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Attenuation of quorum sensing-mediated virulence of Acinetobacter baumannii by Glycyrrhiza glabra flavonoids Nidhi Bhargava1, Sukhvinder P Singh2, Anupam Sharma3, Prince Sharma4 & Neena Capalash*,1

Aim: To develop an alternative quorum quenching therapy against multidrug-resistant Acinetobacter baumannii. Methods & results: Activity-guided partially purified fraction (F1) from Glycyrrhiza glabra significantly (p < 0.05) reduced quorum sensing regulated virulence factors of A. baumannii viz. motility, biofilm formation and production of antioxidant enzymes. Mechanistically, F1 downregulated the expression of autoinducer synthase gene, abaI, and consequently reduced (92%) the production of 3-OH-C12-HSL as determined by ESI–MS. Q-TOF and Q-TRAP analyses suggested the presence of flavonoids viz. licoricone, glycyrin and glyzarin as the active ingredients. Conclusion: This is the first report on quorum quenching activity of G. glabra linked to its flavonoids that downregulated the expression of abaI and attenuated quorum sensing regulated virulence of A. baumannii. Acinetobacter baumannii is an opportunistic pathogen affecting patients with compromised immune systems. It is associated with a wide spectrum of infections such as nosocomial pneumonia, skin and soft tissue infections, burn wound infections, urinary tract infections and meningitis [1,2] . Endocarditis is also caused by A. baumannii and although its occurrence is rare, according to a recent report, it is clinically important [3] . With the increasing incidence of antibiotic-resistant A. baumannii infections [4] and lack of new antibiotics in the development pipeline, treatment options are becoming limited. Hence, fresh perspectives and novel methodologies are needed to address the management of this emerging pathogen. A. baumannii exhibits N-acyl homoserine lactone (AHL)-mediated, cell density dependent quorum sensing (QS) and involves chromosomally encoded transcriptional activator (AbaR) that forms a complex with abal-generated signal, N-(3-hydroxydodecanoyl)-L-homoserine lactone (3-OH-C12HSL) [5] . QS in A. baumanni has been associated with biofilm formation [5] , twitching motility [6] and the production of catalase and superoxide dismutase (SOD) [7] , which are involved in its virulence. Attenuation of QS will result in the impairment of virulence determinants, without building selection pressure on the pathogen, thus limiting the possibility of resistant strains emerging [8] . Plants have been used in traditional medicine for thousands of years [9] as they exhibit novel bioactivities effective against bacterial infections [10] . The quorum quenching potential of phytochemicals such as flavanones, flavonoids [11] , polyphenols [12] , furacoumarins [13] and hydrolysable tannins  [14] has been reported to disrupt QS system in pathogens and help ameliorate infections. Glycyrrhiza glabra Linn. (family Papilionaceae), commonly known as licorice, is a traditional herb with ancient medicinal history in Ayurveda. It has been used to treat arthritis, mouth ulcers and liver Department of Biotechnology, Panjab University, Chandigarh 160014, India NSW Department of Primary Industries, Central Coast Primary Industries Centre, Locked Bag 26, Gosford NSW 2250, Australia 3 University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India 4 Department of Microbiology, Panjab University, Chandigarh 160014, India *Author for correspondence: Tel.: +91 0172 253 4085; Fax: +91 0172 254 1409

Keywords

• Acinetobacter baumannii • biofilm • catalase • flavonoids • Glycyrrhiza glabra • motility • quorum

sensing inhibition • superoxide dismutase

1 2

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ISSN 1746-0913

Research Article  Bhargava, Singh, Sharma, Sharma & Capalash detoxification  [15] . Anti-Helicobacter pylori and antibacterial activities of G. glabra flavonoids have also been reported [16,17] . This study aims to explore the uncharted quorum quenching potential in G. glabra for attenuation of QS-mediated virulence of A. baumannii. Methodology ●●Organisms & culture conditions

Cultures used in the study are listed in Table 1. The antimicrobial susceptibility profile of A. baumannii strains is provided in Supplementary Table 1, which was generated in accordance with Clinical and Laboratory Standards Institute guidelines (2012) [19] using disc diffusion method. Isolates resistant to at least three classes of antimicrobial agents were described as multidrug resistant [20] . ●●Bioassays for quorum quenching activity

