Colostrum Hexasaccharide, a Novel Staphylococcus aureus Quorum-Sensing Inhibitor

A. Srivastava,a,b B. N. Singh,a D. Deepak,b A. K. S. Rawat,a B. R. Singhc

Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, Indiaa; Chemistry Department, Lucknow University,Lucknow, Uttar Pradesh, Indiab; Centre of Excellence in Materials Science (Nanomaterials), Z.H. College of Engineering & Technology, Aligarh Muslim University, Aligarh,Uttar Pradesh, Indiac

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novelopportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria.But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has beenpublished. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterizedCHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquidchromatography evaporative light-scattering detection (RP-HPLC-ELSD), 1H and 13C nuclear magnetic resonance (NMR), andelectrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interferencewith bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by micro-scopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrad-ing acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. Theresults revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreadingability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicatedthat CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere withbacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibioticresistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generationantibacterial.

Staphylococcus aureus is a prevalent and highly adaptable Gram-positive bacterium causing numerous clinical infections (1). It

is a well-documented fact that the occurrence of these infections ismuch more frequent in humans (impetigo) and in dairy cattle(mastitis) than in others (2). The development of antibiotic resis-tance in S. aureus and other species has therefore become a seriousconcern in the medical community (3). One attractive approachto this problem is to target bacterial virulence systems rather thanessential cellular processes. It is hoped that this tactic can reduceselective survival pressures and also slow down the emergence ofresistance. It is well established that S. aureus and other species usequorum sensing (QS), a chemical communication process to ac-tivate defense mechanisms, such as secretion of virulence factorsand biofilm formation (1, 4, 5). These defenses pose a greaterthreat to the host than a common commensal. The discovery of aglobal regulatory QS system for virulence in S. aureus mediated bysmall signaling peptides has provided an appealing opportunityfor inactivating these defenses (5–7).

Numerous studies have demonstrated that QS inhibitors, pro-duced by eukaryotes and prokaryotes, are able to interrupt QS-regulated behaviors of bacteria (8, 9). Unfortunately, most ofthese QS inhibitors, for example, halogenated furanones, are un-suitable for human use, due to their toxicity issues. Therefore,there is an increasing need for the identification of novel nontoxicinhibitors with dual properties, such as anti-QS and antibiofilm.Identification of such natural inhibitors from a dietary sourcecould present us with new opportunities for the development ofnovel drugs to combat bacterial infections. The development ofapproaches that interfere with proper microbial QS signaling hasreceived significant attention, particularly for problematic multi-

antibiotic-resistant isolates like S. aureus (10). Thus, looking fordietary molecules with anti-QS activity may also be an attractivealternative to antibiotics, considering that in recent investigations,dietary compounds have shown the ability to disrupt bacterial QSsystems (1, 11). In this context, colostrum, or first milk, which is aform of milk produced by the mammary glands of mammals inlate pregnancy whose main function is to protect the newbornagainst pathogen invasion, could be a good dietary source of an-timicrobial substances. It has been previously demonstrated thatbovine colostrum contains dietary substances which reduce theinvasion of many human pathogens, including Escherichia coli,Klebsiella pneumonia, Cryptosporidium parvum, Shigella flexneri,Salmonella, and Staphylococcus (12). Before the discovery of anti-biotics, colostrum was the main source of antimicrobials used tofight infections. These reported properties could be explained,

Received 26 June 2014 Returned for modification 3 August 2014Accepted 23 August 2014

Accepted manuscript posted online 2 February 2015

Citation Srivastava A, Singh BN, Deepak D, Rawat AKS, Singh BR. 2015. Colostrumhexasaccharide, a novel Staphylococcus aureus quorum-sensing inhibitor.Antimicrob Agents Chemother 59:2169 –2178. doi:10.1128/AAC.03722-14.

Address correspondence to B. N. Singh, brahmasingh99@gmail.com, or A. K. S.Rawat, rawataks@rediffmail.com.

A.S. and B.N.S. contributed equally.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.03722-14

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among other factors, by a possible interruption of the colostrumin these bacterial QS systems.

Taking this into account, it seemed reasonable that the dietarypolysaccharides isolated from milk colostrum could have anti-QSand antibiofilm properties against bacteria. With this assumption,the purpose of the present investigation was to examine whetherisolated colostrum hexasaccharide (CHS) could inhibit QS signal-ing, production of virulence factors (VFs), and acyl-homoserinelactone (AHL) degradation, and also whether it is able to preventor destroy the biofilm formation in S. aureus.

