Original article

Antibiofilm formation and anti-adhesive (to HEp-2 cells) effects of

rosemary water extract against some food-related pathogens

Hesham Elhariry,1,2* Abeer A. Abuzaid,1,3 Ghada M. Khiralla4 & Youssuf Gherbawy1,5

1 Department of Biology, Faculty of Science, Taif University, PO Box 888, Taif, Saudi Arabia

2 Department of Food Science, Faculty of Agriculture, Ain Shams University, PO Box 68-Hadayek Shoubra, Cairo 11241, Egypt

3 Agriculture Research Center, Food Technology Research Institute, 9 Cairo University St., Giza, Egypt

4 National Organization for Drug Control and Research (NODCAR), 6-7, AboHazem Street, Pyramids, PO Box 29, Giza, Egypt

5 Department of Botany, Faculty of Science, South Valley University, 83523 Qena, Egypt

(Received 5 July 2013; Accepted in revised form 6 October 2013)

Summary The present work aimed to determine the bioactive compounds in two rosemary water extracts (RWE1

and RWE2) and to assess their antimicrobial, anti-adhesive and antibiofilm potentials against the food-

related Bacillus and Pseudomonas species at concentrations; 4, 8, 12, 16 and 20 mg mL�1. Phenolic com-

pounds and isoflavones in the RWEs were determined using HPLC. The concentrations of most bioactive

compounds of RWE1 (benzoic, ellagic, gallic and rosmarinic acids, daidzein and genistein) were higher

than that of RWE2. The MIC90 of RWE1 and RWE2 against all tested bacteria was 12 and 16 mg mL�1,

respectively. The anti-adhesive and antibiofilm doses were higher than MIC90. RWE1 and RWE2 showed

potential reduction in the bacterial cell adhesion to HEp-2 cells – 17.5–64.7 and 12.2–52.9%, respectively.

In conclusion, this study emphasises the effective use of RWE as a natural anti-adhesive and antibiofilm

agent against Bacillus and Pseudomonas, without difficult extraction procedure.

Keywords Antibiofilm, antimicrobial, bioactive compounds, pathogens, rosemary, water extract.

Introduction

Pathogenic bacteria develop a number of mechanismsto cause disease in their hosts. They express a widerange of molecules that adhere to host cell-surfacetargets (Wilson et al., 2002). Adhesion of pathogenicbacteria to host cell surface is a crucial event in coloni-sation and infection (Sharon, 2006). After initial adhe-sion to host tissues, bacteria begin to grow as amonolayer on the surface to form microcolonies thatcan consequently develop and form biofilm. A biofilmmay be described as a microbial-derived sessilecommunity characterised by cells that attach to aninterface, embedded in a matrix. Depending on thespecies involved, the microcolony may be composed of10–25% cells and 75–90% extracellular polymeric sub-stances (EPS) matrix (Garrett et al., 2008). Biofilms ofBacillus spp. and Pseudomonas spp. are recognised asa serious problem and important contaminants inmany food industry settings such as fresh produce,poultry, dairy and red meat processing. They are

among the most well-studied microorganisms capableof biofilm formation and commonly found as part ofbiofilms in food-processing environments (O’Tooleet al., 2000). These microorganisms are considered asprimary colonisers of surfaces and provide the biofilmbackbone to which other microorganisms can adhere(Cloete & Jacobs, 2001; Manuzon, 2009). Presence ofpathogenic bacteria in biofilm matrix provides themseveral advantages and increases their virulence andresistance to disinfectant (Kreske et al., 2006; Wojniczet al., 2012). In this respect, it has been establishedthat the resistance of Bacillus cereus and Pseudomonasin biofilms to antibiotics is increased compared withwhat is normally seen with planktonic cells (Drenkard& Ausubel, 2002). Moreover, Bacillus cereus spores areboth highly resistant to a large number of stresses andvery hydrophobic, which causes them to adhere easilyto biotic and abiotic surfaces (Lindsay et al., 2002).The adherent character of bacterial cells is caused byseveral outer membrane structures such as pili, flagella,proteins and lipopolysaccharides (Camesano & Logan,2000; Hamza et al., 2010). In addition, adhesion ofbacteria and consequently biofilm formation is man-aged by several factors such as electrostatic

*Correspondent: Fax: +202444 4460;

e-mails: h.elhariry@agr.asu.edu.eg, h.elhariry@hotmail.com

International Journal of Food Science and Technology 2013

doi:10.1111/ijfs.12409

© 2013 Institute of Food Science and Technology

1

interactions, van der Waals, acid–base, hydrogenbonding, biospecific interactions (Fletcher, 1996),bacterial surface roughness, solid surface chemicalstructure (Abu-Lail & Camesano, 2003) and hydro-phobicity (Elhariry, 2008). All these surface structuresand properties are well known to be affected by treat-ment with plant extracts rich in phenolic compoundsand isoflavones (Huttunen et al., 2011).

Down the ages, essential oils and other extracts ofplants have evoked interest as sources of natural prod-ucts (Taguri et al., 2004; Klan�cnik et al., 2009; Paivaet al., 2010). They have been screened for their poten-tial uses as alternative remedies for the treatment ofmany infectious diseases (Al-Sereiti et al., 1999). Rose-mary (Rosmarinus officinalis L.) is a woody, perennialherb with fragrant, evergreen needle-like leaves. It is acommon household plant grown in many parts of theworld and belongs to the mint family Lamiaceae(Al-Sereiti et al., 1999). Rosemary leaves have bitter,astringent taste and volatile compounds. Therefore, itis used in food industry as flavouring agents, antioxi-dants and antimicrobials as well as in cosmetics (Liuet al., 2009). Moreover, rosemary extract relaxessmooth muscles of trachea and intestine and has chole-retic, hepatoprotective and antitumorigenic activity.Therefore, in folk-medicine, the hot-water extract offresh and dried-powdered leaves of different medicalplants are used frequently as an antispasmodic in renalcolic, dysmenorrhoea and in relieving respiratory dis-orders (Tsai et al., 2007). Rosemary leaves contain avariety of phenolic acids such as carnosol, carnosicacid and rosmarinic acid, bitter diterpenes, glycosidesand several flavonoids (Angioni et al., 2004; Tsaiet al., 2007). Four diterpenoids (carnosic acid, rosma-nol, carnosol and epirosmanol), isolated from driedleaves of rosemary by bioassay-directed fractionation,inhibited superoxide anion production in the xanthineoxidase system, showing to be protective against oxi-dative stresses (Erkan et al., 2008). One of the mostimportant constituents of rosemary is caffeic acid andits derivatives such as rosmarinic acid. These com-pounds have antioxidant effect. The phenolic com-pound, rosmarinic acid, obtains one of its phenolicrings from phenylalanine via caffeic acid and the otherfrom tyrosine via dihydroxyphenyl-lactic acid (Al-Sere-iti et al., 1999). Due to their phenolic compounds,rosemary extracts prepared using different solventshave been shown to have antimicrobial and antioxi-dant activities (Erkan et al., 2008; Ruiz-Bustos et al.,2009). Moreno et al. (2006) stated that carnosic acidand rosmarinic acid may be the main bioactive antimi-crobial compounds present in rosemary methanolextract and water extract, respectively, which showeddifferent efficacy as antimicrobial agent. Recent find-ings indicate that some natural phenolic compoundsfound in plants have an antibiofouling effect on

