjected to LC-MS profile, MS profile confirmed the presence of rhamnolipids and pyocyanin in crude extract than by using semi-preparative RP-HPLC to obtain the pure rhamnolipid, pyocyanin and phenazines. The peaks were collected and dried by lyophilization further confirmed by LC-MS/MS analysis The purity of the isolated standard markers were in the range of 95-100% and quantity was 1-2 mg (Supporting Information S4 for pyocyanin & phenazines, Supporting Information S8 for rhamnolipids, Supporting

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Information S23, S24, S25 and S26 for purity analysis and Supporting Information S32 and S33 for HNMR of 1-HP and PYO). Quantification of rhamnolipid, pyocyanin, phenazines and lactones. Optimal method development: The crude mixture was initially used to separate the various peaks corresponds to the rhamnolipids, pyocyanin, phenazines, and lactones. Quantification experiments for all QS molecules were carried out on Agilent 6410B QqQ system equipped with Agilent 1260 series. The QS molecules were eluted through HiBer RP-18e, 4.6 × 250 mm, 5 µm column using 0.5 mL.min-1 flow with the gradient program; Eluent A was composed of 0.1% Formic acid in Water and Eluent B was composed of acetonitrile. Gradient programme was as follows: 10-60% of B in 0-10 min, 60-90% of B in 10-20 min, hold at 90% B for 20-30 min, 90-10% of B in 30-32 min and hold for 3 min at the initial composition of the mobile phase. The MS acquisition parameter used was as follows: capillary voltage 4 KV, gas temperature 300 °C, drying gas 12 L.min-1 and nebulizer pressure 35 psi. The scan source parameter skimmer, fragmented and octopole RF peak were 65, 175 V and 700 V respectively. The data was acquired with the mass scan range of 100-2000 m/z. MS/MS acquisition of pyocyanin and phenazines was operated in the same parameter using fix collision energy 30 eV in positive mode. (For calibration curve MRM transition (qualifier and quantifier) and line equation (Supporting Information S13 and S12 for the selection of daughter ions). All standard curves of QS molecules were prepared using serial dilution method . Method validation The optimized LC-MS/MS (LC-MRM) method was validated using parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ) and precision. The linearity of the method was calculated by analyzing a series of the serially diluted standard mixture. Standard mixture solutions were prepared by weighing the proper amount of all 9 markers listed in (Supporting Information S13). Calibration curves were constructed by injecting standard mixture solution of different concentration level. The peak areas were calculated from the extracted MRM transition against the corresponding 12 ACS Paragon Plus Environment

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concentration using linear regression (Supporting Information S14, S15 and S16). The parameters of the linear equations were used to obtain concentration values from the sample. The LOD and LOQ were defined as the lowest concentration with a signal-to-noise (S/N) ratio of 3 and 10, respectively. The precision of the method was evaluated by determining the intraday and interday relative standard deviations (RSD) of the measured concentrations of nine standard mixtures. (Supporting Information S22). For the calibration curves of all 9 markers, the deviations of the measured values from their true values for at least 6 out of 8 concentrations were observed to be within ±20%, whereas the difference was higher than 15% but within 20%. Static biofilm assay To observe the effect of 6-gingerol and capsaicin on biofilm inhibition, static biofilm formation assay was performed in a 96-well microtiter plate.38 Culture of P. aeruginosa (OD at 595 nm = 1) was diluted in fresh AB medium (1:20). The microtiter plate wells containing either 6-gingerol or capsaicin (12.5100 µg mL-1) were aliquoted with the fresh inoculum as prepared in AB medium. After 24 h the OD of the suspended cells within each sample well was measured at 595 nm using Thermo ScientificTM MultiskanTM GO spectrophotometer. The cells attached to the sample well surface were then stained using crystal 0.1% crystal violet in water violet for 30 min. subsequently, washed with deionized water to remove the unbound crystal violet and further eluted with 30% acetic acid in water to remove the bounded crystal violet. The OD of the eluted samples were measured at 545 nm.

