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Food Control 42 (2014) 125e131

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Food Control

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Polyphenolic extract from Rosa rugosa tea inhibits bacterial quorumsensing and biofilm formation

Juanmei Zhang a,b, Xin Rui a, Li Wang a, Ying Guan a, Xingmin Sun c, Mingsheng Dong a,*

aCollege of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095, Jiangsu, People’s Republic of Chinab Shangqiu Vocational and Technical College, 566 Shenhuo Road, Shangqiu 476000, Henan, People’s Republic of ChinacDepartment of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, MA 01536, USA

a r t i c l e i n f o

Article history:Received 4 November 2013Received in revised form29 January 2014Accepted 1 February 2014Available online 11 February 2014

Keywords:Rosa rugosaPolyphenolFlavonoidBiofilmAnti-quorum sensingPseudomonas aeruginosa

Abbreviations: QS, quorum sensing; RTP, Rosa rugacyl-homoserine lactones; HSL, homoserine lactoneconcentration; CLSM, confocal laser scanning microsc* Corresponding author. Tel./fax: þ86 25 84399090

E-mail address: dongms@njau.edu.cn (M. Dong).

http://dx.doi.org/10.1016/j.foodcont.2014.02.0010956-7135/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Quorum sensing (QS) is an intercellular signaling and gene regulatory mechanism, which is implicated inbacterial pathogenicity and food spoilage. Therefore, blocking bacterial QS system may prevent QS-controlled phenotypes responsible for food spoilage. In the present study, we aimed to investigate theanti-biofilm and quorum sensing inhibitory potentials of Rosa rugosa tea polyphenol (RTP) extract, whichis rich in polyphenols (87.52%) and flavonoids (61.03%). The RTP specifically inhibited QS-controlledviolacein production in Chromobacterium violaceum 026 with 87.56% reduction without significantlyaffecting its growth. Moreover, RTP exhibited inhibition in swarming motility (84.90% and 78.03%) andbiofilm formation (67.02% and 72.90%) of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in aconcentration-dependent manner, respectively. These findings strongly suggest that RTP potentiallycould be developed as a new QS inhibitor and/or anti-biofilm agent to enhance the shelf life and increasefood safety.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Quorum Sensing (QS) is an intercellular signaling system thatallows bacteria to monitor their population density and to control avariety of physiological processes, by releasing and receiving smallsignal molecules called autoinducers (Brackman, Hillaert, Van-Calebergh, Nelis, & Coenye, 2009). In Gram-negative bacteria themost common autoinducers are N-acyl-homoserine lactones(AHLs) whereas Gram-positive bacteria usually utilize peptides asautoinducers (Kalia, 2012). Various food spoilage and pathogenicorganisms such as Escherichia coli O157: H7, Salmonella enteric andPseudomonas aeruginosa employed QS systems to regulate theirphenotypes, including pigmentation, swarming motility, biofilmformation, secretion of virulence factors and production of degra-dative enzymes (Høiby, Bjarnsholt, Givskov, Molin, & Ciofu, 2010;Kalia, 2012; Kim, Oh, Park, Seo, & Kim, 2008; Wang et al., 2013).

The concept of quorum sensing has encouraged us to engage inthe development of a novel food preservation strategy using QS

osa tea polyphenol; AHL, N-; MIC, minimum inhibitoryopy..

inhibitor (Choo, Rukayadi, & Hwang, 2006; Packiavathy, Priya,Pandian, & Ravi, 2012; Sybiya Vasantha Packiavathy,Agilandeswari, Musthafa, Karutha Pandian, & Veera Ravi, 2012;Zeng et al., 2008). The ideal QS inhibitors have been defined aschemically stable and highly effective low-molecular-mass mole-cules, which exhibit a high degree of specificity for the QS-controlled phenotypes without toxic side effects (Rasmussen &Givskov, 2006). In recent years, obtaining non-toxic anti-QS activesubstances from natural plant resources takes much attention ofresearchers. Some studies have identified QS inhibitors in a varietyof medicinal and dietary plants, including garlic, vanilla (Choo et al.,2006; Vattem, Mihalik, Crixell, & McLean, 2007), Medicago sativa(Gao, Teplitski, Robinson, & Bauer, 2003), Scorzonera sandrasica(Adonizio, Downum, Bennett, & Mathee, 2006), pea seedlings(Puupponen-Pimia et al., 2005), pomegranate extract (Truchado,Tomás-Barberán, Larrosa, & Allende, 2012) and mushroom suchas Auricularia auricula (Li, Li, Chen, Jiang, & Dong, 2012).