Plate bioassay was performed [21] with slight modifications. Wells were bored into LB agar (0.75%), spread plated previously with Agrobacterium tumefaciens A136 (OD600 ∼1.0). X-gal (80 μg/ml), 3-OH-C12-HSL (5 μM in dimethyl formamide) and the methanol or aqueous plant extract (1 mg/ml) were added into the wells and incubated for 16 h at 30°C. Appearance of blue zone indicated quorum sensing while its absence indicated quorum

quenching activity. Curcumin (30 μg/ml) served as positive control [22] . For quantitation of quorum quenching activity, A. tumefaciens A136 was grown in LB containing 5 μM of 3-OH-C12-HSL in the presence or absence of plant extract (1 mg/ml) at 30°C for 16 h. β-galactosidase activity was determined according to Miller [23] using ortho-nitrophenylβ-galactoside (5 μg/ml) as the substrate and was defined as Miller units (MU). The percentage reduction in activity due to quorum quenching by plant extract was quantitated using the ­formula: ([MU control - MU test]/MU control) × 100. ●●Dietary & medicinal plants

Ten dietary plant materials having medicinal properties were screened for quorum quenching potential (Supplementary Table 2). The plant materials were purchased locally and identified at Herbarium-cum-Museum, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. ●●Preparation of extract

The dried powder of the plant material (30 g) was subjected to exhaustive extraction with methanol using soxhlet apparatus [21] . Water extract was prepared by soaking the dried plant material in water (1:10) at room temperature under

Table 1. Bacterial strains used in the study. Strain

Characteristics

Acinetobacter baumannii    M2 Clinical isolate M2 (abal::Km) Autoinducer synthase (abal) mutant of M2 strain; KanR M2(Pabal-lacZ) lac Z gene fused to the promoter of autoinducer synthase (abaI) C1-C4 Clinical isolates from endoscopic tracheal secretions maintained in laboratory ATCC 19606 Standard strain

Ref.

  LB, 37°C, 180 rpm LB + kanamycin (30 μg/ml), 37°C, 180 rpm LB, 37°C, 180 rpm

  [5] [5] [5]

LB, 37°C, 180 rpm

This study

LB, 37°C, 180 rpm

American type culture collection American type culture collection

ATCC 17978

Standard strain

LB, 37°C, 180 rpm

Agrobacterium tumefaciens A136

Biosensor strain for detection of broad range of AHLs harboring (i) pCF372 encoding traR expression; SpRand (ii) pCF218 encoding PtraI-lacZ reporter; tetR

LB + spectinomycin (50 μg/ ml) + tetracycline (4.5 μg/ ml), 30°C, 180 rpm  

 

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Growth conditions

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[18]

 

Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids  shaking conditions for 16 h followed by filtration through Whatman no. 1 filter paper [14] . The extracts were obtained by evaporating methanol and water using a rotary evaporator. The stock solution of the extract was prepared in DMSO and stored at room temperature as there was no detectable change in quorum quenching activity of the extract stored at room temperature in amber bottle even after 3 years. ●●Purification & identification of quorum

quenching compound(s) Fractionation of methanol extract

Methanol extract of G. glabra was subjected to refluxing for 30 min using solvents of increasing polarity (petroleum ether, chloroform and water) (Figure 1) . The water fraction was further partitioned with ethyl acetate. Fractions were dried using the rotary evaporator, their yields were calculated and quorum quenching activity of each fraction was quantitated. Silica gel chromatography

Fraction of G. glabra extract which showed the best quorum quenching activity was further purified on silica gel column (mesh 60–120, HiMedia). Silica slurry was prepared by mixing 50 g of silica with chloroform and packed in a glass column (200 × 1 cm). The extract (1.4 g), dissolved in chloroform was loaded on the column after passing it through a 0.4 μm filter and eluted with chloroform followed by methanol (1 to 8% in chloroform). Fractions (30 ml each) were collected and dried.

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Q-TOF MS

Subfraction eluted from the HPLC showing maximum quorum quenching activity was directly infused into a high resolution Q-TOF MS system (Triple TOF 5600, ABSciex India) with electrospray ionization in negative and positive modes. Identification of compounds was done by matching high resolution molecular masses of the compounds with the library of bioactive compounds (KNApSAcK database [54]) in G. glabra using peak view software. Matches with ppm error ≤15% and intensity >900 were selected for further analysis [24] . Q-TRAP MS/MS

MS/MS profile of predicted compounds present in the subfraction was generated on an ion trap mass spectrometer (QTRAP 5500 AB SCIEX) to obtain the fragmentation pattern of the compound(s). Peak View fragment analyzer software was used to match the fragmentation pattern of the predicted compounds present in the active fraction. ●●Effect of active fraction on A. baumannii

growth

Effect of different concentrations of active fraction, exhibiting quorum quenching activity, on growth of A. baumannii was determined by growing the cells in the presence and absence of active fraction at 37°C, 180 rpm. Optical density (OD600 nm) was monitored at different time intervals (2, 4, 6, 8, 10 and 24 h). ●●Effect of active fraction on