biofilm formation by Pseudomonas aeruginosa as amodel microorganism of Gram-negative bacteria(Jagani et al., 2009; Plyuta et al., 2013).For our knowledge, although essential oils of rose-

mary have been extensively studied (Angioni et al.,2004; Prabuseenivasan et al., 2006; Erkan et al., 2008),few literatures have interested in the inhibition effectsof rosemary water extract on formation of microbialbiofilm (Quave et al., 2008). It is well known that theessential oils of rosemary are rich in monoterpenes,diterpenes, ketones and alcohols, but there are no phe-nols in the essential oils of rosemary (Mathlouthiet al., 2012). To avoid toxicity and possible negativeeffects caused by essential oils and ethanol extracts,distilled water was used as the extraction agent in thepresent study (Wang et al., 2012). Therefore, the pres-ent work aimed to (i) determine the bioactive com-pounds in the rosemary water extract includingphenolic compounds and isoflavones and (ii) evaluatethe antimicrobial, anti-adhesive and antibiofilm effectof rosemary water extract against some biofilm-form-ing strains of Bacillus and Pseudomonas; where block-ing bacterial adhesion to host surfaces providespotential approach to control the microbial infections.

Materials and methods

Bacterial strains

Twelve bacterial strains belonging to Bacillus andPseudomonas were used in the present study. Tworeference strains – B. cereus DSMZ 345 and P. aeru-ginosa ATCC 10145 – were obtained from stock cul-tures maintained in the microbial culture collection,Department of Food Science, Faculty of Agriculture,Ain Shams University, Cairo, Egypt. The other strainsare characterised as biofilm producers that have beenisolated from slime layer of microbial biofilms indrinking water network in Taif City, KSA. Thesestrains have been molecularly identified as B. cereusTUB8, TUB30, TUB31, TUB32 and TUB33 (Bacillusgroup), P. aeruginosa TUB14, P. alcaligenes TUB12,P. alcaligenes TUB13, P. monteilii TUB15 and P. par-afulva TUB16 (Pseudomonas group) (Elhariry et al.,2012). All strains were cultivated routinely on trypticsoy broth (TSB) for overnight at 37 °C and stored at4 °C on tryptic soy agar (TSA). Stock cultures weremaintained in 15% v/v glycerol/TSB at �70 °C.

Preparation of rosemary water extract

Two fresh rosemary (R. officinalis L.) leaf sampleswere purchased from the local markets of Taif City,Kingdom of Saudi Arabia and Cairo City, Egypt.Rosemary water extracts (RWE1 and RWE2) wereprepared from the fresh rosemary leafs collected from

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al.2

Taif and Cairo, respectively, according to the methodof Wojnicz et al. (2012), with some modifications.Rosemary leaves were dried at 37 °C for 48 h (toreach about 9.6% moisture content; dry basis). Driedleaves were ground into powder in an electric blender.20 g of each leaves powder was dissolved in 180 mLof distilled water in a glass bottle, heated to 85 °C in awater bath and maintained at this temperature withshaking for 8 h. After cooling, the liquid was filteredthrough the Whatman No. 1 filter paper (WhatmanInternational Ltd., Maidstone, UK). To obtain thedried total soluble solids (DTSS) of these extracts, thefiltrates were dried in smaller glass bottles at 37 °C for48 h.

Determination of bioactive compound

Total phenolsThe total phenolic content of the hot-water extracts ofrosemary (12 mg DTSS per mL) was determinedaccording to the method described Amarowicz et al.(2004). Briefly, 18 lL of each extract was transferredinto a 96-well microtiter plate (Biotek, Winooski, VT,USA) and mixed with 36 lL Folin–Ciocalteu reagent(10%) and 145 lL sodium carbonate (700 mmol). Theabsorbance of the mixture was measured at 750 nmafter 60 min incubation in the dark at room tempera-ture. The total phenolic content was calculated as agallic acid equivalent (GAE) from a calibration curveof GA standard solutions (ranging from 25 to800 mg L�1) and expressed as mg of GAE per gramof dry extracts residue. All measurements were done intriplicate (n = 3).

HPLC analysis of phenolic compounds and isoflavonesPhenolic compounds of RWEs were determinedaccording to the method described by Goupy et al.(1999). Briefly, 3 mL of each RWE was filteredthrough an Acrodisc filter (0.20 mm) before HPLCanalysis. Phenolics quantification was performed byreverse-phase HPLC (RP-HPLC)/diode array detection(DAD) using a C18-column, 5 mm (150 9 4.6 mm id).The solvent system used was a gradient of A(CH3COOH 2.5%), B (CH3COOH 8%) and C (aceto-nitrile), with the following gradient: at 0 min, 5% B;at 20 min, 10% B; at 50 min, 30% B; at 55 min, 50%B; at 60 min, 100% B; at 100 min, 50% B and 50%C; at 110 min, 100% C until 120 min. The solventflow rate was 1 mL min�1, and separation wasperformed at 35°C. Phenolic compounds were assayedby external standard calibration at 280 nm (except ros-marinic acid that measured at 330 nm) and expressedin ppm dry matter of equivalent.

Isoflavones were determined by the methoddescribed by Coward et al. (1993). Briefly, separationof isoflavones was achieved by HPLC on reversed-

phase C18-column with a mobile phase consisting of agradient of 0–46.4% acetonitrile in 0.1% (v/v) aqueoustrifluoroacetic acid at a flow rate of 1.5 mL min�1.The concentration of acetonitrile was increased by2.25% min�1. The eluting compounds were detectedfrom their absorbance at 262 nm. Concentrations ofthe isoflavones were calculated from standard curvesof the area responses for authentic isoflavone stan-dards normalised to the constant amount of fluores-cein added to each sample. The concentrations wereexpressed in ppm dry matter of equivalent.