In a previous study, we found for the first time that R. rugosa teaextract exhibited maximum anti-QS activity among 12 Chineseflower teas and 19 spices tested, suggesting R. rugosa tea as a po-tential QS inhibitors (Wang & Dong, 2010). However, no furtherstudy has been done which compounds were responsible for anti-QS activity of R. rugosa tea. It is reported that R. rugosa tea containsabundant polyphenol compounds such as gallic acid, epicatechin,

J. Zhang et al. / Food Control 42 (2014) 125e131126

anthocyanin, kaempferol and quercetin, which are associated withantioxidant and antiaging activities (Baydar & Baydar, 2013; Ding &Peng, 2008; Taganna & Rivera, 2008; Yu et al., 2006). More recently,polyphenol has been reported to inhibit QS-controlled phenotypesat sub-inhibitory concentrations (Truchado et al., 2012). Therefore,in the present study we aimed to investigate the anti-quorumsensing and anti-biofilm potentials of R. rugosa tea polyphenol(RTP) extract against Chromobacterium violaceum 026, E. coli K-12and Pseudomonas aeruginosa POA1 at sub-inhibitory concentration.

2. Material and methods

2.1. Bacterial strains and culture conditions

C. violaceum 026 (a double mini-Tn5 mutant of the wild-typestrain C. violaceum (ATCC 31532) with a kanamycin resistancethat was unable to synthesize its own C6-HSL, but it retains theability to respond C4-AHL or C6-AHL and produces violacein) waskindly provided by Professor Robert J. C. (Texas State University,USA)whichwas used as biomonitor strain. It was routinely culturedaerobically in LuriaeBertani (LB) medium (1% tryptone, 0.5% yeastextract, 0.5% NaCl) with 120 rpm agitation in a shaking incubator at28 �C. The medium for C. violaceum 026 was supplemented with20 mg/ml kanamycin.

The test organism P. aeruginosa PAO1 was purchased fromFisheries Research Institute, Shanghai, China. E. coli K-12 was sup-plied by Nanjing Center for Disease Control and Prevention, Jiangsuprovince, China. P. aeruginosa PAO1 and E. coli K-12 were used todetermine biofilm formation and cultivated in LB medium at 37 �C.

2.2. Preparation of RTP extract and active components assay

RTP was prepared from R. rugosa tea. The 10 g dried R. rugosabuds were purchased from the local supermarket and extractedwith 200 ml of deionized water at room temperature for 30 min byultrasonic treatment. The samples were filtered and then centri-fuged for 10 min, 12,000 rpm. The attained supernatant was addedto the glass Chromatogram column (17 � 300 mm) with activatedmacroporous resin HPD600 (Qinshi Technology, Ltd., Zhengzhou,China) and eluted by 60% ethanol with the flow rate of 1 min/ml.Eluted fraction was removed solvent by a rotary evaporator and re-dissolved with an appropriate amount of deionized water, then,lyophilized (At 108 � 5 Pa chamber pressure and �83 � 1 �C coldcollector temperature after pre-freezed at �20 �C for 24 h) andstored at �20 �C for further experiments.

The active components of RTP were determined as follows. Theconcentration of total polyphenol was analyzed according to theFolineCiocalteau method (Pothitirat, Chomnawang, Supabphol, &Gritsanapan, 2009) with a little modifications. 100 ml of RTP(1 mg/ml) was diluted with 900 ml of distilled water in a test tube,and then the FolineCiocalteu reagent (1 M, 0.5 ml) was added andmixed thoroughly. After an interval of 3 min, 3 ml of 7.5% Na2CO3solution was added, and the mixture was allowed to stand for 2 hwith intermittent mixing. The absorbance of the mixture at 750 nmwas measured on a U-3210 spectrophotometer (Hitachi, Tokyo,Japan). A standard curve using gallic acid (aqueous solution) wasalso prepared by the same method. Total flavonoid content wasdetermined by the previously method (Wu, Chang, Chen, Fan, & Ho,2009) with slight modifications. Next, 0.5 ml of the RTP (1 mg/ml)was diluted with 1.25 ml of distilled water. Then, 100 ml of a 5%NaNO2 solution was added to the mixture. After 6 min, 150 ml of a10% AlCl3$6H2O solution was added, and the mixture was allowedto stand for another 5 min. Afterwards, 0.5 ml of 1 M NaOH and2.5 ml of distilled water were added. The solution was mixed, andthe absorbance was measured immediately against the prepared