MATERIALS AND METHODSBacterial strains and culture conditions. The bacterial biomarker strainChromobacterium violaceum ATCC 12472, S. aureus MTCC3160, andEscherichia coli MTCC1089 were obtained from the Microbial Type Cul-ture Collection (MTCC), India. C. violaceum 12472 was characterized byhigh violacein production and has previously been used in anti-QS studies(5, 13). C. violaceum 12472 was cultivated in Luria-Bertani (LB) medium,while tryptone soy broth (TSB)-0.25% glucose medium (pH 7.0 � 0.2)was used for cultivation of S. aureus and maintained at 37°C. E. coli,Pseudomonas fluorescens P3/pME6863, and P. fluorescens P3/pME6000were grown overnight at 30°C in nutrient medium. For experimentalanalysis, the optical densities of the cultures were adjusted to the respec-tive sterile culture medium to an optical density at 600 nm (OD600) of 0.5.

Apparatus for isolation and identification. The 1H and 13C nuclearmagnetic resonance (NMR) spectra were recorded at 300 MHz with te-tramethylsilane as the internal standard on a Bruker Avance instrument.Chemical shifts are given in parts per million. Correlation spectroscopy(COSY), total correlation spectroscopy (TOCSY), and heteronuclear sin-gle quantum coherence (HSQC) were performed using standard Brukerpulse programs. Mass spectra were recorded on an API 3000 triple qua-drupole mass spectrometer (Applied Biosystems, USA). Sephadex G-25column chromatography (GE Healthcare, India) was used for purificationof the targeted molecule. C18 reverse-phase high-performance liquidchromatography (C18-RP-HPLC; Shimadzu, Japan) consisting of a col-umn (4.6 by 250 mm), pump LC-8A, and a UV detector was used forisolation and purification of a reference compound. High-performancethin-layer chromatography (HPTLC) was performed on CAMAG TLCScanner 3 at a �max of 450 nm in UV absorbance mode for acetylatedcompound by using winCATS software.

Collection and processing of colostrum. Colostrum (250 � 20 ml/day up to 2.0 liters) was collected from female mares (Equus caballus) atDirectorate of Animal Husbandry, Lucknow, India, and kept at �20°Cuntil used. Colostrum was thawed at room temperature, and then thesolidified upper layer was removed by filtration at 0°C. The remainingcolostrum was increased to 2.0 liters by adding 50% ethanol and then leftat 4°C for 8 h. The sample was decanted slowly to remove lactose andprotein, and the obtained supernatant was centrifuged at 6,500 � g (Remi,India). Residual ethanol was removed with the help of a rotary evaporator(Buchi, Switzerland). The remaining supernatant was filtered through a0.22-�m-pore-size filter apparatus (Pall, USA) and lyophilized (Lab-conco, USA) to get a crude oligosaccharide mixture.

Purification, isolation, and characterization of CHS. The lyophilizedcrude mixture of oligosaccharides was packed on a Sephadex G-25 chroma-tography column (1.6 by 40 cm; void volume of 25 ml) and eluted with tripledistilled water (TDW) at a rate of 10 ml/30 min. A total of 320 fractions werecollected (see Table S1 in the supplemental material at https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit). The UV spectra ofthese fractions showed seven peaks (see Fig. S1 posted at https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit). Fractions (48 to 271)under peaks II, III, IV, and V gave a positive phenol-sulfuric acid test, therebyconfirming the presence of oligosaccharides. These fractions were pooled andlyophilized together. Due to the highly polar nature of oligosaccharide, de-rivatization to acetylate them was done as described earlier (14). Separation ofthe acetylated product (150 mg) was carried over a silica gel (50 g) by eluting

with CHCl3 and MeOH, thereby resulting in 10 fractions (50 ml each). Frac-tion 8 showed pure compound, which was chromatographed by CAMAG-HPTLC (CHCl3-MeOH, 9:1) and C18-RP-HPLC-ELSD (acetonitrile-aceticacid, 9:1, 1 ml/min), which yielded hexasaccharide compound (denoted asCHS) with an Rf value of 0.51 (Fig. 1A) and a tR (retention time) value of 5.56min, respectively (Fig. 1B) (15). The 1H and 13C NMR data for the markercompound are presented in Table 1 and Fig. 1C and D. The structure eluci-dation of the compound was performed with the help of UV, 1H NMR, 13CNMR, two-dimensional (2D) experiments (heteronuclear single quantumcoherence [HSQC], correlation spectroscopy [COSY], and total correlationspectroscopy [TOCSY]), and electrospray ionization mass spectrometry(ESI-MS) analysis. Obtained data confirmed the presence of branched hex-asaccharide having one reducing-end Glc, one �-galactosidase (�-Gal), one�-GlcNAc, two �-GalNAc, and one �-GalNAc moiety and interpreted ac-cording to IUPAC nomenclature as ��,D-glucopyranosyl-1¡4)-�,D-galac-topyranosyl-(1¡6)-�,D-glucosamine-(1¡3)-�,D galactosamine-[(1¡4)-�,D galactopyranosyl]-(1¡3)-�,D-galactosamine (Fig. 2). (The results aredescribed in detail in Tables S1 and S2 and Fig. S1 to S5, posted at https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit.) The CHS wasthen dissolved in double-distilled water to prepare working solutions of 1, 2,3, 4, and 5 mg/ml.