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ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge via the internet at http://pubs.acs.org. Supplementary results, discussion, and methods; supporting figures and Tables (S1 to S33) (PDF) AUTHOR INFORMATION Corresponding Authors Ram A. Vishwakarma Email: [email protected] Sundeep Jaglan Email: [email protected] ORCID Sundeep Jaglan: https://orcid.org/0000-0002-5691-7980 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors are thankful to E.P. Greenberg for providing the bacterial strain. We acknowledge R. Anand for providing the analytical instrumentation facility. This work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, India CSIR 12th FYP project (BSC-0108) and DST SERB Grant No. ECR/2017/001381. REFERENCES 1. Van Delden, C., and Iglewski, B. H. (1998) Cell-to-cell signaling and Pseudomonas aeruginosa infections, Emerging Infect. Dis.4, 551-560. 2. Quinn, R. A., Phelan, V. V., Whiteson, K. L., Garg, N., Bailey, B. A., Lim, Y. W., Conrad, D. J., Dorrestein, P. C., and Rohwer, F. L. (2016) Microbial, host and xenobiotic diversity in the cystic fibrosis sputum metabolome, ISME J. 10, 1483-1498.

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3. Sharma, A., Jansen, R., Nimtz, M., Johri, B. N., and Wray, V. (2007) Rhamnolipids from the rhizosphere bacterium Pseudomonas sp. GRP(3) that reduces damping-off disease in Chilli and tomato nurseries, J. Nat. Prod. 70, 941-947. 4. Struss, A. K., Nunes, A., Waalen, J., Lowery, C. A., Pullanikat, P., Denery, J. R., Conrad, D. J., Kaufmann, G. F., and Janda, K. D. (2013) Toward implementation of quorum sensing autoinducers as biomarkers for infectious disease states, Anal. Chem. 85, 3355-3362. 5. Wilson, R., Sykes, D. A., Watson, D., Rutman, A., Taylor, G. W., and Cole, P. J. (1988) Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium, Infect. Immun. 56, 25152517. 6. Lau, G. W., Hassett, D. J., Ran, H., and Kong, F. (2004) The role of pyocyanin in Pseudomonas aeruginosa infection, Trends Mol. Med. 10, 599-606. 7. Muller, M. (2006) Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin, Free Radical Biol. Med. 41, 1670-1677. 8. Diaz De Rienzo, M. A., Stevenson, P. S., Marchant, R., and Banat, I. M. (2016) Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel, Appl. Microbiol. Biotechnol. 100, 5773-5779. 9. Diaz De Rienzo, M. A., Stevenson, P. S., Marchant, R., and Banat, I. M. (2016) Pseudomonas aeruginosa biofilm disruption using microbial surfactants, J. Appl. Microbiol. 120, 868-876. 10. Boles, B. R., Thoendel, M., and Singh, P. K. (2005) Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms, Mol. Microbiol. 57, 1210-1223. 11. Dusane, D. H., Nancharaiah, Y. V., Zinjarde, S. S., and Venugopalan, V. P. (2010) Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms, Colloids Surf., B 81, 242-248. 12. Kim, L. H., Jung, Y., Yu, H. W., Chae, K. J., and Kim, I. S. (2015) Physicochemical interactions between rhamnolipids and Pseudomonas aeruginosa biofilm layers, Environ. Sci. Technol. 49, 3718-3726. 13. Vinckx, T., Wei, Q., Matthijs, S., and Cornelis, P. (2010) The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin, Microbiology156, 678-686. 14. El-Fouly, M. Z., Sharaf, A. M., Shahin, A. A. M., El-Bialy, H. A., and Omara, A. M. A. (2015) Biosynthesis of pyocyanin pigment by Pseudomonas aeruginosa, J. Radiat. Res. Appl. Sci. 8, 3648. 15. Cheluvappa, R. (2014) Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin, Methods X1, 67-73. 16. Koch, A. K., Kappeli, O., Fiechter, A., and Reiser, J. (1991) Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants, J. Bacteriol.173, 4212-4219. 17. Schenk, T., Schuphan, I., and Schmidt, B. (1995) High-performance liquid chromatographic determination of the rhamnolipids produced by Pseudomonas aeruginosa, J. Chromatogr. A 693, 7-13. 18. Rutledge, P. J., and Challis, G. L. (2015) Discovery of microbial natural products by activation of silent biosynthetic gene clusters, Nat. Rev. Microbiol. 13, 509-523. 19. Wang, M., and Carver, J. J., and Phelan, V. V., and Sanchez, L. M., and Garg, N., and Peng, Y., and Nguyen, D. D., and Watrous, J., and Kapono, C. A., and Luzzatto-Knaan, T., and Porto, C., and Bouslimani, A., and Melnik, A. V., and Meehan, M. J., and Liu, W. T., and Crusemann, M., and Boudreau, P. D., and Esquenazi, E., and Sandoval-Calderon, M., and Kersten, R. D., and Pace, L. A., and Quinn, R. A., and Duncan, K. R., and Hsu, C. C., and Floros, D. J., and Gavilan, R. G., and Kleigrewe, K., and Northen, T., and Dutton, R. J., and Parrot, D., and Carlson, E. E., and Aigle, B., and Michelsen, C. F., and Jelsbak, L., and Sohlenkamp, C., and Pevzner, P., and 15 ACS Paragon Plus Environment