Antimicrobial activity

Antimicrobial activity of RWE1 and RWE2 was deter-mined as minimum inhibitory concentration (MIC90)using a microdilution technique in 96-well microtiterplates (Eloff, 1998). The stock extracts (RME1 andRME2 obtained from R1 and R2 samples, respec-tively) were prepared by dissolving 50 mg DTSS permL distilled water and sterilised by filtration through0.22-lm filters. Different amounts; 15, 20, 40, 60, 80and 100 lL of the concentrated stock RWE1 orRWE2 were added to each well of sterile 96-well poly-styrene plates. 10 lL activated culture of each testedstrain (about 106 CFU mL�1) was added. The totalvolume in each well was adjusted to 250 lL usingTSB. The final tested concentrations of DTSS were 4,8, 12, 16 and 20 mg DTSS per mL. After 18 h of incu-bation at 37 °C, bacterial growth inhibition was deter-mined by monitoring the optical density (OD) at595 nm. The MIC90 was defined as the lowest concen-tration of rosemary extract inhibiting >90% of bacte-rial growth. All tests were carried out in triplicate(n = 3), and the results were averaged.

Anti-adhesive and antibiofilm effect

Inhibitory effects of the studied RWEs on bacterialadhesion and biofilm formation were determined in vitrousing the commonly used 96-well polystyrene microtiterplates method. The steps of the first part of this experi-ment were done as described above in determination ofMIC90. After incubation at 37 °C for 4 (to determineanti-adhesive effect) and 24 h (to determine antibiofilmeffect), content of the microtiter plates was poured off,and the wells were washed three times with 300 lL ofphosphate-buffered saline (PBS, pH 7.2). The remainingadhered bacteria were fixed with 250 lL of methanol perwell. After 15 min, microtiter plates were emptied andair-dried. The microtiter plates were stained with 250 lLper well of 1% crystal violet used for Gram staining for5 min. The excess of stain was rinsed off by placing themicrotiter plates under running tap water. After dryingthe microtiter plates, the dye bound to the adherent cellswas extracted with 250 lL of 33% (v/v) glacial acetic

© 2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al. 3

acid per well. The absorbance of each was measured at570 nm using a spectrophotometer. Based on the absor-bance (A570 nm) produced by bacterial films, strains wereclassified into four categories according to the classifica-tion of Christensen et al. (1985), which was modified byStepanovi�c et al. (2000). Briefly, the cut-off absorbance(Ac) was the mean absorbance of the negative control.Strains were classified as follows: A = Ac = no biofilmproducer (0); Ac < A ≤ (2 9 Ac)= weak biofilm pro-ducer (+); (2 9 Ac) < A ≤ (49 Ac) = moderate biofilmproducer (++); (4 9 Ac) < A = strong biofilm producer(+++). All tests were carried out in triplicate (n = 3),and the results were averaged.

The anti-adhesive effect was calculated from theabsorbance values after 4 h. Here, the anti-adhesivedose was considered as the minimum concentration ofRWE required for complete prevention of bacterialcell adhesion to microtiter plates substratum, whenA = Ac after 4 h. The antibiofilm dose was definedhere as the lowest RWE concentration that caused pre-vention of biofilm formation after 24 h by each testedbacterium (to be in the category of no biofilmproducer).

Bacterial adherence to HEp-2 cells and anti-adhesiveeffect

The adherence assay has previously been described by(Boddicker et al., 2002) with some modifications.Briefly, washed HEp-2 cells were incubated with bacte-ria (1 9 108 CFU mL�1) in a bacteria-to-cell ratio of1000:1 in the absence (control) or presence of the mostpotent RME concentrations of both rosemary againstbiofilm formation by the tested bacteria (20 mg mL�1).Following incubation with gentle agitation, nonadher-ent organisms were removed by repeated washing, andafter a final wash, the cells were resuspended in 200 lLof PBS (pH 7.2) and fixed onto glass microscope slides.After staining with Giemsa, the cells were examined bymicroscopy and adherence was recorded as the meannumber of bacteria per cell after examination of at least100 different cells from five separate fields (Boddickeret al., 2002). All tests were carried out in triplicate(n = 3), and the results were averaged. Here, the reduc-tion percentage of adhesive was calculated as [numberof adhered bacteria per HEp-2 cell ( in the presence ofRWEs) – number of adhered bacteria per HEp-2 cell ofcontrol (in the absence of RWEs)]/number of adheredbacteria per HEp-2 cell of control.

Statistical analysis

The data obtained from three replicates were analysedby a two-way ANOVA using ‘Proc Mixed’ (SAS 8.2,Cary, NC, USA). In all cases, the level of statisticalsignificance was of P < 0.05.

Results and discussion

Bioactive compounds of rosemary water extracts

Total phenols of the studied rosemary water extracts(RWEs) were determined and expressed as gallic acidequivalent (GAE). The RWE1 (collected from Taif)showed high phenolic content 85.24 GAE per mL com-pared with that of RWE2 (collected from Cairo)(Table 1).The obtained total phenolic values were inagreement with those recorded previously in rosemarywater extract (Tsai et al., 2007). The bioactive com-pounds including phenolic compounds and isoflavoneswere determined in both extracts using HPLC (Table 1).In general, the dominant phenolic and isoflavone com-pounds were rosmarinic acid and daidzein, respectively.Rosmarinic acid concentration was 161.3 and122.5 ppm in RWE1 and RWE2, respectively, whereasthe concentration of daidzein was 151.57 and129.80 ppm. It is clearly noticed that both rosmarinicacid and daidzein in RWE1 were higher than those

Table 1 Bioactive compounds of rosemary hot-water extractsa

Compoundsb RWE1c RWE2c

Total phenols (mg g�1) 85.24 � 6.21 45.97 � 2.14

Phenolic group (ppm)