control at 510 nm. A standard curve using rutin (methanol solution)was also prepared. All determinations were carried out in tripli-cates. The content was expressed as the number of equivalents ofgallic acid for total polyphenol and rutin for total flavonoid,respectively.

2.3. HPLC analysis for RTP

The analysis of RTP was performed using a HPLC system (AgilentTechnologies, Wilmington, DE, USA) with a G1311 Quat Pump,AutoSampler, G1314A VWD detector and Star Work Station soft-ware (version Rev.A.10.02). A ZORBAX SB-C18 reverse-phase col-umn, 4.6 � 250 mm, 5 mm particle size (Eclipse Plus, AgilentTechnologies, Wilmington, DE, USA) was used. Preceding theanalytical column was a ZORBAX SB-C18 guard column(4.6 � 12.5 mm, 5 mm particle size, Eclipse Plus, Agilent Technolo-gies, Wilmington, DE, USA), used to prevent any non-soluble resi-dues of samples from contaminating the analytical column.

The mobile phase consisted of deionized water as solvent A(contain 0.1% formic acid) and 100% acetonitrile (contain 0.1% for-mic acid) as solvent B. The time program started at 5% B for 5 minand then increased to 10% in 5 min and maintained for 5 min, andthen increased to 13% in 15 min. Solvent B sequentially increased to18% in 2 min and then ramped to 35% in 20 min, then increased to80% in 3 min. Finally, it increased to 100% B over 2 min and main-tained for 3 min, and then returned to 5% B in 5 min. The flow ratewas 0.8ml/min. An auto injector was used to inject 20ml of the testsolution into the HPLC system. The wavelength was monitored at280 nm. The temperature of the column oven was set at 25 �C.

2.4. Determination of the minimum inhibitoryconcentration (MIC) of RTP

MIC of the RTP was determined against tested pathogens bybroth macrodilution method as recommended by the Clinical andLaboratory Standards Institute, USA (2006). The tested pathogenswas inoculated into 20 ml of LB medium supplemented with thediluted extracts to attain the final concentrations ranging from 0.1to 3.0 mg/ml and incubated at their optimum temperature for 24 h.Before and after incubation, the absorbance of the mediums wasmeasured by spectrophotometer at wavelength of 600 nm. MIC isdefined as the lowest concentration of RTP which showed inhibi-tion of visible growth of the tested strains (Khan, Zahin, Hasan,Husain, & Ahmad, 2009). The sub-MIC concentration was selectedfor the assessment of anti-biofilm and anti-QS activity.

2.5. Quorum sensing inhibition assays

Standard disk-diffusion assay was used to detect anti-QS activityof RTP by using biomonitor strain of C. violaceum 026 by themethod of Adonizio et al. (2006). C. violaceum 026 was grown in LBmedium solidified with 0.75% agar when required and supple-mented with 20 mg/ml kanamycin. Five milliliter of molten LB agar(0.75% w/v) was seeded with 200 ml of overnight LB culture ofC. violaceum 026, together with 5 ml of 40 mM C6-HSL as an exog-enous AHL source. Then, it was gently mixed and poured imme-diately over the surface of a solidified LB agar plate as an overlay.20 ml of RTP to be tested was pipetted on sterile paper discs whichwere placed on the solidified agar. 20 ml of deionized water wasused as a negative control. A paper disks loaded with 20 ml of 50 mg/ml C10-HSL (N-decanoyl-L-homoserine lactone, DHL), a reportedantagonist of violacein synthesis (McLean, Pierson, & Fuqua, 2004)was used as a positive control. The plates were incubated overnightat 28 �C and examined for violacein production. QS inhibition wasdetected by a colorless, opaque, but viable halo around the discs.