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Edlund, A., and McLean, J., and Piel, J., and Murphy, B. T., and Gerwick, L., and Liaw, C. C., and Yang, Y. L., and Humpf, H. U., and Maansson, M., and Keyzers, R. A., and Sims, A. C., and Johnson, A. R., and Sidebottom, A. M., and Sedio, B. E., and Klitgaard, A., and Larson, C. B., and P, C. A. B., and Torres-Mendoza, D., and Gonzalez, D. J., and Silva, D. B., and Marques, L. M., and Demarque, D. P., and Pociute, E., and O'Neill, E. C., and Briand, E., and Helfrich, E. J. N., and Granatosky, E. A., and Glukhov, E., and Ryffel, F., and Houson, H., and Mohimani, H., and Kharbush, J. J., and Zeng, Y., and Vorholt, J. A., and Kurita, K. L., and Charusanti, P., and McPhail, K. L., and Nielsen, K. F., and Vuong, L., and Elfeki, M., and Traxler, M. F., and Engene, N., and Koyama, N., and Vining, O. B., and Baric, R., and Silva, R. R., and Mascuch, S. J., and Tomasi, S., and Jenkins, S., and Macherla, V., and Hoffman, T., and Agarwal, V., and Williams, P. G., and Dai, J., and Neupane, R., and Gurr, J., and Rodriguez, A. M. C., and Lamsa, A., and Zhang, C., and Dorrestein, K., and Duggan, B. M., and Almaliti, J., and Allard, P. M., and Phapale, P., and Nothias, L. F., and Alexandrov, T., and Litaudon, M., and Wolfender, J. L., and Kyle, J. E., and Metz, T. O., and Peryea, T., and Nguyen, D. T., and VanLeer, D., and Shinn, P., and Jadhav, A., and Muller, R., and Waters, K. M., and Shi, W., and Liu, X., and Zhang, L., and Knight, R., and Jensen, P. R., and Palsson, B. O., and Pogliano, K., and Linington, R. G., and Gutierrez, M., and Lopes, N. P., and Gerwick, W. H., and Moore, B. S., and Dorrestein, P. C., and Bandeira, N. (2016) Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking, Nat. Biotechnol. 34, 828-837 20. Hou, Y., Tianero, M. D., Kwan, J. C., Wyche, T. P., Michel, C. R., Ellis, G. A., Vazquez-Rivera, E., Braun, D. R., Rose, W. E., Schmidt, E. W., and Bugni, T. S. (2012) Structure and biosynthesis of the antibiotic bottromycin D, Org. lett.14, 5050-5053 21. Hou, Y., Braun, D. R., Michel, C. R., Klassen, J. L., Adnani, N., Wyche, T. P., and Bugni, T. S. (2012) Microbial strain prioritization using metabolomics tools for the discovery of natural products, Anal. Chem. 84, 4277-4283. 22. Law, S. K., Dodds, A. W., and Porter, R. R. (1984) A comparison of the properties of two classes, C4A and C4B, of the human complement component C4, EMBO J. 3, 1819-1823. 23. Loo, C. Y., Lee, W. H., Young, P. M., Cavaliere, R., Whitchurch, C. B., and Rohanizadeh, R. (2015) Implications and emerging control strategies for ventilator-associated infections, Expert Rev. Anti-Infect. Ther. 13, 379-393. 24. Simões, M., Simões, L. C., and Vieira, M. J. (2010) A review of current and emergent biofilm control strategies, LWT - Food Sci. Technol. 43, 573-583 25. Badave, G. K., and Kulkarni, D. (2015) Biofilm Producing Multidrug Resistant Acinetobacter baumannii: An Emerging Challenge, J. Clin. Diagn. Res. JCDR9, DC08-10 26. Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B. E., and Singh, P. K. (2011) Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria, Science 334, 982-986. 27. Lewis, K. (2007) Persister cells, dormancy and infectious disease, Nat. Rev. Microbiol. 5, 48-56. 28. Kim, H. S., Lee, S. H., Byun, Y., and Park, H. D. (2015) 6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition, Sci. Rep 5, 8656. 29. Choi, H., Ham, S. Y., Cha, E., Shin, Y., Kim, H. S., Bang, J. K., Son, S. H., Park, H. D., and Byun, Y. (2017) Structure-Activity Relationships of 6- and 8-Gingerol Analogs as Anti-Biofilm Agents, J. Med. Chem.DOI.10.1021/acs.jmedchem.7b01426. 30. Marini, E., Magi, G., Mingoia, M., Pugnaloni, A., and Facinelli, B. (2015) Antimicrobial and Anti-Virulence Activity of Capsaicin Against Erythromycin-Resistant, Cell-Invasive Group A Streptococci, Front. Microbiol. 6, 1281. ACS Paragon Plus Environment