Benzoic acid 103.80 � 11.12 32.97 � 2.4

Caffeic acid 7.80 � 0.60 10.90 � 1.51

Caffein 12.85 � 0.48 11.02 � 0.98

Catechin 71.10 � 9.13 44.40 � 2.56

Catechol 42.02 � 6.66 17.57 � 1.02

Chlorogenic acid 47.70 � 7.08 35.90 � 1.93

Coumarin 4.70 � 0.22 6.93 � 0.68

Ellagic acid 73.10 � 8.56 54.50 � 3.14

Ferulic acid 12.80 � 1.11 19.97 � 1.22

Gallic acid 35.02 � 6.23 4.53 � 0.07

P-Coumaric acid 8.20 � 0.22 5.40 � 0.36

Protocatechuic acid 10.70 � 6.20 34.90 � 3.51

Pyrogallol NDd 95.40 � 9.21

Rosmarinic acid 161.30 � 18.22 122.50 � 16.42

Salicylic acid 49.80 � 4.01 59.97 � 3.56

Vanillic acid 9.50 � 0.56 17.13 � 1.57

Isoflavones (ppm)

Biochanin NDd 0.60 � 0.01

Daidzein 151.57 � 12.33 129.80 � 11.54

Formononetin 0.88 � 0.06 NDd

Genistein 24.55 � 2.33 12.78 � 1.08

Isorhamnetin 6.35 � 0.64 17.23 � 0.97

aThe examined extract was selected as the minimum concentration

inhibited biofilm formation by tested bacteria (of RWEs per mL).bValues are means � SD, n = 3.cRWE1 and RWE2 are rosemary water extracts of plant leaves collected

from Taif and Cairo, respectively, which were prepared by dissolving

12 mg DTSS per mL; DTSS is the dried total soluble solids obtained

after soaking rosemary dry leaves in hot water (20:180 w/v) at 80 °C for

8 h.dND, not detectable under studied conditions.

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al.4

determined in RWE2. RWE1 and RWE2 were alsocharacterised by their high content of benzoic acid(103.8 ppm) and pyrogallol (95.4 ppm), respectively.Both extracts contained high amount of ellagic acid(73.1 and 54.5 ppm), catechin (71.1 and 44.4 ppm) andsalicylic acid (49.8 and 59.97 ppm). Also, chlorogenicacid (47.7 and 35.9 ppm) and catechol (42 and17.57 ppm) were also found in considerable amounts inboth tested extracts, respectively. Following daidzein,genistein (24.55 ppm) and isorhamnetin (17.24 ppm)were presented in high level in RWE1 and RWE2,respectively (Table 1). In more detail, the susceptibilityof bacteria to polyphenols also depends on the bacterialspecies and polyphenol type and structure (Taguri et al.,2004). Beside the high content of rosmarinic acid, cate-chin, ellagic acid, chlorogenic acid and catechol in bothtested RWEs, RWE1 is distinguished by its high content

of benzoic acid, while RWE2 by high content of pyro-gallol (Table 1). Rosmarinic acid is known to be a natu-ral phenolic compound widely distributed in Labiataeherbs such as rosemary. It is a water-soluble polypheno-lic compound that has been reported to have antioxida-tive, anti-inflammatory and antidepressive activities(Paiva et al., 2010). Moreover, the total phenols andflavonoids in the aqueous extract were higher than thosedetermined in methanolic extract (Tsai et al., 2007).However, the methanolic extract has conferred strongerantibacterial activity than does its aqueous extract (Tsaiet al., 2007; Rajkumar & Kanimozhi, 2010).

Antimicrobial activity of rosemary water extract

Rosemary leaf extracts are proposed as importanthuman dietary factors and have been investigated as

(A) (B)

(C) (D)

Figure 1 Minimum inhibitory concentration (black column) of rosemary water extracts RWE1 (A and B) and RWE2 (C and D) against Bacil-

lus (A and C) and Pseudomonas (B and D) strains. RWEs were prepared by dissolving per 1 mL: 0, 4, 8, 12, 16 or 20 mg of the dried total sol-

uble solids (DTSS) obtained after soaking rosemary dry leaves in hot water (20:180 w/v) at 80 °C for 8 h. Values are means � SD, n = 3.

Columns with the same letter within each group are insignificantly different (P > 0.05).

© 2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al. 5

potential therapeutic against several diseases (Morenoet al., 2006). However, very little is known regardingtheir application in human oral health to achievecertain advantages such as inhibiting growth of patho-gens and their adhesive ability to host cells. The esti-mated concentration of ‘a cup’ of rosemary infusion(3 g of leaves per 200 mL of boiling water) was2.45 mg of water-soluble extract per mL of infusion(Tsai et al., 2007). Therefore, different dry total solu-ble solid (DTSS) concentrations (0, 4, 8, 12, 16 and20) of two rosemary water extracts (RWE1 andRWE2) were tested for their potential inhibition activ-ity against different Bacillus and Pseudomonas strains(Fig. 1). Significant differences in growth of mosttested strains were recorded when the DTSS of RWE1and RME2 was used at level of 4 mg mL�1. Morereduction in the growth of all tested strains wasnoticed due to increasing RWE1 concentration to

8 mg mL�1. However, insignificant reduction in thebacterial growth was obtained by increasing RWE2concentration from 4 to 8 mg mL�1. The MIC90 ofRWE1 and RWE2 against all studied bacteria was 12and 16 mg DTSS per mL, respectively. In previousstudies, MIC90 of the rosemary water extract againstB. cereus has been reported to be 20 mg mL�1

(Klan�cnik et al., 2009). The slight differences betweenour results and results of the previous studies could bedue to extraction procedure and the tested strains.Remarkably, all Pseudomonas strains as Gram-nega-tive bacteria exhibited more resistance to the RWEscomparing with the other tested Gram-positive bacte-ria (Bacillus group). This may be due to the outermembrane surrounding the cell wall in Gram-negativebacteria, which restricts diffusion of compoundsthrough its lipopolysaccharide covering (Vaara, 1992).In previous reports, rosmarinic acid and ellagic acid

(A) (B)

(C) (D)

Figure 2 Anti-adhesive effect of rosemary water extracts RWE1 (A and B) and RWE2 (C and D) against Bacillus (A and C) and Pseudomonas

(B and D) strains. RWEs were prepared as described in Fig. 1. Negative (wells with TSB) and positive (wells with TSB and tested strain) con-

trols were not treated with RWEs. Values are means � SD, n = 3. Anti-adhesive dose * was defined when A570 of the treatment = A570 of the

negative control. Columns with the same letter within each group are insignificantly different (P > 0.05).

