Colostrum Hexasaccharide, a Novel Staphylococcus aureus QuorumSensing 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

S

taphylococcus aureus is a prevalent and highly adaptable Grampositive bacterium causing numerous clinical infections (1). It is a well-documented fact that the occurrence of these infections is much more frequent in humans (impetigo) and in dairy cattle (mastitis) than in others (2). The development of antibiotic resistance in S. aureus and other species has therefore become a serious concern in the medical community (3). One attractive approach to this problem is to target bacterial virulence systems rather than essential cellular processes. It is hoped that this tactic can reduce selective survival pressures and also slow down the emergence of resistance. It is well established that S. aureus and other species use quorum sensing (QS), a chemical communication process to activate defense mechanisms, such as secretion of virulence factors and biofilm formation (1, 4, 5). These defenses pose a greater threat to the host than a common commensal. The discovery of a global regulatory QS system for virulence in S. aureus mediated by small signaling peptides has provided an appealing opportunity for inactivating these defenses (5–7). Numerous studies have demonstrated that QS inhibitors, produced by eukaryotes and prokaryotes, are able to interrupt QSregulated behaviors of bacteria (8, 9). Unfortunately, most of these QS inhibitors, for example, halogenated furanones, are unsuitable for human use, due to their toxicity issues. Therefore, there is an increasing need for the identification of novel nontoxic inhibitors with dual properties, such as anti-QS and antibiofilm. Identification of such natural inhibitors from a dietary source could present us with new opportunities for the development of novel drugs to combat bacterial infections. The development of approaches that interfere with proper microbial QS signaling has received significant attention, particularly for problematic multi-

April 2015 Volume 59 Number 4

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

Received 26 June 2014 Returned for modification 3 August 2014 Accepted 23 August 2014 Accepted manuscript posted online 2 February 2015 Citation Srivastava A, Singh BN, Deepak D, Rawat AKS, Singh BR. 2015. Colostrum hexasaccharide, 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, [email protected], or A. K. S. Rawat, [email protected]. A.S. and B.N.S. contributed equally. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03722-14

Antimicrobial Agents and Chemotherapy

aac.asm.org

2169

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity 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 been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), 1H and 13C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

Srivastava et al.

among other factors, by a possible interruption of the colostrum in these bacterial QS systems. Taking this into account, it seemed reasonable that the dietary polysaccharides isolated from milk colostrum could have anti-QS and antibiofilm properties against bacteria. With this assumption, the purpose of the present investigation was to examine whether isolated colostrum hexasaccharide (CHS) could inhibit QS signaling, production of virulence factors (VFs), and acyl-homoserine lactone (AHL) degradation, and also whether it is able to prevent or destroy the biofilm formation in S. aureus. Bacterial strains and culture conditions. The bacterial biomarker strain Chromobacterium violaceum ATCC 12472, S. aureus MTCC3160, and Escherichia coli MTCC1089 were obtained from the Microbial Type Culture Collection (MTCC), India. C. violaceum 12472 was characterized by high 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/pME6000 were grown overnight at 30°C in nutrient medium. For experimental analysis, the optical densities of the cultures were adjusted to the respective sterile culture medium to an optical density at 600 nm (OD600) of 0.5. Apparatus for isolation and identification. The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded at 300 MHz with tetramethylsilane 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 single quantum coherence (HSQC) were performed using standard Bruker pulse programs. Mass spectra were recorded on an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, USA). Sephadex G-25 column chromatography (GE Healthcare, India) was used for purification of the targeted molecule. C18 reverse-phase high-performance liquid chromatography (C18-RP-HPLC; Shimadzu, Japan) consisting of a column (4.6 by 250 mm), pump LC-8A, and a UV detector was used for isolation and purification of a reference compound. High-performance thin-layer chromatography (HPTLC) was performed on CAMAG TLC Scanner 3 at a ␭max of 450 nm in UV absorbance mode for acetylated compound 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) at Directorate of Animal Husbandry, Lucknow, India, and kept at ⫺20°C until used. Colostrum was thawed at room temperature, and then the solidified upper layer was removed by filtration at 0°C. The remaining colostrum was increased to 2.0 liters by adding 50% ethanol and then left at 4°C for 8 h. The sample was decanted slowly to remove lactose and protein, 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 a 0.22-␮m-pore-size filter apparatus (Pall, USA) and lyophilized (Labconco, USA) to get a crude oligosaccharide mixture. Purification, isolation, and characterization of CHS. The lyophilized crude mixture of oligosaccharides was packed on a Sephadex G-25 chromatography column (1.6 by 40 cm; void volume of 25 ml) and eluted with triple distilled water (TDW) at a rate of 10 ml/30 min. A total of 320 fractions were collected (see Table S1 in the supplemental material at https://docs.google .com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit). The UV spectra of these 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, thereby confirming the presence of oligosaccharides. These fractions were pooled and lyophilized together. Due to the highly polar nature of oligosaccharide, derivatization to acetylate them was done as described earlier (14). Separation of the acetylated product (150 mg) was carried over a silica gel (50 g) by eluting