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31. Ballok, A. E., and O'Toole, G. A. (2013) Pouring salt on a wound: Pseudomonas aeruginosa virulence factors alter Na+ and Cl- flux in the lung, J. Bacteriol. 195, 4013-4019. 32. Lau, G. W., Hassett, D. J., and Britigan, B. E. (2005) Modulation of lung epithelial functions by Pseudomonas aeruginosa, Trends Microbiol. 13, 389-397. 33. Kasper, S. H., Bonocora, R. P., Wade, J. T., Musah, R. A., and Cady, N. C. (2016) Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression, ACS Chem. Biol. 11, 1106-1117. 34. Das, T., Kutty, S. K., Tavallaie, R., Ibugo, A. I., Panchompoo, J., Sehar, S., Aldous, L., Yeung, A. W., Thomas, S. R., Kumar, N., Gooding, J. J., and Manefield, M. (2015) Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation, Sci. Rep.5, 8398. 35. Chang, C. Y., Krishnan, T., Wang, H., Chen, Y., Yin, W. F., Chong, Y. M., Tan, L. Y., Chong, T. M., and Chan, K. G. (2014) Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target, Sci. Rep. 4, 7245. 36. Wang, Y., Zhang, X., Wang, C., Fu, L., Yi, Y., and Zhang, Y. (2017) Identification and Quantification of Acylated Homoserine Lactones in Shewanella baltica, the Specific Spoilage Organism of Pseudosciaena crocea, by Ultrahigh-Performance Liquid Chromatography Coupled to Triple Quadrupole Mass Spectrometry, J. Agric. Food Chem. 65, 4804-4810. 37. Williams, P., and Camara, M. (2009) Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules, Curr. Opin. Microbiol12, 182-191. 38. Merritt, J. H., Kadouri, D. E., and O'Toole, G. A. (2005) Growing and analyzing static biofilms, Curr Protoc Microbiol.Chapter 1, Unit 1B 1 39. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. CLSI document M100-S16 CLSI, Wayne, PA (2006).

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Table 1. List of compounds identified by LC HRMS. Molecular family

Identified compound

In form

Calculated mass

Observed mass

Formula

∆ ppm

Phenazine

1-HP PYO PCA HHQ NHQ dbNHQ NONQ HQNQ db-UHQ Rha-Rha-(C10-C10) Rha-Rha-(C8-C10.) Rha-Rha-(C10-C10.1.) Rha-Rha-(C10-C12) Rha-Rha-(C10-C12.1) Rha-Rha-(C12-C12.1.) Rha-Rha-(C12-C12.) Rha-(C10-C10) Rha-(C8-C10), Rha-(C10-C10.1) Rha-(C10-C12) Rha-(C10-C12:1) 3-oxo-C12-HSL