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al.6

have been previously reported to have a high potentialfor being a promising antimicrobial compound againstboth Gram-positive and Gram-negative pathogens(Miguel et al., 2010). In this respect, rosmarinic acidand ellagic acid have been previously reported as pow-erful phenolic compounds against B. cereus, whereMICs were about 10 ppm (Cetin-Karaca, 2011). Theantimicrobial effect of RWEs could be explained bythose mentioned by Zhang et al. (2008). Theymentioned that the phenolic compounds and flavo-noids may act through inhibiting cytoplasmic mem-brane function as well as by inhibition of certainenzymes such as DNA gyrase. Moreover, the isoflav-one genistein was able to inhibit the synthesis of theDNA and RNA (Ulanowska et al., 2006).

Anti-adhesive and antibiofilm activity of rosemary waterextract

Adhesion and biofilm-forming ability of Bacillus andPseudomonas strains were evaluated using 96-wellsmicrotiter plate technique in the absence and presenceof rosemary water extracts (RWE1 and RWE2). Fig-ure 2 shows the anti-adhesive activity after 4 h ofexposure to different concentrations of RWE1 andRWE2. In the absence of RWEs (positive control), allstudied strains showed high ability to adhere to poly-styrene substratum. Both tested extracts impaired butdid not prevent adhesion of all tested strains when theextracts were used at concentration of 4, 8 and12 mg mL�1. The anti-adhesive dose (that causedcomplete prevention of cell adhesion) of RWE1 was16 and 20 mg mL�1 against Bacillus and Pseudomonasstrains, respectively (Fig. 2A, B), whereas the anti-adhesive dose of RWE2 was 20 mg mL�1 against alltested strains (Fig 2C, D).

Antibiofilm activity was studied after 24 h of expo-sure to different concentrations of RWE1 and RWE2(Table 2). In the absence of RWE, the referencestrains B. cereus and P. aeruginosa were characterisedas moderate biofilm producers (++), while the biofilm-forming strains of both studied genera were strongbiofilm producer (+++). No changes in the ability oftested bacteria to form biofilm were noticed wheneither RWE1 or RWE2 was incorporated in incuba-tion medium up to 4 mg mL�1 (Table 2). All testedpathogens displayed remarkable reduction in biofilmformation when 8 mg mL�1 of RWEs were used, butthey still have ability to form biofilm. By increasingDTSS concentration of RWE1 to 12 mg mL�1,biofilm-forming ability of all tested strains was reducedto be weak producer (+), however, they still were mod-erate biofilm producers (++) when RWE2 was appliedat the same concentration. All Bacillus strains losttheir ability to form biofilm and became no biofilmproducer (0) when RWE1 was used at concentration

of 16 mg mL�1. Therefore, this concentration wasconsidered as antibiofilm dose of RWE1 against Bacil-lus strains. At this concentration, Pseudomonas strainsstill form biofilm (+). By increasing the concentrationof RWE1 to 20 mg mL�1, all studied Pseudomonasstrains became no biofilm producers. On the otherhand, antibiofilm dose of RWE2 against all Bacillusand Pseudomonas strains was 20 mg mL�1. From theabovementioned results, it can be noticed that theantibiofilm dose values were in harmony with the anti-

Table 2 Antibiofilm activitya of rosemary water extracts against

some Bacillus and Pseudomonas strains

Strains

Biofilm formationb

RWE1c (mg DTSSd per mL)

0 4 8 12 16 20

B. cereus DSMZ 345 ++ ++ ++ + 0 0

B. cereus TUB8 +++ +++ ++ + 0 0

B. cereus TUB30 +++ +++ ++ + 0 0

B. cereus TUB31 +++ +++ ++ + 0 0

B. cereus TUB32 +++ +++ ++ + 0 0

B. cereus TUB32 +++ +++ ++ ++ 0 0

P. aeruginosa ATCC 10145 ++ ++ ++ + 0 0

P. aeruginosa TUB14 +++ +++ ++ + + 0

P. alcaligenes TUB12 +++ +++ ++ + + 0

P. alcaligenes TUB13 +++ +++ ++ + + 0

P. monteilii TUB15 +++ +++ ++ + + 0

P. parafulva TUB16 +++ ++ ++ + + 0

Strains

Biofilm formationb

RWE2c (mg DTSSd per mL)

0 4 8 12 16 20

B. cereus DSMZ 345 ++ ++ ++ ++ + 0

B. cereus TUB8 +++ +++ ++ ++ + 0

B. cereus TUB30 +++ +++ ++ ++ + 0

B. cereus TUB31 +++ +++ ++ ++ + 0

B. cereus TUB32 +++ +++ ++ ++ + 0

B. cereus TUB32 +++ +++ ++ ++ + 0

P. aeruginosa ATCC 10145 ++ ++ ++ ++ + 0

P. aeruginosa TUB14 +++ +++ ++ ++ + 0

P. alcaligenes TUB12 +++ +++ ++ ++ + 0

P. alcaligenes TUB13 ++ +++ ++ ++ + 0

P. monteilii TUB15 +++ +++ ++ ++ + 0

P. parafulva TUB16 +++ ++ ++ ++ + 0

aAntibiofilm dose was defined when the tested strain became no

biofilm producer (0).bStrains were classified as no biofilm producer (0), weak biofilm

producer (+), moderate biofilm producer (++) or strong biofilm pro-

ducer (+++); n = 3.cRWE1 and RWE2 are rosemary water extracts of plant leaves collected

from Taif and Cairo, respectively, which were prepared by dissolving

per 1 mL: 0, 4, 8, 12, 16 or 20 mg of DTSS.dDTSS is the dried total soluble solids obtained after soaking rosemary

dry leaves in hot water (20:180 w/v) at 80 °C for 8 h.

© 2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al. 7

adhesive doses. These finding emphasises that the ini-tial adhesion is the first and indispensable stage in theformation of biofilm.