2170

aac.asm.org

Antimicrobial Agents and Chemotherapy

April 2015 Volume 59 Number 4

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

MATERIALS AND METHODS

with CHCl3 and MeOH, thereby resulting in 10 fractions (50 ml each). Fraction 8 showed pure compound, which was chromatographed by CAMAGHPTLC (CHCl3-MeOH, 9:1) and C18-RP-HPLC-ELSD (acetonitrile-acetic acid, 9:1, 1 ml/min), which yielded hexasaccharide compound (denoted as CHS) with an Rf value of 0.51 (Fig. 1A) and a tR (retention time) value of 5.56 min, respectively (Fig. 1B) (15). The 1H and 13C NMR data for the marker compound are presented in Table 1 and Fig. 1C and D. The structure elucidation of the compound was performed with the help of UV, 1H NMR, 13C NMR, two-dimensional (2D) experiments (heteronuclear single quantum coherence [HSQC], correlation spectroscopy [COSY], and total correlation spectroscopy [TOCSY]), and electrospray ionization mass spectrometry (ESI-MS) analysis. Obtained data confirmed the presence of branched hexasaccharide having one reducing-end Glc, one ␤-galactosidase (␤-Gal), one ␤-GlcNAc, two ␤-GalNAc, and one ␣-GalNAc moiety and interpreted according to IUPAC nomenclature as ␣␤,D-glucopyranosyl-1¡4)-␣,D-galactopyranosyl-(1¡6)-␣,D-glucosamine-(1¡3)-␣,D galactosamine-[(1¡4)␣,D galactopyranosyl]-(1¡3)-␣,D-galactosamine (Fig. 2). (The results are described in detail in Tables S1 and S2 and Fig. S1 to S5, posted at https://docs .google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit.) The CHS was then 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 was determined as per the modified guidelines of the Clinical and Laboratory Standards Institute, USA (16). Briefly, in a sterile, flat-bottom plate (Tarsons, 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-fold serially diluted CHS to attain final concentrations ranging from 2 to 10 mg/ml. The plate was covered with a BREATHseal along with the lid and kept at 37°C under static conditions for 12 h. Turbidity was examined by reading the OD600 using a UV-visible (UV-Vis) spectrophotometer (Thermo Scientific, USA). The MIC was recorded as the lowest concentration which showed complete inhibition of visible growth of the bacteria. The readings were performed in two independent assays and in duplicate per trial. Anti-QS activity assay. To examine the anti-QS activity of CHS, a standard 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 an overnight-grown culture of C. violaceum 12472. This agar-culture solution was immediately poured on an LB agar plate. Various concentrations of 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 examined by a colorless and opaque halo zone of inhibition around the disk. Violacein inhibition assay. Violacein inhibition assay was carried out according 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 of CHS. After incubation for 24 h, the culture was treated with 10% sodium dodecyl sulfate (SDS; HiMedia) and incubated at room temperature for 5 min to lyse the cells. For fractionation of the violacein pigment, water-saturated n-butanol was added, and the mixture was vortexed and centrifuged at 13,000 rpm for 10 min. The upper layer, containing violacein, was collected and quantified at A585 using a UVVis spectrophotometer. Determination of virulence factors. For hemolysis assay, TSB containing S. aureus (OD600 of 0.5) was added to each well of the 96-well microtiter plate. The CHS (dissolved in TSB) was also added, and then plates were incubated statically at 37°C for 6 h. The cultures were then tested for hemolytic activity. Rat red blood cells (RBCs) were centrifuged at 3,000 ⫻ g for 5 min. The supernatant was discarded and the cells were resuspended in saline phosphate buffer (PBS; pH 7.2 ⫾ 02). The suspended RBCs were added and incubated at 37°C for 20 min. The plates were centrifuged to pellet down the cells at 450 ⫻ g for 5 min. The supernatant was transferred to a fresh microtiter plate, and absorbance was measured at A420 using a plate reader (18). The spreading ability assay was performed on TSB, containing 0.3%