Preparative TLC

The active fraction was further resolved on preparative TLC (Silica GF254, Merck, 20 ×20 cm) with ethyl acetate and toluene (4.5:5.5). Spots were visualized with H 2 SO 4 -anisaldehyde, scrapped and eluted with ethyl acetate. Quorum quenching activity in each spot was quantitated by liquid bioassay against 5μM 3-OH-C12-HSL using A. tumefaciens A136 as the biosensor strain. HPLC

Twenty microliter of the fraction (100 ppm) was fractionated on C-18 column (250 × 4.6 mm; particle size 5 μm; Waters). Water-acetonitrile (35:65) was used as the mobile phase with 1 ml/ min flow rate for 0–14 min followed by linear gradient (10:90) for 14–23 min. The fractions were collected based on absorbance ranging from 190 to 800 nm.

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quorum sensing mediated virulence factors Motility

LB agar (0.3%) supplemented with active fraction (0.5 mg/ml) was used for surface motility. A. baumannii cells were grown to early stationary phase (OD600 ∼1.0) and washed with PBS. One microliter of the cell suspension was inoculated in the center of the motility plate and incubated at 37°C [25] , followed by measurement of surface growth zone. For twitching motility, an overnight grown colony was stabbed through LB agar (1%) to the bottom of the petri dish with a sterile toothpick [26] . Plates were incubated overnight at 37°C. LB agar was removed and the plate was stained with 1% crystal violet for 1 min followed by washing with sterile distilled water. The diameter of twitching zone was measured.

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash

G. glabra rhizome powder Methanol extract Partitioning Petroleum Chloroform Water ether extract extract extract (Yield: 18.8%, (Yield: 3.3%, (Yield: 15 ± 1.7%, QQ activity: 5 ± 0.3%) QQ activity: 36 ± 4.2%) QQ activity: 40.2 ± 1.7%)

Ethyl acetate extract (Yield: 21.7%, QQ activity: 56 ± 6.1%)

Residue (Yield: 7.0%, QQ activity: 10 ± 0.8%)

Silica gel column chromatography

Elution with chloroform and methanol (1–8% in chloroform) + bioactivity determined with A. tumefaciens A136

Motility

Biofilm Virulence assays

F1 (Active fraction) (eluted in chloroform)

Catalase Preparative TLC Superoxide dismutase Spots

S1

S2

S3 (QQ activity↑) HPLC

HPLC fractions

S3a

S3b

S3c

S3d

S3e (QQ activity↑)

Q-TOF and Q-TRAP

Flavonoids (Licoricone, glycyrin, gylzyrin)

Figure 1. Schematic representation of bioactivity-guided purification and identification of quorum quenching compounds from Glycyrrhiza glabra rhizome.

Biofilm formation

Biofilm was grown in LB (200 μl) containing glycerol (1%) in polypropylene microcentrifuge tubes in the presence and absence of active

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fraction (0.5, 1.0, 2.0 mg/ml) at 37°C under stationary conditions. Glycerol was added to medium to enhance the biofilm formation [27] . Planktonic growth in the medium was measured

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Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids  at 600 nm before draining it off. Biofilm was washed three times with sterile distilled water to remove loosely adhered bacterial cells and stained with 200 μl of 0.5% crystal violet for 30 min followed by three washings with distilled water to remove excess stain. The bound stain was extracted with 200 μl of 33% glacial acetic and quantitated at 570 nm. Biofilm formed was expressed as biofilm index (Ab570 /OD600 ) [28] . For confocal laser scanning microscopy, biofilm was formed on a coverslip by incubating A. baumannii in the presence of active fraction (2.0 mg/ml) under conditions as described above. Biofilm was stained with 10 μl of live (Syto 9) and dead (propidium iodide) stain for 30 min in dark. Coverslip was given three washings with phosphate buffer (50 mM, pH 7.0), air dried and observed under 100× oil immersion. Syto 9 (green) signal was recorded at 517 nm (emission) after excitation at 488 nm, while propidium iodide (red) signal was captured at 636 nm (emission) after excitation at 493 nm. Three independent biofilm images were analyzed for their thickness by taking Z stack at 0.5 μm distance and bacterial death using Neiss viewer image analysis software [29] . Biofilm formed in the absence of active fraction served as control. Antioxidant enzymes

Effect of active fraction on the expression of N-acyl homoserine lactones in A. baumannii ●● A. baumannii was grown in the presence (0.5

mg/ml) and absence of active fraction. AHLs were extracted from cell free supernatant as described earlier and quantitated. ●● A. baumannii M2 (PabaI-LacZ) was grown for

16 h at 37°C in LB containing 5 μM 3-OHC12-HSL in the presence and absence of active fraction at various concentrations (0, 0.1, 0.5, 1.0 and 2.0 mg/ml) and β-galactosidase activity was determined according to Miller (1972) [23] .