Table 1The composition of RTP identified by HPLC analysis.

Peak number Polyphenolic compounds Rt (min) RTP (%)

1 Gallic acid 9.845 8.3202 Catechin 28.085 8.0843 Epicatechin 32.242 18.0804 Epigallocatechin gallate 37.044 13.1025 Epicatechin gallate 39.472 2.8046 Quercetin 42.930 3.6647 Benzoic acid 45.165 6.8828 Quercetin glucoside 51.204 0.3849 Tannin 53.944 3.43910 Kaempferol 55.127 0.814Total polyphenol (Folin-Ciocalteu method) 87.52

(875.2 mg GA/g)Total flavonoid (Wu et al. method) 61.03

(610.3 mg rutin/g)

J. Zhang et al. / Food Control 42 (2014) 125e131 127

For violacein production quantitative assay, C. violaceum 026was inoculated into 4 ml of LB medium and incubated for 18 h at28 �C. The culture was diluted 1:50 in 4 ml of fresh LB mediumcontaining 40 nM N-hexanoyl-L-homoserine lactone (C6-HSL) anda series of different concentrations of RTP (0.1 mg/ml, 0.2 mg/ml,0.6 mg/ml and 1.2 mg/ml, respectively). The sample without RTPwas used as control, and then incubated for 48 h at 28 �C. Aftervortexing the tubes to resuspend any layer of adherent cells, 300 mlof each culture was placed in a 1.5 ml microtube. Violacein wasextracted using the previously described method with slightmodifications (Adonizio et al., 2006). The cells were lysed by adding150 ml of 10% sodium dodecyl sulfate (SDS), followed by mixing for10 s with a vortex mixer. Violacein was extracted from the celllysate by adding 600 ml of water-saturated butanol, vortexing for5 s, and centrifuged for 5 min at 12,000 rpm. The butanol phase(upper) containing the violacein was transferred into 96-well mi-crotiter plates, with 150 ml of extract per well. Then, analyzedextract at 585 nm using a microplate reader (Winooski, VT, USA).The effects of RTP were evaluated based on the relative levels ofviolacein production, and the control value was set to 100%.

2.6. Swarming motility inhibition assay

Fivemicroliters ofmolten soft top agar (0.6 g agar, 2.0 g tryptone,1.0 g Yeast extract powder, 1.0 g sodium chloride, 200 ml deionizedwater) with the final concentrations of RTP were 80 mg/ml, 160 mg/ml, 320 mg/ml and 640 mg/ml, respectively. Then, poured it imme-diately over the surface of a solidified LB agar plate as an overlay. Theplate center point was inoculatedwith an overnight culture of E. coliK-12 and P. aeruginosa PAO1. Once the overlaid agar had solidifiedand incubated at 37 �C for 30 h. Following swim and swarm zones ofthe bacterial cells, swimming and swarming migration wereobserved. The migration distance of E. coli K-12 and P. aeruginosaPAO1weremeasuredwhile theywere incubated for 30 h at differentconcentrations of RTP. All the experiments were performed in trip-licate, and data are presented as means � standard deviation.

2.7. Biofilm formation assay

The quantification of biofilm formation using regular 96 wellmicrotiter plates was performed as described previously(Girennavar et al., 2008) with minor modifications. Overnight cul-ture of tested bacteria E. coli K-12 and P. aeruginosa PAO1 wereinoculated (1:100) in LB medium. Aliquots of the diluted culture(95 ml) were added to each well of sterile round-bottom 96-wellmicrotiter plates. To assess the impact of the RTP on biofilm for-mation, 5 ml RTP was added to these culture suspensions. Use 5 ml ofdeionized water added to 95 ml LB medium as control. The plateswere incubated at 37 �C for 24 h without shaking. After incubation,the suspension culture was removed (50 ml of suspension culturewas pipetted to determine the total biomass on LB agar plates), andthe plates were washed for 3 times with deionized water. The bio-films were stained with 125 ml of 0.3% crystal violet per well for15 min. The excess dye was removed by washing the plates 3 timeswith deionized water. Dye associated with attached biofilm wasdissolved with 200 ml of 95% ethanol. Aliquots of the crystal violet/ethanol solution from each well (125 ml) were transfer to a separatewell in anopticallyclearflat-bottom96-wellmicrotiterplate and theOD value was measured at 595 nm. Each data point was averagedfrom four replicate wells and the standard errors were calculated.