Determination of MIC of CHS. The MIC of CHS against S. aureus wasdetermined as per the modified guidelines of the Clinical and LaboratoryStandards Institute, USA (16). Briefly, in a sterile, flat-bottom plate (Tar-sons, India), 1% (100 �l) of overnight culture of S. aureus (OD600 of 0.5)was added to TSB-0.25% glucose (100 �l) and supplemented with 2-foldserially diluted CHS to attain final concentrations ranging from 2 to 10mg/ml. The plate was covered with a BREATHseal along with the lid andkept at 37°C under static conditions for 12 h. Turbidity was examined byreading the OD600 using a UV-visible (UV-Vis) spectrophotometer(Thermo Scientific, USA). The MIC was recorded as the lowest concen-tration which showed complete inhibition of visible growth of the bacte-ria. The readings were performed in two independent assays and in du-plicate per trial.

Anti-QS activity assay. To examine the anti-QS activity of CHS, astandard disk diffusion assay was used as described elsewhere (13). Briefly,5 ml of molten LB agar (0.25% [wt/vol]) was inoculated with 50 �l of anovernight-grown culture of C. violaceum 12472. This agar-culture solu-tion was immediately poured on an LB agar plate. Various concentrationsof CHS (20 �l of each) to be tested were applied on sterilized paper disks(Axiva, India) and incubated overnight at 37°C. Anti-QS activity was ex-amined by a colorless and opaque halo zone of inhibition around the disk.

Violacein inhibition assay. Violacein inhibition assay was carried outaccording to the method of Singh et al. (17). Overnight culture of C.violaceum 12472 (OD600 of 0.5) was added into sterilized culture tubes(Borosil) containing LB broth and incubated at 37°C in the presence ofCHS. After incubation for 24 h, the culture was treated with 10%sodium dodecyl sulfate (SDS; HiMedia) and incubated at room tem-perature for 5 min to lyse the cells. For fractionation of the violaceinpigment, water-saturated n-butanol was added, and the mixture wasvortexed and centrifuged at 13,000 rpm for 10 min. The upper layer,containing violacein, was collected and quantified at A585 using a UV-Vis spectrophotometer.

Determination of virulence factors. For hemolysis assay, TSB con-taining S. aureus (OD600 of 0.5) was added to each well of the 96-wellmicrotiter plate. The CHS (dissolved in TSB) was also added, and thenplates were incubated statically at 37°C for 6 h. The cultures were thentested for hemolytic activity. Rat red blood cells (RBCs) were centrifugedat 3,000 � g for 5 min. The supernatant was discarded and the cells wereresuspended in saline phosphate buffer (PBS; pH 7.2 � 02). The sus-pended RBCs were added and incubated at 37°C for 20 min. The plateswere centrifuged to pellet down the cells at 450 � g for 5 min. The super-natant was transferred to a fresh microtiter plate, and absorbance wasmeasured at A420 using a plate reader (18).

The spreading ability assay was performed on TSB, containing 0.3%

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(wt/vol) agar-agar powder and various concentrations of CHS. The over-night culture of S. aureus (OD600 of 0.5) was point inoculated at the centerof the medium and incubated for 24 h at 37°C. Levels of the spreadingability were determined by measuring diameters of the spreading and thencompared with the control. For proteolytic assay, TSB culture mediumsupplemented with 2% casein (Sigma-Aldrich, USA) was used. After in-

oculation of S. aureus (OD600 of 0.5) and with various concentrations ofCHS, the mixture was incubated at 37°C for 24. The liberated peptides byproteinase action were determined according to reference 19. Agar plateassay was applied to determine lipolytic activity. For this, the CHS (1 to 5mg/ml) was added to TS agar containing 1% tributyrin (Fluka, Buchs,Germany). S. aureus was subcultured and incubated at 37°C for 72 h. The

FIG 1 Characterization of CHS. (A) HPTLC plate photograph; (B) RP-HPLC-ELSD chromatogram; (C) 1H-1H COSY NMR spectra; (D) 1H-13C HSQC spectra.MO, mixture of oligosaccharide; F-8, fraction 8.