●● 3-OH-C12-HSL was extracted from the cell

free supernatant of A. baumannii grown in the presence (0.5 mg/ml) and absence of active fraction with equal volumes of acidified (0.01% v/v, acetic acid) ethyl acetate [32] . The extract was dried using a rotary evaporator and resuspended in methanol (500 μl) containing 0.1% (v/v) formic acid followed by ESI–MS analysis [33] . Signal peaks were analyzed using MassLynx 4.22 software (Waters) and comparison of peaks corresponding to 3-OHC12-HSL (m/z: 301.3 [M+H]) was done. Restoration of virulence factors on exogenous addition of 3-OH-C12-HSL in the presence of active fraction (F1)

Overnight grown culture of A. baumannii was diluted (1:100) with 10 ml LB containing active fraction (0.5 mg/ml) and incubated at 37°C, 180 rpm for 16 h. Culture without active fraction was used as control. The cells were centrifuged, washed and sonicated in PBS. Catalase and SOD activities were expressed as units per mg of protein. One unit of catalase activity was the amount of enzyme that degraded 1mM H2O2 per min at room temperature [30] and one unit of SOD activity was the amount of enzyme that resulted in 50% inhibition of NBT [31] .

Cells were grown in the presence of active fraction alone and in combination with 5 μM 3-OH-C12-HSL. Surface motility, twitching, biofilm formation, catalase and SOD activities were performed as described earlier.

●●Mechanism of quorum quenching

●●Screening of dietary medicinal plants for

Effect of active fraction on quorum sensing signal molecule

3-OH-C12-HSL (5 μM) was incubated with active fraction (0.5 mg/ml) for 16 h at 37°C. AHLs were extracted from the reaction mixture with acidified ethyl acetate [32] and quorum sensing activity was quantitated by ortho-nitrophenylβ-galactoside assay with A. tumefaciens A136 as biosensor.

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Statistical analysis Data are presented as mean ± SD, and all experiments were repeated three times in at least three biological replicates. Student’s t-test was used to determine the significance and p ≤ 0.05 was ­considered statistically significant. Results quorum quenching activity

Aqueous extracts of Eugenia caryophyllus, Coriandrum sativum, Foeniculum vulgare, Amomum subulatum, Cinnamomum zeylanicum and methanol extract of Origanum vulgare showed quorum quenching activity (QQ) in plate bioassay with A. tumefaciens A136 (Supplementary Table 2) . Glycyrrhiza glabra showed QQ activity both in aqueous and

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash methanol extracts (Supplementary Figure 1) . Methanol extract showed activity even at lower concentration (0.5 mg/ml). Hence methanol extract of G. glabra was selected for further work. Trigonella foenum-graceum, Allium cepa and Capsicum annuum did not show QQ activity.

quorum quenching activity ranging from 50 to 70%. F1 fraction (elution volume 300–330 ml) showed maximum quorum quenching activity (70 ± 4.7%) against 3-OH-C12-HSL. Fraction F1 was further resolved into three spots with Rf values 0.56, 0.7 and 0.86 on preparative TLC. Quorum quenching activity was observed in all but maximum activity (90%) was in spot S3 which was 1.64-times higher than that of ethyl acetate extract (Supplementary Figure 2A &  B) . Compounds present in spot (S3) were further fractionated into five subfractions (S3a–e) on HPLC. Fraction (S3e) showed maximum QQ activity (54.8 ± 3.6%) whereas no activity was observed in fraction S3c. S3a, S3b and S3d showed 5 ± 0.3, 12 ± 0.92 and 52 ± 4.2% reduction in quorum sensing activity respectively with A. tumefaciens A136 at 50μg/ml against 5 μM of 3-OH-C12-HSL (Supplementary Figure 3) . Fraction S3e was further analyzed by a high resolution Q-TOF mass spectrometer. Sixty and 90 peaks were obtained in the negative and positive modes of ionization, respectively, corresponding to molecular masses ranging

●●Bioactivity-guided purification &

identification of quorum quenching compounds (Figure 1)

Methanol extract was partitioned with petroleum ether, chloroform and water and their quorum quenching activity was checked at 0.5 mg/ml concentration by liquid bioassay against 5 μM of 3-OH-C12-HSL using A. tumefaciens A136 biosensor. The water extract showed maximum quorum quenching activity (40.2 ± 1.7%) where 15 MU of β-galactosidase activity was taken as equivalent to 100% QS activity. Partitioning of water extract with ethyl acetate resulted in ethyl acetate fraction that showed higher quorum quenching activity (56 ± 6.1%) and was further fractionated on silica-gel column. Seven out of 11 pooled fractions showed