2.8. Confocal laser scanning microscopy (CLSM) analysis

The biofilms weremonitored under a CLSM (Leica TCS SP2; LeicaMicrosystems, Heidelberg, Germany) as described previously

(Berney, Hammes, Bosshard, Weilenmann, & Egli, 2007;Rasmussen, et al., 2005). Overnight cultures of tested strainsincubated in LB medium were diluted to a final density of1.0 � 106 CFU/ml with fresh medium. 1 ml of cell suspensionssupplemented with 640 mg/ml RTP and a piece of plastic coverslip(0.2 mm thick and 13 mm in diameter; Nunc, Roskilde, Denmark)were dispensed into the wells of 24-well microtitre plates, using1 ml of cell suspensions without RTP as control. Plates were stati-cally incubated at 37 �C for 48 h. CLSM was performed on biofilmsformed on the plastic coverslips. At the end of incubation, thecoverslips were gently washed with sterile distilled water andstained with the LIVE/DEAD BacLight Bacterial Viability Kit (Invi-trogen, USA) according to the manufacturer’s instruction. Stainedcoverslips were gently washed twice with sterile distilled waterand observed with CLSM. The Leica confocal software was used foranalysis of biofilm images, which allowed for collection of z-stacksthree-dimensional reconstruction. Images were acquired fromrandom positions of biofilms formed on the upper side of thecoverslips. The thicknesses of biofilms were also determineddirectly from the confocal stack images.

2.9. Statistical analysis

The results were expressed as means � standard deviation (SD).Analysis of variance was conducted and differences between vari-ables were tested for significance by one-way ANOVA with Tukeytest using the SPSS V16.0 program. Differences at P < 0.05 wereconsidered statistically significant.

3. Results and discussion

3.1. Composition analysis of RTP

The component contents of the RTPwere summarized in Table 1.The data confirmed that RTP was rich in polyphenol (87.52%) andflavonoid (61.03%). Ten kinds of polyphenols present in RTP,including gallic acid, catechin, epicatechin, epigallocatechin gallate(EGCG), epicatechin gallate, quercetin, benzoic acid, quercetinglycoside, tannins and kaempferol were identified by HPLC anal-ysis, at approximately 8.32%, 8.08%, 18.08%, 13.10%, 2.80%, 3.66%,6.88%, 0.38%, 3.44%, and 0.81%, respectively (Fig. 1, Table 1). All thepolyphenol compounds in RTP except benzoic acid has been re-ported to have anti-QS and/or anti-biofilm activities (Kalia, 2012;Taganna, Quanico, Perono, Amor, & Rivera, 2011; Taganna & Rivera,2008).

In order to ensure if polyphenols are responsible for anti-QSactivities of RTP, we completely removed polyphenols of RTP byadding a saturated solution of neutral lead acetate, and recovered

Fig. 1. The HPLC chromatogram of the identification of the active compounds in RTP. (A) HPLC chromatogram of 10 kinds of standard polyphenols. Peaks: (1) Gallic acid; (2)Catechin; (3) Epicatechin; (4) Epigallocatechin gallate; (5) Epicatechin gallate; (6) Quercetin; (7) Benzoic acid; (8) Quercetin glucoside; (9) Tannin; and (10) Kaempferol. (B) HPLCchromatograms of polyphenols from RTP.

J. Zhang et al. / Food Control 42 (2014) 125e131128

the polyphenols by adding hydrochloric acid (0.8 M). Disk-diffusionassay using reporter strain C. violaceum 026 indicated that theformer (removed polyphenols) doesn’t have anti-QS activity whilethe latter (recovery polyphenols) exhibited obviously anti-QS ac-tivity (Fig. 2). The observation confirmed that anti-QS activity ofRTP was associated with polyphenol compounds.

Fig. 2. When enough saturated lead acetate in neutral solution was added into RTP, yellowremoved polyphenols by enough saturated lead acetate has no anti-QS activity in C. violaceumeffects reoccurred (C).