Quorum-Sensing Blocker

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

oculation of S. aureus (OD600 of 0.5) and with various concentrations of CHS, the mixture was incubated at 37°C for 24. The liberated peptides by proteinase action were determined according to reference 19. Agar plate assay was applied to determine lipolytic activity. For this, the CHS (1 to 5 mg/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

TABLE 1 1H and 13C NMR values of CHS Chemical shift (ppm) Sugar

H-1

H-2

H-3

H-4

H-5

H-6

␣-Glc(S1) ␤-Glc(S1) ␤-Gal(S2) ␤-GlcNAc(S3) ␤-Gal(S4) ␣-GalNAc(S5) ␤-GalNAc(S6)

5.26 4.68 4.45 4.65 4.45 5.26 4.36

3.64 3.34 3.55 3.36 3.56 3.68 4.30

3.68 3.61 3.24 3.75 3.66 3.31 3.23

3.96 3.81 3.72 3.69 3.72 3.80 3.68

3.34 3.52 3.58 3.52 3.88 3.66 ND

3.88 3.76 3.75 NDa ND ND ND

a

NAc

C-1

C-2

C-3

C-4

C-5

C-6

-CO

-CH3

2.30

91.3 95.3 102.4 102.7 102.4 102.4 102.4

72.0 74.3 70.5 59.9 70.7 51.9 59.5

72.0 74.9 78.1 73.3 77.8 68.1 70.9

78.1 78.1 69.6 74.9 68.1 70.5 69.6

70.9 77.8 74.3 77.8 74.3 72.0 78.1

62.5 60.5 72.0 59.5 62.5 62.5 60.5

176

20.8

173 173

20.8 20.8

2.30 2.30

ND, not detectable.

April 2015 Volume 59 Number 4

Antimicrobial Agents and Chemotherapy

aac.asm.org

2171

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

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.

Srivastava et al.

FIG 2 Chemical structure of CHS.

2172

aac.asm.org

tions of CHS were added to previously formed biofilms for their eradication. Experiments were conducted in sterile round-bottom 96-well microtiter plates. Plates were placed in the incubator at 37°C for the next 24 h. After incubation, the cover glasses were rinsed with distilled water to remove the planktonic cells. The biofilms adhered on the cover glasses were 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 diluting 1:99 in TSB-0.25% glucose from the overnight culture. One-hundred-microliter aliquots were added to each well, and plates were incubated at 37°C for 36 h. Various concentrations of CHS were prepared in TSB, and their 100-␮l aliquots were transferred to the bacterial plate. The plates were covered and incubated overnight at 37°C for 24 h. The fixation with 0.12 HCl, staining with 0.4% CV, and dissolution with NaOH procedures were the same as those described above for the biofilm inhibition assay. The remaining CV content was measured by UV-Vis spectrophotometer at A650 (5). The positive control (TSB and S. aureus) was considered a good biofilm former when the absorbance exceeded 0.3. Positive and negative controls comprising TSB and CHS for sample background color were included in each assay. Synergistic effect of CHS with antibiotics. One percent of the overnight-grown culture of S. aureus (OD600 of 0.5) was added to 1 ml of LB on a 24-well microtiter plate containing CHS (5 mg/ml) and antibiotics (15 ␮g/ml): clindamycin, linezolid, and erythromycin. The controls were maintained with their respective antibiotic and without CHS. The plates were incubated overnight at 37°C, and growth was measured after 24 h of incubation at A600 nm using a UV-visible spectrophotometer (11). Statistical analysis. Values are presented as means ⫾ standard deviations (SD) and analyzed by using one-way analysis of variance (SPSS 11.5). For all analyses, the criterion for significance was a P value of ⬍0.05.