H+ H+ H+ H+ H+ H+ H+ H+ H+ HHHHHHHHHHHHH+

197.0709 211.0866 225.0656 244.1696 272.2009 270.1852 288.1958 260.1645 298.2165 649.3805 621.3492 647.3638 677.4118 675.3961 703.4274 705.4431 503.3226 475.2913 501.3069 531.3539 529.3882 298.2013

197.0715 211.0868 225.0667 244.1695 272.2009 270.1853 288.1953 260.1638 298.2162 649.3802 621.3487 647.3639 677.4105 675.3952 703.4240 705.4402 503.3235 475.2907 501.3051 531.3534 529.3373 298.2017

C12H8N2O C13H10N2O C13H8N2O2 C16H21NO C18H25NO C18H23NO C18H25NO2 C16H21NO2 C20H27NO C32H56O13 C30H54O13 C30H56O13 C34H62O13 C34H60O13 C36H64O13 C36H66O13 C26H48O9 C24H44O9 C26H46O9 C28H52O9 C28H50O9 C16H27NO4

3.01 -1.12 -4.01 -0.06 0.00 -0.04 1.46 2.41 1.58 0.88 1.27 1.53 2.17 1.76 3.62 3.48 -1.26 1.5 1.5 1.19 2.13 -1.39

Quinolone

Rhamnolipid

Lactones

Abbreviation: 2-nonenyl-4-hydroxyquinolone (db-NHQ), 2-undecenyl-4-hydroxyquinolone (db-UHQ), 1hydroxyphenazine(1-HP),2-hepty-l-4-hydroxyquinolone (HHQ), 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), 2-nonyl-4hydroxyquinolone (NHQ), 2-nonyl-4-hydroxyquinolone N-oxide (NQNO), phenazine-1-carboxylic acid (PCA,), pyocyanin (PYO), 2-undecyl-4-hydroxyquinolone (UHQ), 5-methylphenazine-1-carboxylic acid (5-MPCA).

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Table 2. Identified compounds and their MS/MS

S. No.

m/z

Name

ESI mode

Fragments

1

211.086

positive

168.069, 183.093 and 196.094

2

197.071

PYO 1-HP

positive

169.076, 179.060 and 197.070

3

225.066

PCA

positive

4

244.169

HHQ

positive

5

260.163

HQNQ

positive

6

270.185

dbNHQ

positive

7

272.2

NHQ

positive

8

288.196

NONQ

positive

9

298.216

db-UHQ

positive

10

621.348

Rha-Rha-(C8C10.)

negative

11

647.363

Rha-Rha-(C10-C10.1.)

negative

12

475.29

Rha-(C8-C10)

negative

13

649.38

Rha-Rha-(C10-C10)

negative

14

675.395

Rha-Rha-(C10-C12.1)

negative

15

503.322

Rha-(C10-C10)

negative

16

677.411

Rha-Rha-(C10-C12.)

negative

17

529.337

Rha-(C10-C12:1)

negative

18

703.424

Rha-Rha-(C12-C12.1.)

negative

19

531.353

Rha-(C10-C12)

negative

20

705.44

Rha-Rha-(C12-C12.)

negative

179.059 and 207.053 159.069, 172.060, 186.091 and 200.106 159.068, 172.076,186.092, 200.107, 214.123,228.140 and 242.155 159.068, 172.075, 184.075, 198.091, 212.1056 and 226.104 159.067, 172.075,186.090, 200.105, 214.121 and 228.134 159.067, 172.076,186.092, 200.107, 214.119, 228.139 , 242.154 and 270.182 159.067, 172.076,186.092, 200.107, 214.119, 228.139 , 242.154 and 270.182 115.039, 141.091, 163.060, 169.121, 205.071, 247.082, 291.104, 309.122, 355.139, 451.2180 and 479.248 115.039, 141.091, 163.060, 169.121, 205.071, 247.082, 291.104, 309.122, 355.139, 451.2180 and 479.248 141.091, 169.121, 283.133, 357.051, 370.108 and 391.143 115.039, 163.060, 169.121, 205.071, 247.082, 291.104 and 479.248 115.039, 163.060, 169.121, 205.071, 247.082, 291.104, 309.122, 365.139, 394.335 and 479.248 163.060, 169.121, 303.192, 333.188 and 347.975 115.039, 163.060, 169.121, 205.071, 247.082, 291.104, 309.122, 367.284, 394.335 ,479.248, 507.278 and 658.398 169.122, 333.189, 352.164, 375.175, 402.296 and 469.210 115.039, 143.034, 163.060, 197.153, 205.071, 393.204 and 507.278 169.121, 197.153, 333.188, 361.219, 463.373, 490.211 and 516.779 115.039, 143.034, 163.060, 197.153, 205.071, 339.192, 507.278, 535.309 and 663.297