3.2. RTP inhibits violacein production in C. violaceum 026

The MIC value of RTP against C. violaceum 026 was 1.8 mg/ml.We selected sub-MIC concentrations of RTP to further spectro-photometrically measured anti-QS activity of the RTP on violaceinproduction of C. violaceum 026. As shown in Fig. 3, a gradual

-green precipitate produced, indicating the presence of polyphenols (A); The RTP of026 (B); After RTP precipitate recovered by 0.8 M hydrochloric acid, its anti-QS activity

Fig. 3. The biomass and violacein production of C. violaceum 026 when it was incu-bated in the medium with different concentrations of RTP. Data were represented aspercentage of violacein inhibition. Each bar represents mean and standard deviationsof the mean of all the measurements.

J. Zhang et al. / Food Control 42 (2014) 125e131 129

decrease in the violacein production was observed when treatedwith the increasing concentration of RTP. In quantitative analysis,RTP (at the concentration of 1.20 mg/ml) reduced violacein pro-duction dramatically to the level of 87.56% in C. violaceum 026. Toensure that inhibition of violacein production was not because ofantibacterial activity, the effect of RTP on the growth of C. violaceum026 was further performed. The results showed that RTP concen-trations lower than 1.2 mg/ml did not have effect on the growthrate of C. violaceum 026 (Fig. 3).

The present data corroborates well with the findings of Vattemet al. (2007), who have reported the inhibition of violacein

Table 2The migration distance of E. coli K-12 and P. aeruginosa PAO1 after they were incumeans � standard deviations.

Bacterial strain E. coli K-12

Concentrationsof RTP (mg/ml)

Diameter ofswarm (mm)

Decrease in swarmingmotility over control (%)

Cell viability a(Log CFU/ml)

Control 81.79 � 1.87 0 8.5080 55.02 � 0.76 32.73 8.53160 35.96 � 1.36 56.03 8.51320 26.90 � 0.53 67.11 8.43640 12.35 � 2.18 84.90 8.48

Fig. 4. Effects on migration distance and pigment production against P. aeruginosa PAO1andRTP treatment.

production in C. violaceum 026 by certain spices containing highconcentrations of phenolic compounds to the level of about 41%.Similarly, other authors have also demonstrated that polyphenolcompounds, such as gallic acid, epigallocatechin gallate and cur-cumin can interfere with bacterial QS (Kalia, 2012; Packiavathyet al., 2012; Taganna et al., 2011; Taganna & Rivera, 2008).

3.3. RTP inhibits swarming motility of E. coli K-12 and P. aeruginosaPAO1

The MICs of RTP against E. coli K-12 and P. aeruginosa PAO1 were1.6 mg/ml and 1.2 mg/ml, respectively. As shown in Table 2, the RTPsignificantly (P < 0.05) inhibited QS dependent swarming migra-tion in a concentration-dependent manner in E. coli K-12 andP. aeruginosa PAO1. When the concentration of RTP was 640 mg/ml,the inhibition rates in swarming-migration of E. coli K-12 andP. aeruginosa PAO1 were 84.90% and 78.03%, respectively. Itinhibited swarming motility of the two strains without inhibitingtheir growth (Table 2), thus proving that the swarming inhibitionby RTP was not due to an antibacterial effect. Interestingly, RTPinhibited swarming motility of P. aeruginosa PAO1 but promoted itsgreen pigment production, a QS-controlled phenotype (Fig. 4).

3.4. RTP inhibits biofilm formation in E. coli K-12 and P. aeruginosaPAO1

In biofilm biomass quantification assay, a significant (p < 0.01)decrease in biofilm formation was observed when bacterial strainsgrown in the presence of the RTP. At the concentration of 640 mg/ml, RTP showed a maximum of 67.02% and 72.90% reduction inbiofilm biomass of E. coli K-12 and P. aeruginosa PAO1, respectively(Fig. 5). It was clear that RTP did inhibit the biofilm formation of thetest strains without inhibiting its biomass. It proved that the biofilminhibition activity of RTP was not due to the antibacterial effect.

bated for 30 h at different concentrations of RTP. Results are expressed as the

P. aeruginosa PAO1

ssay Diameter ofswarm (mm)