By comparing MIC90 with anti-adhesive dose and an-tibiofilm dose obtained in this study, it can be noticedthat the growth of studied bacteria may be inhibitedwhile they still have ability to adhere to polystyrenesurface and form biofilm. This could be explained bythose mentioned by Plyuta et al. (2013). They statedthat, at low concentrations that suppressed bacterialgrowth, plant phenolic compounds including gallic,ferulic and chlorogenic acids increased the capacity ofP. aeruginosa to adhesion and formation of biofilms.However, at higher concentrations, these compoundsdisplayed a biofilm-inhibiting effect. The high contentof several phenolic compounds (especially rosmarinicacid, benzoic acid, pyrogallol, ellagic acid, ferulic andgallic acids) and isoflavones (especially daidzein, geni-stein and isorhamnetin) in RWEs (Table 1) may explaintheir anti-adhesive and antibiofilm activity against thestudied bacteria. Most of these compounds have beenrecognised to affect the adhesive proteins and structuralproperties of the outer membrane and cell wall ofbacteria. For instance, plant extracts with high contentof ellagic acid have been strongly recommended fordevelopment as a botanical drug for use in theprevention and treatment of Staphylococcus aureus bio-film-associated infections (Quave et al., 2012). Isoflav-ones – daidzein and genistein – are known as inhibitorsof protein kinase activity. Moreover, genistein has beenreported to be able to change cell morphology and formfilamentous cells (Ulanowska et al., 2006). Anotherinstance, coumarins act on microorganism membranesas well as bind to polysaccharides, which play an impor-

tant role in microbial cell adhesion (Cowan, 1999).Ferulic and gallic acids promote reductions in biofilmactivity (>70%) for biofilms formed different Gram-positive and Gram-negative bacteria including P. aeru-ginosa (Borges et al., 2012). These two phenolic acidswere detected in considerable amount in RWE1 com-pared with that of RWE2 (Table 1).

Anti-adhesive effect (on HEp-2 cells) of rosemary waterextract

Adhesion of Bacillus and Pseudomonas cells has beenstudied using cell lines of human origin in culture as invitro models for intestinal epithelium. In the presentstudy, adhesion of Bacillus and Pseudomonas strains toHEp-2 cells in the absence and presence of the anti-adhesive dose, which has been recorded against eachbacterial group (Bacillus and Pseudomonas), was inves-tigated (Table 3). In the absence of RWEs, theadhered bacterial cells of Bacillus and Pseudomonasranged from 38 � 9 to 50 � 11 and from 34 � 10 to41 � 13 cells per HEp-2 cell, respectively. The anti-adhesive activity of RWEs was expressed as a reduc-tion percentage of adhesion (R%) and presented inTable 3. Inhibition in the bacterial cell adherence toHEp-2 by the reference strains B. cereus and P. aeru-ginosa was recorded at level of 63.1 and 64.7%, respec-tively, when RWE1 was used; and 52.6 and 52.9%,respectively, when RWE2 was applied. RWE1 wasmore effective for inhibiting bacterial cell adherencethan RWE2, where the respective reduction percent-ages of adhesion (R%) ranged from 17.5 to 64.7%and from 12.2 to 52.9%, respectively On the otherhand, both studied RWEs showed high anti-adhesive

Table 3 Anti-adhesive activity of RWEsa against some Bacillus and Pseudomonas strains

Strains

Number of adhered bacteria per HEp-2 cellb

Control RWE1a % Reductionc RWE2a % Reductionc

Bacillus cereus DSMZ 345 38.0 � 9 14.0 � 2 63.1 18.0 � 2 52.6

B. cereus TUB8 53.0 � 7 22.0 � 2 58.5 32.0 � 3 39.6

B. cereus TUB30 49.0 � 10 30.0 � 4 38.7 37.0 � 2 24.5

B. cereus TUB31 44.0 � 10 24.0 � 2 45.4 30.0 � 2 31.8

B. cereus TUB32 43.0 � 11 26.0 � 2 39.5 30.0 � 2 30.2

B. cereus TUB32 50.0 � 11 25.0 � 2 50.0 33.0 � 4 34.0

P. aeruginosa ATCC 10145 34.0 � 10 12.0 � 3 64.7 16.0 � 4 52.9

P. aeruginosa TUB14 36.0 � 10 28.0 � 4 22.2 30.0 � 3 16.6

P. alcaligenes TUB12 40.0 � 12 33.0 � 5 17.5 32.0 � 2 20.0

P. alcaligenes TUB13 33.0 � 8 26.0 � 4 21.2 26.0 � 2 18.2

P. monteilii TUB15 39.0 � 10 30.0 � 4 30.0 32.0 � 2 17.9

P. parafulva TUB16 41.0 � 13 39.0 � 5 29.3 36.0 � 4 12.2

aRWEs (RWE1 and RWE2) are rosemary water extracts of plant leaves collected from Taif and Cairo, respectively, which were prepared by dissolving

20 mg DTSS per mL; DTSS is the dried total soluble solids obtained after soaking rosemary dry leaves in hot water (20:180 w/v) at 80 °C for 8 h.bThe mean number (n = 3) of bacteria � SD per HEp-2 cell after examination of at least 100 different cells from five separate fields.cReduction percentage of adhesive; [number of adhered bacterial cells per HEp-2 cell (in the presence of RWEs) – number of adhered bacterial cells

per HEp-2 cell of control (in the absence of RWEs)]/number of adhered bacterial cells per HEp-2 cell of control.

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al.8

potential against Bacillus strains (R% ranged from24.5 to 58.5%) compared with Pseudomonas strains (R% ranged from 12.2 to 30%). This effect is in agree-ment with those mentioned previously (Rasooli et al.,2008; Jagani et al., 2009; Wojnicz et al., 2012; Plyutaet al., 2013). The authors mentioned that many plantextracts including rosemary led to reduced adhesionand consequently biofilm formation by Bacillus andPseudomonas. This may be due to the high phenoliccontent of the rosemary water extracts (Table 1). Onthe other hand, the environmental strains may acquiresome characteristics as a result of their presence in bio-film layers in the environment, where the exposure toenvironmental stresses leads to alteration of their sur-face properties (Elhariry et al., 2012). This explanationhas been demonstrated in our previous work, wherethe biofilm-forming strains of B. cereus (that havebeen isolated from slime layer of microbial biofilms indrinking water network and used in the present study)had showed high ability to form biofilm on green-leafyvegetables compared with the reference strains(Elhariry, 2011). This may explain the obtained resultswhich showed clearly that the biofilm-forming strainsof Bacillus and Pseudomonas displayed high resistanceto the anti-adhesive effect of studied RWEs comparedwith the reference strains (Table 3).

In conclusion, the study reveals that the hot-waterextract of rosemary leaves contained various bioactivecompounds, especially benzoic, ellagic, gallic and ros-marinic acids, daidzein and genistein. Significant anti-microbial, antibiofilm and anti-adhesive activities ofrosemary water extract were demonstrated againstBacillus and Pseudomonas. Practically, as anti-adhesiontherapy may become feasible for attenuating infectiousdisease, rosemary hot-water extract can be developedas an effective inhibitor to prevent bacterial cell adhe-sion to biotic (HEp-2 cells) or abiotic (polystyrene)surfaces and subsequently inhibiting biofilm formation.This may provide natural protection from the infectionby some pathogenic bacteria.