TABLE 1 1H and 13C NMR values of CHS

Sugar

Chemical shift (ppm)

H-1 H-2 H-3 H-4 H-5 H-6 NAc C-1 C-2 C-3 C-4 C-5 C-6 -CO -CH3

�-Glc(S1) 5.26 3.64 3.68 3.96 3.34 3.88 91.3 72.0 72.0 78.1 70.9 62.5�-Glc(S1) 4.68 3.34 3.61 3.81 3.52 3.76 95.3 74.3 74.9 78.1 77.8 60.5�-Gal(S2) 4.45 3.55 3.24 3.72 3.58 3.75 102.4 70.5 78.1 69.6 74.3 72.0�-GlcNAc(S3) 4.65 3.36 3.75 3.69 3.52 NDa 2.30 102.7 59.9 73.3 74.9 77.8 59.5 176 20.8�-Gal(S4) 4.45 3.56 3.66 3.72 3.88 ND 102.4 70.7 77.8 68.1 74.3 62.5�-GalNAc(S5) 5.26 3.68 3.31 3.80 3.66 ND 2.30 102.4 51.9 68.1 70.5 72.0 62.5 173 20.8�-GalNAc(S6) 4.36 4.30 3.23 3.68 ND ND 2.30 102.4 59.5 70.9 69.6 78.1 60.5 173 20.8a ND, not detectable.

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inhibition of clear zones was considered an indicator of antilipolytic ac-tivity of CHS (20).

Degradation of AHLs by CHS. The AHL degradation potential ofCHS was assessed by the method of Medina-Martinez et al. (21). The CHSwas mixed with 5 �g/ml of C4-AHL and C12-AHL (Sigma-Aldrich, USA)in the presence of E. coli after 6 h of incubation. The recombinant strainsof P. fluorescens, P3/pME6863 and P3/pME6000, were used as the de-grader strain and nondegrader strain, respectively. The reaction mixturewas incubated for 24 h at 30°C with constant shaking at 180 rpm. After-ward, the mixture was centrifuged at 4,000 � g for 10 min (1). The super-natant obtained was extracted with acidified ethyl acetate (1% [vol/vol]acetic acid) (supernatant-acidified ethyl acetate, 7:3 [vol/vol]) and finallyconcentrated and dried using a rotary evaporator at 40°C (22). One mi-croliter of ethyl acetate extract was spotted onto TLC plates (silica gel 60F254; Merck). The separation of AHLs was obtained on a mobile phaseconsisting of methanol-water-acetic acid (8:1.5:0.5) and photographedunder UV light, and the content of AHLs was quantified using HPTLC,with a densitometer (CAMAG, Switzerland).

Biofilm inhibition assay. The effect of CHS on S. aureus biofilm for-mation was determined by biofilm inhibition assay. This protocol is basedon our group’s methodology to stain the biofilm with crystal violet (CV)and analyze it by light microscopy and confocal laser scanning microscopy(CLSM) analyses. Experiments were performed on 1- by 1-cm coverglasses on 24-well microtiter plates. One percent of overnight bacterialculture was diluted in TSB-0.25% glucose, and a 1-ml aliquot of it addedwas added to each well. Subsequently, various concentrations of CHSwere added. Plates were closed with BREATHseal along with the lid andplaced in an incubator maintained at 37°C for 24 h. After incubation, thecover glasses were rinsed with distilled water to remove the planktoniccells. The biofilms adhered on the cover glasses were stained with 1% CVsolution and SYTO-9 fluorescent dyes and then examined under a lightmicroscope (Leica, Germany) and by CLSM (LSM 510; Germany), re-spectively, at a magnification of �20 (11).

Biofilm inhibition by CHS and the different fractions thereof was alsoquantified by using a round-bottom 96-well-polystyrene-plate assay (23).Each sample and dilution was tested in triplicate in three independentassays. The bacterial suspension (200 �l in each well) and treatment withCHS were as described above. After 24 h of incubation, the media wereaspirated carefully and rinsed twice with distilled water, and the biofilmwas fixed with 200 �l of 0.12 M HCl for 80 min at room temperature. Thefixative was removed and 200 �l of 0.4% CV was added to stain the biofilmfor 5 min. The stain was removed and the wells were washed two timeswith distilled water to remove excess solution. The stain was further sol-ubilized with 0.2 ml of 95% ethanol. The biomass of the biofilm wasquantified by measuring the intensity of CV in ethanol at OD650 by usinga UV-visible spectrophotometer (Bio-Rad, USA). The positive control(TSB and S. aureus) was considered to show good biofilm formation whenthe optical density was more than 0.3. Positive and negative controls com-prising TSB and CHS for sample background color were included in eachassay.

Biofilm eradication assay. The biofilm eradication assay was a mod-ified version of the biofilm inhibition assay in which first S. aureus wasallowed to form the biofilm inside the wells for 36 h. Different concentra-

tions of CHS were added to previously formed biofilms for their eradica-tion. Experiments were conducted in sterile round-bottom 96-well mi-crotiter plates. Plates were placed in the incubator at 37°C for the next 24h. After incubation, the cover glasses were rinsed with distilled water toremove the planktonic cells. The biofilms adhered on the cover glasseswere stained with SYTO-9 fluorescent dyes and then examined by CLSM(LSM 510; Germany) at a magnification of �20.