RESULTS

MIC of CHS. First of all, MIC was assessed to throw away any possible inhibitory effect of CHS on S. aureus growth using the doubling dilution method with the concentrations varying from 2 to 10 mg/ml. The data obtained revealed no inhibition during incubation until 12 h with CHS (ⱖ5 mg/ml) (see Fig. S6 posted at https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRu UDA/edit). Anti-QS activity of CHS. Production of QS-regulated purple-colored violacein pigmentation in C. violaceum 12472 provides a naturally occurring model system without the need for additional substrate to evaluate anti-QS activity of molecules (5). Using disk diffusion assay, the anti-QS activity of CHS was determined. The turbid zone of violacein inhibition around the disk in C. violaceum 12472 by CHS in a concentration-dependent manner was detected, indicating the anticipated inhibition of QS (Fig. 3A). This might be an important attribute, as QS inactivation is focused on the attenuation of bacterial signaling. At all concentrations used, the CHS and halogenated furanone (HF; 1.5 ␮g/ml) did not show a clear halo zone of the test organism (see Fig. S7A posted at

Antimicrobial Agents and Chemotherapy

April 2015 Volume 59 Number 4

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

inhibition of clear zones was considered an indicator of antilipolytic activity of CHS (20). Degradation of AHLs by CHS. The AHL degradation potential of CHS was assessed by the method of Medina-Martinez et al. (21). The CHS was 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 strains of P. fluorescens, P3/pME6863 and P3/pME6000, were used as the degrader strain and nondegrader strain, respectively. The reaction mixture was incubated for 24 h at 30°C with constant shaking at 180 rpm. Afterward, the mixture was centrifuged at 4,000 ⫻ g for 10 min (1). The supernatant obtained was extracted with acidified ethyl acetate (1% [vol/vol] acetic acid) (supernatant-acidified ethyl acetate, 7:3 [vol/vol]) and finally concentrated and dried using a rotary evaporator at 40°C (22). One microliter of ethyl acetate extract was spotted onto TLC plates (silica gel 60 F254; Merck). The separation of AHLs was obtained on a mobile phase consisting of methanol-water-acetic acid (8:1.5:0.5) and photographed under 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 formation was determined by biofilm inhibition assay. This protocol is based on 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 cover glasses on 24-well microtiter plates. One percent of overnight bacterial culture was diluted in TSB-0.25% glucose, and a 1-ml aliquot of it added was added to each well. Subsequently, various concentrations of CHS were added. Plates were closed with BREATHseal along with the lid and placed in an incubator maintained at 37°C for 24 h. After incubation, the cover glasses were rinsed with distilled water to remove the planktonic cells. The biofilms adhered on the cover glasses were stained with 1% CV solution and SYTO-9 fluorescent dyes and then examined under a light microscope (Leica, Germany) and by CLSM (LSM 510; Germany), respectively, at a magnification of ⫻20 (11). Biofilm inhibition by CHS and the different fractions thereof was also quantified by using a round-bottom 96-well-polystyrene-plate assay (23). Each sample and dilution was tested in triplicate in three independent assays. The bacterial suspension (200 ␮l in each well) and treatment with CHS were as described above. After 24 h of incubation, the media were aspirated carefully and rinsed twice with distilled water, and the biofilm was fixed with 200 ␮l of 0.12 M HCl for 80 min at room temperature. The fixative was removed and 200 ␮l of 0.4% CV was added to stain the biofilm for 5 min. The stain was removed and the wells were washed two times with distilled water to remove excess solution. The stain was further solubilized with 0.2 ml of 95% ethanol. The biomass of the biofilm was quantified by measuring the intensity of CV in ethanol at OD650 by using a UV-visible spectrophotometer (Bio-Rad, USA). The positive control (TSB and S. aureus) was considered to show good biofilm formation when the optical density was more than 0.3. Positive and negative controls comprising TSB and CHS for sample background color were included in each assay. Biofilm eradication assay. The biofilm eradication assay was a modified version of the biofilm inhibition assay in which first S. aureus was allowed to form the biofilm inside the wells for 36 h. Different concentra-