Acknowledgments

This work was supported by a grant (Contract No.1-433-1920) from Taif University, Kingdom of SaudiArabia.

Conflict of interest

The authors declare that there is no conflict of inter-est.

References

Abu-Lail, N.I. & Camesano, T.A. (2003). Role of ionic strength onthe relationship of biopolymer conformation, DLVO contributions,

and steric interactions to bioadhesion of Pseudomonas putidaKT2442. Biomacromolecules, 4, 1000–1012.

Al-Sereiti, M.R., Abu-Amer, K.M. & Sen, P. (1999). Pharmacol-ogy of rosemary (Rosmarinus officinalis Linn.) and its therapeu-tic potentials. Indian Journal of Experimental Biology, 37, 124–130.

Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B. &Weil, J.A. (2004). Free-radical scavenging capacity and antioxidantactivity of selected plant species from the Canadian prairies. FoodChemistry, 84, 551–562.

Angioni, A., Barra, A., Cereti, E., Barile, D., Coisson, J.D. &Arlorio, M. (2004). Chemical composition, plant genetic difference,antimicrobial and antifungal activity investigation of the essentialoil of Rosmarinus officinalis L. Journal of Agricultural and FoodChemistry, 52, 3530–3535.

Boddicker, J.D., Ledeboer, N.A., Jagnow, J., Jones, B.D. & Clegg,S. (2002). Differential binding to and biofilm formation on, HEp-2cells by Salmonella enterica serovar Typhimurium is dependentupon allelic variation in the fimH gene of the fim gene cluster.Molecular Microbiology, 45, 1255–1265.

Borges, A., Saavedra, M.J. & Sim~oes, M. (2012). The activity offerulic and gallic acids in biofilm prevention and control of patho-genic bacteria. Biofouling, 28, 755–767.

Camesano, T.A. & Logan, B. (2000). Probing bacterial electrostericinteractions using atomic force microscopy. Environmental Scienceand Technology, 34, 3354–3362.

Cetin-Karaca, H. (2011). Evaluation of natural antimicrobial pheno-lic compounds against foodborne pathogens. MSc Thesis, Nutri-tion and Food Science Department, University of Kentucky, USA.

Christensen, G.D., Simpson, W.A. & Younger, J.J. (1985). Adher-ence of coagulase-negative staphylococci to plastic tissue cultureplates: a quantitative model for the adherence of staphylococci tomedical devices. Journal of Clinical Microbiology, 22, 996–1006.

Cloete, T.E. & Jacobs, L. (2001). Surfactants and the attachment ofPseudomonas aeruginosa to 3CR12 stainless steel and glass. WaterSA, 27, 21–26.

Cowan, M.M. (1999). Plant products as antimicrobial agents. Clini-cal Microbiology Reviews, 12, 564–582.

Coward, L., Barnes, N.C., Setchell, K.D.R. & Barnes, S. (1993).Genistein, daidzein, and their b-glycoside conjugates: antitumorisoflavones in soybean foods from American and Asian diets. Jour-nal of Agricultural and Food Chemistry, 41, 1961–1967.

Drenkard, E. & Ausubel, F.M. (2002). Pseudomonas biofilm forma-tion and antibiotic resistance are linked to phenotypic variation.Nature, 416, 740–743.

Elhariry, H.M. (2008). Biofilm formation by endospore-formingBacilli on plastic surface under some food-related and environmen-tal stress conditions. Global Journal of Biotechnology and Biochem-istry, 3, 69–78.

Elhariry, H.M. (2011). Attachment strength and biofilm formingability of Bacillus cereus on green-leafy vegetables: cabbage and let-tuce. Food Microbiology, 28, 1266–1274.

Elhariry, H., Gherbawy, Y., El-Deeb, B. & Altalhi, A. (2012).Molecular identification and biofilm-forming ability of culturableaquatic bacteria in microbial biofilms formed in drinking waterdistribution networks. Geomicrobiology Journal, 29, 561–569.

Eloff, J.N. (1998). A sensitive and quick microplate method to deter-mine the minimal inhibitory concentration of plant extracts forbacteria. Planta Medica, 64, 711–713.

Erkan, N., Ayranci, G. & Ayranci, E. (2008). Antioxidant activitiesof rosemary (Rosmarinus officinalis L.) extract, blackseed (Nigellasativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol.Food Chemistry, 110, 76–82.

Fletcher, M. (1996). Bacterial Adhesion: Molecular and EcologicalDiversity. New York: Wiley.

Garrett, T.R., Bhakoo, M. & Zhang, Z. (2008). Bacterial adhesionand biofilms on surfaces. Progress in Natural Science, 18, 1049–1056.

© 2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al. 9

Goupy, P., Hugues, M., Boivin, P. & Amiot, M.J. (1999). Antioxi-dant composition and activity of barley (Hordeum vulgare) andmalt extracts and of isolated phenolic compounds. Journal of theScience of Food and Agriculture, 79, 1625–1634.

Hamza, S.J., Al-Zubaidy, S.H. & Al-Amir, L.A.K. (2010). Role ofPili and Polysaccharide in adherence of Pseudomonas aeruginosa tomammalian epithelial cells. Journal of Biotechnology ReserchCenter, 4, 111–117.

Huttunen, S., Toivanen, M., Arkko, S., Ruponen, M. & Tikkanen-Kaukanen, C. (2011). Inhibition activity of wild berry juicefractions against Streptococcus pneumoniae binding to human bron-chial cells. Phytotherapy Research, 25, 122–127.

Jagani, S., Chelikani, R. & Kim, D.S. (2009). Effects of phenol andnatural phenolic compounds on biofilm formation by Pseudomonasaeruginosa. Biofouling, 25, 321–424.

Klan�cnik, A., Guzej, B., Kolar, M.H., Abramovi�c, H. & Mo�zina,S.S. (2009). In vitro antimicrobial and antioxidant activity of com-mercial rosemary extract formulations. Journal of Food Protection,72, 1744–1752.

Kreske, A.C., Ryu, J.-H., Pettigrew, C.A. & Beuchat, L.R. (2006).Lethality of chlorine, chlorine dioxide, and a commercial producesanitizer to Bacillus cereus and Pseudomonas in a liquid detergent,on stainless steel, and in biofilm. Journal of Food Protection, 14,2621–2634.