For quantification, S. aureus culture suspension was prepared by di-luting 1:99 in TSB-0.25% glucose from the overnight culture. One-hun-dred-microliter aliquots were added to each well, and plates were incu-bated at 37°C for 36 h. Various concentrations of CHS were prepared inTSB, and their 100-�l aliquots were transferred to the bacterial plate. Theplates were covered and incubated overnight at 37°C for 24 h. The fixationwith 0.12 HCl, staining with 0.4% CV, and dissolution with NaOH pro-cedures were the same as those described above for the biofilm inhibitionassay. The remaining CV content was measured by UV-Vis spectropho-tometer at A650 (5). The positive control (TSB and S. aureus) was consid-ered a good biofilm former when the absorbance exceeded 0.3. Positiveand negative controls comprising TSB and CHS for sample backgroundcolor were included in each assay.

Synergistic effect of CHS with antibiotics. One percent of the over-night-grown culture of S. aureus (OD600 of 0.5) was added to 1 ml of LB ona 24-well microtiter plate containing CHS (5 mg/ml) and antibiotics (15�g/ml): clindamycin, linezolid, and erythromycin. The controls weremaintained with their respective antibiotic and without CHS. The plateswere incubated overnight at 37°C, and growth was measured after 24 h ofincubation at A600 nm using a UV-visible spectrophotometer (11).

Statistical analysis. Values are presented as means � standard devia-tions (SD) and analyzed by using one-way analysis of variance (SPSS11.5). For all analyses, the criterion for significance was a P value of 0.05.

RESULTSMIC of CHS. First of all, MIC was assessed to throw away anypossible inhibitory effect of CHS on S. aureus growth using thedoubling dilution method with the concentrations varying from 2to 10 mg/ml. The data obtained revealed no inhibition duringincubation until 12 h with CHS (�5 mg/ml) (see Fig. S6 posted athttps://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit).

Anti-QS activity of CHS. Production of QS-regulated pur-ple-colored violacein pigmentation in C. violaceum 12472 pro-vides a naturally occurring model system without the need foradditional substrate to evaluate anti-QS activity of molecules (5).Using disk diffusion assay, the anti-QS activity of CHS was deter-mined. The turbid zone of violacein inhibition around the disk inC. violaceum 12472 by CHS in a concentration-dependent mannerwas detected, indicating the anticipated inhibition of QS (Fig. 3A).This might be an important attribute, as QS inactivation is focusedon the attenuation of bacterial signaling. At all concentrationsused, the CHS and halogenated furanone (HF; 1.5 �g/ml) did notshow a clear halo zone of the test organism (see Fig. S7A posted at

FIG 2 Chemical structure of CHS.

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https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit), except gentamicin (GMN; 10 �g/ml), which showed aclear halo zone (see Fig. S7B posted at https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit). The turbid zonesproduced on lawns of C. violaceum could be the result of eitherinhibition of cell growth or quenching of QS systems. To confirm,we performed growth kinetic studies. The growth curves of C.violaceum 12472 showed that the CHS at 10 mg/ml did not inhibitgrowth and inhibited only violacein production (Fig. 3B).

Inhibition of violacein by CHS. CHS was subjected to quali-tative analysis to find out its anti-QS potential against C. violaceum12472. The bacterium is reported to produce the violet-coloredpigment violacein by responding to its QS signal molecule AHLs.The AHLs bind to its receptor LasR, and this complex triggers theproduction of violacein. In qualitative analysis, CHS showed aconcentration-dependent inhibition of violacein production in C.violaceum 12472. Inhibitory activity of CHS on violacein produc-tion was further measured spectrophotometrically. Concentra-tion- and time-dependent reduction in violacein content of C.violaceum 12472 was observed. Inhibition of up to 85.7% and100% was determined when treated with CHS at 5 mg/ml for 24and 48 h of incubation, respectively (Fig. 3C).

Inhibition of VF secretion by CHS. Numerous studies havedocumented that the production of hemolysins is regulated by theQS system in S. aureus (24). Therefore, any compound that inhib-

its the QS system should also inhibit the production of hemoly-sins, and we reasoned that this outcome could be readily quanti-tated by bacterial hemolysis assay. Inhibition of hemolysin wasmeasured as 2.91, 16.8, 40.9, 67.9, and 88.3% in the samplestreated with CHS at 1, 2, 3, 4, and 5 mg/ml, respectively (Fig. 4A).Next, we evaluated the inhibition of colony spreading ability of S.aureus by CHS, and we found concentration-dependent reduc-tion by 17, 34, 63, 88, and 100% at 1, 2, 3, 4, and 5 mg/ml CHS,respectively (Fig. 4C).