Abbreviations: 2-nonenyl-4-hydroxyquinolone (db-NHQ), 2-undecenyl-4-hydroxyquinolone (dbUHQ), 1-hydroxyphenazine (1-HP), 2-hepty-l-4-hydroxyquinolone (HHQ), 2-heptyl-4hydroxyquinolone-N-oxide (HQNO), 2-nonyl-4-hydroxyquinolone (NHQ), 2-nonyl-4hydroxyquinolone N-oxide (NQNO), phenazine-1-carboxylic acid (PCA,), pyocyanin (PYO), 2undecyl-4-hydroxyquinolone (UHQ), 5-methylphenazine-1-carboxylic acid (5-MPCA).

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Figure 1. LCESI +/-ve MS/MS-based LCMRM chromatogram. (a) Standard mixture. (b) Sample treated with 6-gingerol. (c) Sample treated with capsaicin 128 µg/ mL.

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Figure 2. Extracted ion chromatogram (EIC). (a) 3-oxo-C12-HSL. (b) Rhamnolipid, Rha-Rha-(C8C10); Rhamnolipid, Rha-Rha-(C10-C10); Rhamnolipid, Rha-Rha-(C10-C12.1); Rhamnolipid, Rha-Rha(C10-C12). (c) Pyocyanin (PYO), 1-hydroxyphenazine (1-HP), 2-undecenyl-4-hydroxyquinolone (dbNHQ) and 2-nonyl-4-hydroxyquinolone (NHQ). ACS Paragon Plus Environment

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Figure 3. Dynamics of production of signaling molecules in the presence of different concentrations of 6-gingerol. (a) Rhamnolipid, Rha-Rha-(C8-C10.). (b) Rhamnolipid, Rha-Rha-(C10-C10). (c) Rhamnolipid, Rha-Rha-(C10-C12). (d) Rhamnolipid, Rha-Rha-(C10-C12.1). (e) 1-hydroxyphenazine ACS Paragon Plus Environment

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(1-HP). (f) Pyocyanin, PYO. (g) 2-nonenyl-4-hydroxyquinolone (db-NHQ). (h) 2-nonyl-4hydroxyquinolone (NHQ). (i) 3-oxo-C12-HSL. BDL (below detection limit)

Figure 4. +ESI-MRM, peaks are assigned with numbers. (a) Control: 1 (Pyocyanin, tR= 4.3, 1× 103); 2 (1-hydroxyphenazine, tR= 17.5, 2.2 × 104); 3 (2-nonenyl-4-hydroxyquinolone, tR = 21.5, 5.0 × 103); 4 (2nonyl-4-hydroxyquinolone, tR = 22.6, 3.5 × 104). (b) Treatment with 6-gingerol (4 µg/ mL): 1 (Pyocyanin, below detection limit); 2 (1-hydroxyphenazine, tR= 17.5, 0.8 × 103); 3 (2-nonenyl-4hydroxyquinolone, tR = 21.5, 0.25× 103); 4 (2-nonyl-4-hydroxyquinolone, tR = 22.6, 4 × 103). (c) Treatment with capsaicin (32 µg/ mL): (Pyocyanin, below detection limit); 2 (1-hydroxyphenazine, tR= 17.5 ,3 × 103); 3 (2-nonenyl-4-hydroxyquinolone, tR = 21.5, 0.5 × 103); 4 (2-nonyl-4-hydroxyquinolone, tR = 22.6, 9 × 103).

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Graphical Abstract 81x60mm (300 x 300 DPI)

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