Lindsay, D., Br€ozel, V.S., Mostert, J.F. & Von Holy, A. (2002). Dif-ferential efficacy of a chlorine dioxide-containing sanitizer againstsingle species and binary biofilms of a dairy-associated Bacillus cer-eus and a Pseudomonas fluorescens isolate. Journal of AppliedMicrobiology, 92, 352–361.

Liu, D.-C., Tsau, R.-T., Lin, Y.-C., Jan, S.-S. & Tan, F.-J. (2009).Effect of various levels of rosemary or Chinese mahogany on thequality of fresh chicken sausage during refrigerated storage. FoodChemistry, 117, 106–113.

Manuzon, M.Y. (2009). Investigation of Pseudomonas biofilm devel-opment and removal on dairy processing equipment surfacesusingFourier Transform Infrared (FT-IR) spectroscopy. PhD, The OhioState University, Columbus, Ohio, USA.

Mathlouthi, N., Bouzaienne, T., Oueslati, I. et al. (2012). Use ofrosemary, oregano, and a commercial blend of essential oils inbroiler chickens: in vitro antimicrobial activities and effects ongrowth performance. Journal of Animal Science, 90, 813–823.

Miguel, M., Neves, M. & Antunes, M. (2010). Pomegranate (Punicagranatum L.): a medicinal plant with myriad biological properties –a short review. Journal of Medicinal Plant Research, 4, 2836–2847.

Moreno, S., Scheyer, T., Romano, C.S. & Vojnov, A.A. (2006).Antioxidant and antimicrobial activities of rosemary extractslinked to their polyphenol composition. Free Radical Research, 40,223–231.

O’Toole, G., Kaplan, H.B. & Kolter, R. (2000). Biofilm formationas microbial development. Annual Review of Microbiology, 54, 49–79.

Paiva, P.M.G., Gomes, F.S., Napole~ao, T.H., S�a, R.A., Correia,M.T.S. & Coelho, L.C.B.B. (2010). Antimicrobial activity of sec-ondary metabolites and lectins from plants. In: Current Research,Technology and Education Topics in Applied Microbiology andMicrobial Biotechnology (edited by A. M�endez-Vilas). Pp. 396–406.Badajoz, Spain: FORMATEX, Microbiology Series No 2.

Plyuta, V., Zaitseva, J., Lobakova, E., Zagoskina, N., Kuznetsov, A.& Khmel, I. (2013). Effect of plant phenolic compounds on biofilm

formation by Pseudomonas aeruginosa. APMIS: Acta Pathologica,Microbiologica, et Immunologica Scandinavica, doi:10.1111/apm.12083 [Epub ahead of print].

Prabuseenivasan, S., Jayakumar, M. & Ignacimuthu, S. (2006). Invitro antibacterial activity of some plant essential oils. BMC Com-plementary and Alternative Medicine, 6, 39–46.

Quave, C.L., Est�evez-Carmona, M., Compadre, C.M., et al. (2012).Ellagic acid derivatives from Rubus ulmifolius Inhibit Staphylococ-cus aureus biofilm formation and improve response to antibiotics.PLoS One, 7, e28737.

Quave, C.L., Plano, L.R.W., Pantuso, T. & Bennett, B.C. (2008).Effects of extracts from Italian medicinal plants on planktonicgrowth, biofilm formation and adherence of methicillin-resistantStaphylococcus aureus. Journal of Ethnopharmacology, 118, 418–428.

Rajkumar, A.P. & Kanimozhi, M. (2010). Phytochemical screeningand antimicrobial activity from five Indian medicinal plant againsthuman pathogens. Middle-East Journal of Scientific Research, 5,477–482.

Rasooli, J., Shayegh, S., Taghizadeh, M. & Astaneh, S.D.A. (2008).Phytotherapeutic prevention of dental biofilm formation. Phyto-therapy Research, 22, 1162–1167.

Ruiz-Bustos, E., Velazquez, C., Garibay-Escobar, A., Garcia, Z. &Plascencia-Jatomea, M. (2009). Antibacterial and antifungal activi-ties of some Mexican medicinal plants. Journal of Medical Food,12, 1398–1402.

Sharon, N. (2006). Carbohydrates as future anti-adhesion drugs forinfection diseases. Biochimica et Biophysica Acta, 1760, 527–537.

Stepanovi�c, S., Vukovi�c, D., Daki�c, I., Savi�c, B. & �Svabi�c-Vlahovi�c,M. (2000). A modified microtiter-plate test for quantification ofstaphylococcal biofilm formation. Journal of Microbiological Meth-ods, 40, 175–179.

Taguri, T., Tanaka, T. & Kouno, I. (2004). Antimicrobial activity of10 different plant polyphenols against bacteria causing food-bornedisease. Biological & Pharmaceutical Bulletin, 27, 1965–1969.

Tsai, P.J., Tsai, T.H. & Ho, S.C. (2007). In vitro inhibitory effects ofrosemary extracts on growth and glucosyltransferase activity ofStreptococcus sobrinus. Food Chemistry, 105, 311–316.

Ulanowska, K., Tkaczyk, A., Konopa, G. & Wezgrzyn, G. (2006).Differential antibacterial activity of genistein arising from globalinhibition of DNA, RNA and protein synthesis in some bacterialstrains. Archives of Microbiology, 184, 271–278.

Vaara, M. (1992). Agents that increase the permeability of the outermembrane. Microbiological Review, 56, 395–411.

Wang, Q.I.N., Ou, Z., Lei, H., Zeng, X., Ying, Y.U.E. & Bai, W.(2012). Antimicrobial activities of a new formula of spice waterextracts against foodborne bacteria. Journal of Food Processing andPreservation, 36, 374–381.

Wilson, J., Schurr, M., Leblanc, C., Ramamurthy, R., Buchanan, K.& Nickerson, C. (2002). Mechanisms of bacterial pathogenicity.Postgraduate Medical Journal, 78, 216–224.

Wojnicz, D., Kucharska, A.Z., Sok�o-etowska, A., Kicia, M. & Tich-aczek-Goska, D. (2012). Medicinal plants extracts affect virulencefactors expression and biofilm formation by the uropathogenicEscherichia coli. Urological Research, 40, 683–697.

Zhang, L., Kong, Y., Wu, D. et al. (2008). Three flavonoids target-ing the b-hydroxyacyl-acyl carrier protein dehydratase from Heli-cobacter pylori: crystal structure characterization with enzymaticinhibition assay. Protein Science, 17, 1971–1978.

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2013

Antibiofilm and anti-adhesive effects of rosemary H. Elhariry et al.10