Table 2. MS/MS profile of putative compounds present in S3e HPLC fraction exhibiting quorum quenching activity. Extraction MS/MS Collision energy (eV), Compound Structure mass†  fragments‡  ppm error†, intensity†  name (empirical formula) 381.13436 [M-H]

169, 125.1, 191.1

-38, 12.8%, 1110

Glycyrin (C22H22O6)

Phytochemical sub-class

Hydroxyisoflavone 87.8

CH3

O

O

H 3C

matches‡ (% Intensity)

O

O CH3

HO

OH

CH3

 

 

 

Licoricone (C22H22O6)

Hydroxyisoflavone 92.1

O

HO

OH

O O

O

CH3

CH3

H3C

295.09649 162.1, 111, 40, 2.2% 910 [M+H] 109, 161, 121, 123, 165.1, 151.1, 125

Glyzarin (C18H14O4)

Isoflavonoid

O

HO

CH3

81.2

O

O

Peak viewer software analyzed data, generated using †Q-TOF/MS and ‡Q-TRAP/MS/MS.

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Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids  from 112 to 419. The high resolution molecular weights of these 150 peaks were matched with the library of 250 bioactive compounds from G. glabra (Knapsack database). Two peaks corresponding to extraction masses of 381.13436 and 295.09649 with ppm error ≤15% and abundance >900 were selected as the most appropriate matches (Table 2) . Structure identification of these compounds was done by MS–MS fragmentation, performed by an ion trap mass spectrometer (Supplementary Figure 8 & 9) . Fragment matching suggested the presence of licoricone (92.1%), glycyrin (87.8%) and glyzarin (81.2%) as the most probable flavonoids that could be responsible for quorum quenching activity of G. glabra against A. baumannii. Photochemical analysis of active fraction F1 was done which showed presence of flavonoids only (Supplementary Table 3) . Due to low yield of HPLC purified fraction S3e, the active fraction F1 was used for all further work on quorum quenching of virulence factors and determining mechanism of action. ●●Effect of active fraction on Acinetobacter

baumannii Growth

The growth profile of A. baumannii ATCC 17978 in the presence of different concentrations (0.25, 0.5 and 2.0 mg/ml) of active fraction F1 clearly showed that there was no effect of the active compounds on growth. Hence, virulence assays were performed within these ­concentrations (Supplementary Figure 4) . Motility

A. nosocomialis M2 and A. baumannii strains (clinical C1–4 and ATCC 17978 and 19606T) showed surface motility in the range of 0.1–2.2 cm and twitching spread was 0.1–0.9 cm (Figure 2 & Supplementary Figure 5) . Treatment with 0.5 mg/ml of F1 led to reduction (20–70%) in surface motility which was significant (p ≤ 0.05) for all the clinical strains. There was reduction in twitching by 20–66% that was significant (p ≤ 0.05) in all the strains. Due to the absence of quorum sensing in A. nosocomialis M2 (abal::Km), no reduction in surface and twitching motility was observed on treatment with F1. Biofilm formation

Biofilm index (BI) for A. baumannii isolates ranged from 0.2 to 1.36. Treatment with F1 decreased the BI in a concentration-dependent manner. Significant reduction in BI was observed

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on treatment with 2.0 mg/ml of F1 which ranged from 30 to 70% (Figure 3) . QS-mutant M2 (abal::Km), impaired in biofilm formation, did not show significant change in BI on treatment with F1. Confocal laser scanning microscopy showed that A. nosocomialis M2 formed 16 ± 0.2 μm thick, mat-like biofilm on glass coverslip while fragmented aggregates of cells were observed with 5 ± 0.7 μm thickness in the presence of 2.0 mg/ml of F1 (Figure 4) . The percentage of dead cells in treated and untreated biofilm did not show significant difference (p = 0.08) with live and dead staining using Syto9 and PI. Antioxidant enzymes

Catalase and SOD activities of Acinetobacter strains ranged from 2 to 25 U and 2 to 14 U per mg protein, respectively. Significant (p < 0.05) decrease in catalase (29.3–85.5%) and SOD activities (29.4–83.6%) in clinical and reference strains was observed (Figure 5A & B) on treatment with 0.5 mg/ml of F1. QS mutant of M2 (abaI::Km) showed three-fold (p ≤ 0.01) and 2.6-fold (p ≤ 0.05) lower levels of catalase and SOD activities respectively as compared with wild-type M2 and there was no change in these activities in the presence of F1 due to the absence of QS in this strain. ●●Mechanism of quorum quenching activity