Decrease in swarmingmotility over control (%)

Cell viabilityassay (Log CFU/ml)

59.35 � 0.92 0 8.2529.59 � 0.81 46.54 8.3026.50 � 1.07 52.12 8.3123.66 � 1.75 57.25 8.2612.16 � 1.46 78.03 8.28

E. coli K-12 by (A) control, (B) 80 mg/ml, (C) 160 mg/ml, (D)320 mg/ml, and (E) 640 mg/ml

Fig. 5. The inhibition of E. coli K-12 (A) and P. aeruginosa PAO1 (B) biofilm in presence of different concentrations of RTP. Data were represented as percentage of biofilm inhibition.Each bar represents mean and standard deviations of the mean of all the measurements.

J. Zhang et al. / Food Control 42 (2014) 125e131130

3.5. In situ image analysis of bacterial biofilms by CLSM

CLSM z-section analyses can provide surface coverage of thebiofilms as well as the entire thickness of bacterial biofilms. CLSMz-section analyses showed that the two strains (E. coli K-12 andP. aeruginosa PAO1) formed thick and compact biofilms whengrown in the absence of RTP. In contrast, RTP at a sub-MIC con-centration of 640 mg/ml resulted in thinner and looser cell aggre-gations on surfaces instead of normal biofilm architecture (Fig. 6).The confocal stack images showed that, in the negative control

Fig. 6. The CLSM images of biofilm formation by E. coli K-12 and P. aeruginosa

group, the thicknesses of biofilms formatted by E. coli K-12 andP. aeruginosa PAO1 were respectively 20.366 � 1.269 mm and27.924 � 1.105 mm. While E. coli K-12 and P. aeruginosa PAO1 wereincubated together with 640 mg/ml of RTP, the biofilms thicknessesdropped to 8.130 � 1.033 mm and 9.318 � 1.014 mm, respectively.

Besides the difference in biofilm thickness, RTP also influencedthe surface area covered. The CLSM top-view images of E. coli K-12and P. aeruginosa PAO1 biofilms showed that the biofilms of controlgroups (without RTP) covered the entire surface of the coverslips.But the test groups with 640 mg/ml of RTP, CLSM assessments of the

PAO1 in the absence of RTP (A) and the presence of RTP (640 mg/ml) (B).

J. Zhang et al. / Food Control 42 (2014) 125e131 131

both bacteria exhibited an obvious decrease in the surface coverageof coverslips, and the density of bacteria was significantlydecreased (Fig. 6).

QS mechanism influences both the initiation and also the matu-ration of bacterial biofilm (Kievit, 2009). The culture medium with640 mg/ml of RTP effectively reduced the thickness of biofilm andformation of microcolonies during the process of biofilm formationby the bacterial pathogens such as P. aeruginosa PAO1 and E. coliK-12.This evidenced that biofilm formation was possibly inhibited at thebeginning of the attachment stage itself. This is in agreement withpreviously published report of Brackman et al. (2009).

4. Conclusions

In the present study, for the first time, we demonstrated theanti-biofilm and anti-QS potentials of RTP, a polyphenol-richextract from R. rugosa tea, and identified the composition of RTPassociated with anti-QS activity. The RTP significantly inhibited QSdependent violacein production in C. violaceum 026 and swarmingmigration in a concentration-dependent manner in E. coli K-12 andP. aeruginosa PAO1. It also significantly inhibited in vitro biofilmformation at sub-MIC concentrations. Furthermore, we want toknow whether the RTP could increase the antimicrobial efficacy ofsanitizers or antimicrobials when used with them in combination.Meanwhile, if the RTP has the activity of anti-biofilm against otherfood spoilage bacteria and pathogens, that is, if the RTP is specific orgeneric type of QSI and the anti-biofilm mechanisms of RTP aredeeply interested us, We will exploit them in our further research.

Acknowledgments

This work was co-financed by National Natural Science Foun-dation of China (No. 31371807 and 31201422), High-Tech Researchand Development Program of China (No. 2011AA100903 and2013BAD18B01-4) was also supported by the Project Funded by thePriority Academic Program Development of Jiangsu Higher Edu-cation Institutions (PAPD).

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