Proteases and lipases also play a major role in the pathogen-esis of S. aureus due to their hydrolytic activities. Epidermal per-meability barrier dysfunction by protease in nude mice was as-sessed (25); therefore, we were quite interested to determinewhether CHS could inhibit protease activity of S. aureus. For this,S. aureus was grown in the presence of 0, 1, 2, 3, 4, and 5 mg/ml ofCHS, and obtained supernatants were used for determination ofprotease and lipase activities. The data revealed that increasing theconcentration from 1 to 5 mg/ml of CHS enhanced the inhibitionof both lytic enzymes (Fig. 4B and D.). Inhibition of protease andlipase activities was observed to be a maximum of 74.2% and52.9%, respectively, when S. aureus cells were treated with 5mg/ml of CHS.

Inhibition of the QS system by CHS via degradation of AHLs.Recent studies have proven that signaling molecule or autoin-ducer AHLs play an important role in regulation of QS-dependent

FIG 3 (A) Disk diffusion assay for anti-QS activity using the C. violaceum 12472 biomonitor strain. A “halo” zone of inhibition of bacteria indicates an anti-QSaction of CHS in a dose-dependent manner. As the untreated control, an H2O-treated sterile paper disk was used. (B) Effect of CHS on the growth of C. violaceum12472. Cells were grown in the presence of different concentrations of CHS (0 to 10 mg/ml). Samples were taken from each culture at OD600 values for theindicated time intervals. The CHS did not inhibit growth of C. violaceum 12472. (C) Quantitative analysis of violacein production inhibition in C. violaceum12472 by CHS at 24 and 48 h. Inhibition values were directly plotted as the means from eight independent experiments � SD against various concentrations ofCHS. * and *** indicate significance at P values of 0.05 and 0.001, respectively.

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expression of virulence genes in Gram-negative and Gram-posi-tive bacteria (5). The potentiality of CHS to degrade C4-AHL andC12-AHL was determined by HPTLC (CAMAG, Switzerland) andquantified by a densitometer. In Fig. 5A and B, the amounts ofAHLs for each treatment were compared with their respectivepositive controls as shown. As anticipated, the treatment for Pseu-domonas fluorescens P3/pME6863 (an AHL degrader control

strain) showed very small amounts of both C4-AHL and C12-AHL,at 4 and 3 �M, respectively, while no degradation of C4-AHL andC12-AHL was observed in the treatment for P. fluorescens P3/pME6000 (an AHL nondegrader control strain). Remarkable deg-radations of AHLs were also observed when samples were treatedwith CHS. The obtained results thus revealed that the intensity ofthe AHL bands was reduced with increasing concentrations of

FIG 4 Effect of CHS on virulence factor (VF) production of S. aureus. Cells were treated with various concentrations of CHS (diluted in TSB) and incubatedstatically at 37°C. The supernatant was used to examine hemolytic activity (A) and proteolytic activity (B). Plate assays were used to determine the inhibition ofspreading ability (C) and lipolytic activity (D) of CHS, and results were expressed as the percent inhibition compared to that of the untreated controls. Inhibitionvalues were directly plotted as the means from six independent experiments � SD against various concentrations of CHS. *, **, and *** indicate a significance atP values of 0.05, 0.01, and 0.001.

FIG 5 AHL degradation potential of CHS. The remaining AHL concentrations of samples were measured after the incubation of 2.7 ml of each CHSconcentration with 0.3 ml of C4-AHL and C12-AHL for 24 h at 37°C in the presence of E. coli (used as a Gram-negative bacterium). The separation of AHLs wasobtained on a mobile phase consisting of methanol-water-acetic acid (8:1.5:0.5) and photographed under UV at 254 nm (A) and 366 nm (B). The contents ofC4-AHL and C12-AHL were quantified using HPTLC, and results were obtained by taking the average from three replicates in three independent assays. ** and*** indicate significance at P values of 0.01 and 0.001. M, marker compound; DS, AHL degrader control strain (P. fluorescens P3/pME6863); NDS, AHLnondegrader strain (P. fluorescens P3/pME6000); Hs, hexasaccharide.

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CHS. At 5 mg/ml, CHS displayed very faint bands of both C4-AHLand C12-AHL, with mean values of 2 �M for both, compared withthe controls by 10 and 7 �M, respectively. The rest of the lowconcentrations tested were also shown to be considerably effectivein a dose-response picture.

Antibiofilm activity of CHS. Direct microscopic observationsof biofilm inhibition and biofilm eradication after exposure toCHS are known to provide valuable information of the action ofantibiofilm compounds; therefore, light microscopy and confocallaser scanning microscopy (CLSM) analyses were performed. In-hibition of biofilm formation in S. aureus showed a clear doseresponse to increasing concentrations of CHS (Fig. 6A). A thickcoating of biofilm was observed in the control, whereas a remark-able decrease in the number of microcolonies was observed in theCHS-treated biofilm. Application of 5 mg/ml CHS exhibited avisible biofilm formation inhibition compared with that of H2O.Moreover, CHS deteriorated the architecture of the biofilm too, asit was more evident from CLSM analysis (Fig. 6B). The CHS wasalso found to be very effective in eradicating the previously formedS. aureus biofilm. The CLSM microphotographs indicated thedeath of cells in the biofilm when treated with CHS (Fig. 6C).