of F1 against Acinetobacter baumannii

The pH of G. glabra active fraction was 7.0, hence there was no nonspecific quorum quenching activity due to inactivation of lactone molecule by lactonolysis at alkaline pH. Approximately 88% activity of 5 μM of 3-OH-C12-HSL was retained after incubation with 0.5 mg/ml of active fraction for 16 h. The reduction in activity could be due to loss of 3-OH-C12-HSL during extraction with ethyl acetate and not due to inactivation of F1. A. nosocomialis M2 and A. baumannii (clinical and reference) strains produced AHLs which ranged from 90 to 120 μM. Treatment with 0.5 mg/ml of F1 led to significant (p < 0.05) decrease (56–86%) in AHLs production (Figure 6) . The effect of F1 on AHL production by A. nosocomialis M2 was also studied by ESI-MS, which showed maximum reduction of AHL production in the presence of F1. There was 92.02% reduction in relative intensity of peak corresponding to 3-OH-C12 HSL (m/z 301.3 [M+H]), the QS signal of Acinetobacter. Peak with m/z 102.2 corresponded to the lactone ring structure

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash

2.5

Absence Presence of fraction F1 of Glycyrrhiza glabra (0.5 mg/ml)

Surface motility (cm)

2.0

1.5

1.0

*

*

0.5

*

* * 0.0

C1

C2

C3

C4

#M2

#M2 (abal::Km)

19606

17978

A. baumannii strains 1.4

Twiching motility (cm)

1.2 1.0

* *

0.8 0.6 0.4

*

*

0.2 * 0.0

C1

C2

C3

C4

#M2

#M2 (abal::Km)

*

*

19606

17978

A. baumannii strains

Figure 2. (A) Surface and (B) twitching motilities of A. baumannii in the absence and presence of fraction F1 of Glycyrrhiza glabra (0.5 mg/ml). *p ≤ 0.05. # A. nosocomialis.

of AHL molecules where as other peaks probably are ethyl acetate soluble cellular metabolites (Figure 7A) and plant metabolites (Figure 7B) present in F1. Decrease in surface motility, twitching, biofilm formation, catalase and SOD activities observed on treatment of A. baumannii ATCC 17978 with active fraction F1 was restored on addition of exogenous 3-OH-C12-HSL (5 μM) (Supplementary Figure 6 & 7) . These results

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supported the effect of F1 on AHL as the ­mechanism of quorum quenching. A concentration-dependent reduction in the expression of β-galactosidase regulated by the promoter of lactone synthase gene (abaI ) was observed when A. nosocomialis M2 (PabaI-lacZ ) was treated with F1 in the presence of inducer 3-OH-C12-HSL. In the presence of 2.0 mg/ ml F1, 94.37% reduction (p ≤ 0.001) in β-galactosidase activity was observed (Figure 8) .

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Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids 

Research Article

1.6 1.4

*

Biofilm index (OD570/OD600)

1.2 1.0 0.8 0.6

*

*

$

* $

¥ ¥

$

$

$

*

*

$

$ * $

¥

*

0.4

* $

0.2 0.0

C1

C2

C3

#M2 (abal::Km) A. baumannii strains C4

#M2

Figure 3. Effect of fraction F1 (0.0 - , 0.5 - ,1.0 - and 2.0 A. baumannii. *p < 0.05, $ p < 0.01 and ¥ p < 0.001. #A. nosocomialis.

19606

17978

mg/ml) on biofilm formation by

Figure 4. CLSM images of live and dead stained biofilm formed by A. nosocomialis M2 on glass coverslip in the absence (A) and presence (B) of fraction F1 (2.0 mg/ml) with quorum quenching activity. Lower panel (C & D) shows intensity graphs of green (Syto 9) and red (propidium iodide) fluorescence quantitated along the line indicated by blue arrow of panel (A and B) using NIS element viewer software. Magnification 100×. CLSM: Confocal laser scanning microscopy

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash

40

Absence Presence of 0.5 mg/ml of fraction F1

Catalase U/mg protein

35 30 25 ** 20 15 10 5 * 0

C1

C2

**

*

*

** C4

C3

* #M2

A. baumannii strains

#M2 (abal::Km)

19606

17978

25

SOD U/mg protein

20

15 *

* 10

*

** *

*

*

5 ** 0

C1

C2

C3

C4

#M2

A. baumannii strains

#M2 (abal::Km)

19606

17978

Figure 5. Catalase (A) and SOD (B) activities of A. baumannii strains grown in the absence and presence of 0.5 mg/ml of fraction F1. *p ≤ 0.05. #A. nosocomialis.

Discussion A. baumannii has emerged as a global nosocomial pathogen that, through QS, regulates its virulence factors like biofilm formation [5] , motility [6] , catalase and SOD [7] and conserves resources by limiting wasteful expression of genes. Attenuation of virulence genes through inhibition of QS with phytocompounds has led to the successful control of bacterial diseases [34] .