Biofilm inhibition and biofilm eradication were also quantifiedusing 96-well-microtiter-plate assays. The attained results showed

a concentration-dependent antibiofilm activity of CHS in S. au-reus. Concentrations of 5 mg/ml efficiently dislodged the biofilmbiomass, inhibiting biofilm formation (P 0.001) by 100% andalso eradicating the preformed biofilm (P 0.001) by 71.5% withrespect to the untreated controls (Fig. 6D). The rest of the lowconcentrations, including 3 and 4 mg/ml, were also found to bestatistically effective (P 0.05). Moreover, at the lowest concen-tration tested (1 mg/ml), inhibition and eradication of biofilmswere recorded to be 8.4% and 3.1%, respectively.

Synergistic activity of CHS with antibiotic used against S.aureus. In the synergistic assay, the enhanced susceptibility of S.aureus to recommended antibiotics, such as clindamycin, lin-ezolid, and erythromycin, was evaluated in the presence of CHS.The results revealed that CHS at 5 mg/ml with 15 �g/ml of eachantibiotic enhanced the susceptibility of S. aureus to cell death, asexamined by CLSM analysis (Fig. 7).

DISCUSSION

This work for the first time explored interference with the cell-to-cell communication system of S. aureus, thereby demonstrat-ing that CHS, a functional oligosaccharide of colostrum, sup-presses expression of the virulence factors, degrades AHLs,prevents biofilm formation, and induces biofilm eradication.

FIG 6 Dose-dependent biofilm inhibitory potential of CHS. Microscopic images of S. aureus biofilms grown in the absence and presence of CHS and examinedunder light microscopy after crystal violet (CV) staining (A) and confocal laser scanning microscopy (CLSM) after staining with and SYTO-9 (B). (C) Theeradication of S. aureus biofilms by various doses of CHS by using SYTO-9 (for live cells) and propidium iodide (for dead cells) staining assay and images ofCLSM. (D) Quantification of biofilm inhibition and eradication by CHS by using microtiter plate assays. The tested concentrations of CHS were 1, 3, and 5mg/ml. Saline water (0.85%) was included as a positive control with which to compare each treatment. Data are results from three independent assays intriplicate. Bars represent SD from the mean. For each concentration, the asterisks show significant differences regarding saline water: *, P 0.05; **, P 0.01;***, P 0.001.

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Moreover, CHS in combination with antibiotics enhanced thesusceptibility of S. aureus to cell death. Colostrum is the first stageof breast milk that occurs during pregnancy and provides essentialprotection from microbial pathogens to very susceptible mam-mary glands through high antimicrobial content (26). Colostrumcontains carbohydrates, proteins, vitamin A, and a low content oflipids. The most pertinent bioactive constituents in colostrum aregrowth factors and antimicrobial factors (12). A study conductedby Zivkovic and Barile has demonstrated that the acidic oligosac-charides of colostrum played an important role in the preventionof adhesion of pathogenic bacteria to the epithelial surface, butthey were unable to describe which mechanism was responsiblefor this effect (27). In our study, a bioactive hexasaccharide com-ponent was identified in colostrum which was found to be ac-countable for regulating the QS system in S. aureus.

Several QS-based VFs, such as lipase, protease, and swarmingmotility, have been found to be associated with staphylococcalpathogenesis (28). Several proteases have been reported to be pro-duced by both health-associated and community-associated S.aureus strains, and some of them have been suggested to have arole in the degree of virulence (19). Moreover, production oflipase has been reported to be upregulated in the biofilm mode ofbacterial growth (29). Protease and lipase have been reported todestroy or deactivate a wide range of biological tissues and immu-nological agents. In this regard, suppression of lipase and proteasecould block or manage S. aureus invasion to fully develop a func-tional biofilm. Previous studies have documented the possible in-volvement of oligosaccharide-based protease and lipase inhibitorin the inhibition of virulence behaviors of several pathogenic bac-teria (30). A study by Alur at al. (31) reported how Hakea gibbosagum containing an oligosaccharide inhibits a model protease, py-roglutamate aminopeptidase. In the present study, significant re-duction in both protease and lipase activities was observed whentreated with CHS, thereby confirming the reduction in pathoge-nicity of S. aureus.

In order to make continued progress toward inhibiting dis-

eases caused by S. aureus, novel therapeutic strategies should bedeveloped, aimed at the VFs involved in the pathogenesis of thisbacterium. The chronic biofilms regulated by QS are consideredan important cause of chronic and recurrent infections by S. au-reus, in particular because of their ability to form and persist onmedical surfaces and indwelling devices (9). Moreover, biofilmsare much more resistant to conventional antibiotics than theirplanktonic counterparts (5, 9, 16). By aiming to prevent or inhibitbiofilm formation, therapeutic tactics for the prevention of bio-film development in S. aureus would be precise, and unlike athigher doses of antibiotics, the body bacterial flora would notnecessarily be inhibited. However, most chemotherapeutic strat-egies are based on killing cells of pathogenic bacteria by antibiot-ics, although growing evidence suggests that the ability of theseorganisms to develop chronic biofilms might be more importantthan their level in targeted tissue. However, recent studies suggestnaturally produced dietary compounds having a nonantibioticnature, such as mannose-containing polysaccharides and others,could also inhibit biofilm development, controlling microbial in-teraction with interfaces (32). Colostrum is rich in oligosaccha-rides, and in particular neutral and acidic anti-infective oligosac-charides have been identified to date (33). It would therefore beplausible that the presence of CHS is related to the inhibition anddestruction of S. aureus biofilms.

The potential of CHS to inhibit the QS system and biofilm forma-tion of bacteria is a very new idea, and it has not been previouslyreported by other authors. It has been reported that colostrum pro-tects the mammary gland from pathogenic microorganisms (34).The mammary gland is very susceptible to microbial infection, be-cause mammary secretions provide an excellent source of nutrientsfor pathogens, and the gland, through the teat opening, has a directexposure to the external environment (26). On the other hand, anumber of studies have been published on anti-QS and antibiofilmactivities of dietary compounds (5, 13, 35, 36).

AHLs are a class of QS signaling molecules, synthesized byLasI-RhlI synthease, which diffuse freely across the cell membrane

FIG 7 Synergistic effect of CHS with antibiotics. S. aureus culture was grown in the presence and absence of CHS (5 mg/ml) and antibiotics (15 �g/ml) whichare related to therapy of S. aureus, such as clindamycin, linezolid, and erythromycin. The plates were incubated overnight at 37°C for 24 h, and growth wasmeasured at A600. Data are results from six independent assays. Bars represent SD from the mean. For each concentration, the asterisks show significantdifferences regarding saline water: *, P 0.05; **, P 0.01; ***, P 0.001.

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upon reaching a putative threshold concentration. The transcrip-tional regulator, an LasR-RhlR receptor protein, binds to the dif-fusing AHLs, thus activating transcription of the target genes (4,35). Many genes encoding virulence factors have been describedto be regulated by AHLs (5, 9, 37). In our study, it was reallyinteresting to see that the CHS was able to degrade both C4-AHLsand C12-AHLs. AHLs are a class of QS signaling molecules, syn-thesized by LasI-RhlI synthetase (36). Numerous studies haveshown that inhibiting the production of AHLs of a pathogen canresult in a significant inhibition of the expression of QS-controlledgenes (37). The bacterial QS circuit can be interfered with by in-hibition of AHL molecule biosynthesis, degradation of AHL mol-ecules by bacterial lactonases, and using small molecules to blockthe activation of the AHL receptor protein (9). In our investiga-tion, the AHL degradation potential of CHS was observed, sug-gesting that CHS has anti-QS activity against Gram-negative bac-teria. Furthermore, CHS has been shown to have synergisticaction with conventional antibiotics at low concentrations to en-hance cell death of S. aureus, possibly due to interference with theQS process. These data thus provide a lead that CHS can be aneffective anti-QS and antibiofilm agent against S. aureus. How-ever, exhaustive studies exploring the exact molecular mecha-nisms pertaining to the antibacterial nature of CHS will certainlymake the picture clearer.

Conclusions. This study reports on dietary hexasaccharidefrom mare colostrum and its newly determined anti-QS activity.CHS attenuated production of virulence factors and biofilm de-velopment, and it also enhanced the susceptibility of S. aureus toantibiotics. Thus, CHS may play an effective role as a novel an-tipathogenic agent or could serve as the lead compound. More-over, this approach is highly attractive, because it does not imposeharsh selective pressure for the emergence of antibiotic resistance,opening the possibility of a new nonantibiotic-based antibacterialtherapy by deactivating the QS of bacteria.

ACKNOWLEDGMENTS

We are thankful to C. S. Nautiyal, Director, CSIR-National BotanicalResearch Institute, Lucknow, India, for providing research facilities.

We are thankful to the Council of Scientific and Industrial Research,New Delhi, India, for providing the research project funds (BSC-0106 andOLP-0089). B.R.S. is also thankful to the CSIR, India, for awarding PoolScientists Scheme [13(8595-A) 2012-Pool].

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