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In a similar attempt, ten dietary medicinal plants were screened for quorum quenching activity against A. baumannii. Glycyrrhiza glabra, which showed this activity in both aqueous and methanol extracts, was selected for the study. Quorum quenching activity has been reported in toluene extract of Allium sativum  [35] and methanol extract of Terminalia chebula  [14] against Pseudomonas aeruginosa. Ethanol

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Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids  extracts of Mangifera indica and Puncia granatum showed quorum quenching activity against Chromobacterium violaceum CV12472 [36] . To the best of our knowledge, this is the first report on quorum quenching by G. glabra, although its anticancer [37] , antioxidant, anti-aflatoxigenic  [38] and antibacterial [39] ­activities have previously been reported. Phytochemical analysis of active fraction F1 of G. glabra extract showed the presence of flavonoids only, which on further purification and characterization based on computational analysis of high resolution MS/MS analysis (Q-TOF and Q-TR AP) revealed licoricone, glycyrin and glyzarin as the identifiable flavonoids. A similar computational approach was adopted by Zhou et al.  [24] for the prediction of bioactive compounds in Lotus nelumbo, while Sarabhai et al.  [14] confirmed the identity of active compounds in methanol extract of Terminalia chebula on the basis of database matches of MS/MS fragmentation patterns. Bioactivity in flavonoids derived from orange peels has been reported to attenuate QS and related physiological processes in Yersinia enterocolitica [11] . Flavonoid rich fraction from Centella asialica (L.) showed antiquorum sensing activity against P. aeruginosa PAO1 [40] , underlying

the importance of flavonoids in the control of pathogens. Biofilm and motility in A. baumannii was significantly decreased by flavonoid-rich active fraction F1 of G. glabra. Involvement of motility in different stages of biofilm formation (initial adhesion, microcolony formation and maturation) has been demonstrated in A. baumannii [41] and other pathogens [42,43] . Dead and live staining of biofilm showed that active fraction F1 had no toxic effect on cells and reduction in biofilm was not due to inhibition of growth. No change in biofilm formation, surface and twitching motility of M2 (abaI::Km), a QS mutant of A. nosocomialis M2, showed specificity of F1. Catalase and superoxide dismutase, which are under the control of QS system in A. baumannii [7] , were also significantly reduced in response to treatment with active fraction F1. Catalase and SOD play an important role in defence against reactive oxygen species produced by host during infections  [44] . In A. baumannii ATCC 17978, inactivation of SOD led to complete loss of virulence in Galleria mellonella caterpillar infection model besides showing defects in motility, decreased resistance to oxidative stress and susceptibility to antibiotics [45] . Low levels of antioxidant enzymes in A. baumannii have been shown to

150

AHL concentration (µm)

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Absence Presence of fraction F1 (0.5 mg/ml)

100

50

*

*

*

* **

** 0

C1

C2

C3

C4

#M2

*

19606

17978

A. baumannii strains

Figure 6. AHL production by A. baumannii grown in the absence and presence of fraction F1 (0.5 mg/ml). *p ≤ 0.05, ** p ≤ 0.001. # A. nosocomialis.

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash

Figure 7. ESI-MS of ethyl acetate extract of cell free supernatant of A. nosocomialis M2 grown in the (A) absence and (B) presence (0.5 mg/ml) of G. glabra active fraction F1. 3-OH-C12-HSL of A. baumannii (M+H; m/z 301.3).

affect its survival against pyocyanin-mediated oxidative stress to which it was exposed in co-culture conditions with P. aeruginosa [7] . Interfering with the ability of the pathogen to withstand oxidative stress with quorum quencher could make them more susceptible to disinfection agents (H2O2 or betadine) and oxidative stress in the host.

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QS circuit in A. baumannii involves the activation of regulator protein (AbaR) on forming a complex with autoinducer 3-OH-C12-HSL which regulates the transcription of various genes including the genes responsible for virulence [5] . Quorum quenching can occur through different mechanisms, the most common being:

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Attenuation of quorum sensing-mediated virulence of A. baumannii by G. glabra flavonoids  signal (AHLs) mimicry as is in case of furanones  [46] ; degradation of quorum sensing signals by lactonase and acylase [47] ; mutations in signal binding site of the receptor protein [48] and inhibition of signal expression [21] , as observed with BuT-DADMe-ImmA [49] and triclosan [50] . Quorum quenching activity of naringenin, a flavone from citrus fruits, was reported to be due to both reduction in production of lactone molecules and defective functioning of the complex between RhlR receptor and C4-HSL in P. aeruginosa [51] . It is proposed that flavonoid-rich active fraction from G. glabra caused quorum quenching in A. baumannii by repressing autoinducer synthase expression leading to significant decrease in 3-OH-C12-HSL production. Inhibition of AHL production would affect AbaR-AHL complex formation which might be responsible for significant reduction in surface motility, twitching, biofilm formation, catalase and SOD activities. Insignificant change in expression of other proteins like urease (data not shown) ruled out generalized suppression of transcription by F1 in A. baumannii. Further, restoration of suppressed phenotypes on addition of exogenous 3-OH-C12-HSL (5 μM) supported the ­proposed hypothesis. The bioactive fraction F1 constituted minimum 0.03% of G. glaba powder and 5% of the

Research Article

crude extract (data not shown). Therefore, to achieve 2 mg of active fraction F1, only 40 mg of the crude extract would be required for in vivo studies. Moreover, pure compounds may be required in less amounts as compared with the crude extracts as Jakobsen et al. [52] showed bioactivity in Ajoene, a sulphur-rich molecule purified from garlic, at 25 mg/kg which was less than the amount (15 g/kg) of crude extract used in the infection model [35] . Conclusion In the post-antibiotic era, alternative therapeutic strategies are needed for multidrug resistant pathogens. Targeting QS systems is an attractive alternative tried in this study. The flavonoid-rich quorum quenching fraction of G. glabra (rhizome) led to a significant reduction in QS-mediated virulence of A. baumanni and acted by downregulating the expression of autoinducer synthase, abaI which reduced the levels of 3-OH-C12-HSL. Licoricone, glycyrin and glyzarin were identified as the flavonoids responsible for quorum quenching. Future perspective It is imperative to develop alternate strategies to combat nosocomial infections caused by MDR pathogens. A. baumannii is an emerging nosocomial pathogen, which regulates the expression

β-galactosidase activity (Miller units)

2000

1500

* 1000 **

500 ** ** 0

0.0

0.1 0.5 1.0 F1 fraction of G. glabra (mg/ml)

2.0

Figure 8. Effect of G. glabra active fraction (F1) on the expression of AbaI in A. nosocomialis M2 (PabaI-lacZ). Values are plotted as mean ± SD for each set. *p ≤ 0.01, **p ≤ 0.001.

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Research Article  Bhargava, Singh, Sharma, Sharma & Capalash of its virulence genes by AHL-mediated QS system. Interruption of QS is an antipathogenic approach the does not challenge the bacterial existence and could be a potential drug target that may render bacteria nonpathogenic without generating selection pressures on them. This study aimed to decipher the quorum quenching potential of G. glabra, which is an easily available dietary plant. Dietary phytochemicals with quorum quenching property, alone or in combination with antibiotics, may be effective to control the infections caused by A. baumannii. In addition, they may be effective against other Gram-negative pathogens like P. aeruginosa, which also exhibit N-acyl homoserine lactone based QS systems for the expression of virulence factors. This assumes significance in the backdrop of overlapping sites of infection for A. baumannii and P. aeruginosa with reports of mixed species infections by them [53] . Further studies are warranted to determine the quorum

quenching activity by pure compounds from G. glabra to realize their actual therapeutic potential. Acknowledgements The authors are grateful to PN Rather for providing Acinetobacter baumannii strains used in the study. N Bhargava acknowledges the Council of Scientific and Industrial Research, New Delhi, India for senior research fellowship.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or ­pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary ●●

ethanol extract of Glycyrrhiza glabra Linn. was successively partitioned by extensive refluxing with solvents of M different polarity namely petroleum ether, chloroform, ethyl acetate and water. Ethyl acetate fraction showing maximum (56.1 ± 6.1%) quorum quenching activity was further purified by fractionation on silica column (mesh size 60–120). Bioactive fraction F1 showed maximum quorum quenching activity (70 ± 4.7%) and was found to be rich in flavonoids.

●●

F urther purification of bioactive fraction (F1) by preparative TLC, HPLC, Q-TOF and Q-TRAP identified licoricone, glycyrin and glyzarin as the quorum quenching flavonoids.

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uorum quenching activity of G. glabra extract acted through the downregulation of autoinducer synthase gene Q (abaI) leading to decreased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone.

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Growth of Acinetobacter baumannii in the presence of active fraction F1 (0.25, 0.5 and 2.0 mg/ml) was not affected. Further live and dead cells staining using confocal laser scanning microscopy confirmed that active fraction effectively decreased biofilm formation in A. baumannii without affecting the viability of biofilm cells.

●●

ioactive fraction (F1) significantly reduced the quorum sensing mediated production of virulence factors like motility, B biofilm formation, catalase and superoxide dismutase.

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