Quorum-Sensing Blocker

https://docs.google.com/file/d/0Bxioy67Ki1RzRlR0akJwYlRu UDA/edit), except gentamicin (GMN; 10 ␮g/ml), which showed a clear halo zone (see Fig. S7B posted at https://docs.google.com/ file/d/0Bxioy67Ki1RzRlR0akJwYlRuUDA/edit). The turbid zones produced on lawns of C. violaceum could be the result of either inhibition 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 inhibit growth and inhibited only violacein production (Fig. 3B). Inhibition of violacein by CHS. CHS was subjected to qualitative analysis to find out its anti-QS potential against C. violaceum 12472. The bacterium is reported to produce the violet-colored pigment violacein by responding to its QS signal molecule AHLs. The AHLs bind to its receptor LasR, and this complex triggers the production of violacein. In qualitative analysis, CHS showed a concentration-dependent inhibition of violacein production in C. violaceum 12472. Inhibitory activity of CHS on violacein production was further measured spectrophotometrically. Concentration- and time-dependent reduction in violacein content of C. violaceum 12472 was observed. Inhibition of up to 85.7% and 100% was determined when treated with CHS at 5 mg/ml for 24 and 48 h of incubation, respectively (Fig. 3C). Inhibition of VF secretion by CHS. Numerous studies have documented that the production of hemolysins is regulated by the QS system in S. aureus (24). Therefore, any compound that inhib-

April 2015 Volume 59 Number 4

its the QS system should also inhibit the production of hemolysins, and we reasoned that this outcome could be readily quantitated by bacterial hemolysis assay. Inhibition of hemolysin was measured as 2.91, 16.8, 40.9, 67.9, and 88.3% in the samples treated 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 reduction 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 pathogenesis of S. aureus due to their hydrolytic activities. Epidermal permeability barrier dysfunction by protease in nude mice was assessed (25); therefore, we were quite interested to determine whether 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 of CHS, and obtained supernatants were used for determination of protease and lipase activities. The data revealed that increasing the concentration from 1 to 5 mg/ml of CHS enhanced the inhibition of both lytic enzymes (Fig. 4B and D.). Inhibition of protease and lipase activities was observed to be a maximum of 74.2% and 52.9%, respectively, when S. aureus cells were treated with 5 mg/ml of CHS. Inhibition of the QS system by CHS via degradation of AHLs. Recent studies have proven that signaling molecule or autoinducer AHLs play an important role in regulation of QS-dependent

Antimicrobial Agents and Chemotherapy

aac.asm.org

2173

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

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-QS action 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. violaceum 12472. 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 the indicated time intervals. The CHS did not inhibit growth of C. violaceum 12472. (C) Quantitative analysis of violacein production inhibition in C. violaceum 12472 by CHS at 24 and 48 h. Inhibition values were directly plotted as the means from eight independent experiments ⫾ SD against various concentrations of CHS. * and *** indicate significance at P values of ⬍0.05 and ⬍0.001, respectively.

Srivastava et al.

expression of virulence genes in Gram-negative and Gram-positive bacteria (5). The potentiality of CHS to degrade C4-AHL and C12-AHL was determined by HPTLC (CAMAG, Switzerland) and quantified by a densitometer. In Fig. 5A and B, the amounts of AHLs for each treatment were compared with their respective positive controls as shown. As anticipated, the treatment for Pseudomonas 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 and C12-AHL was observed in the treatment for P. fluorescens P3/ pME6000 (an AHL nondegrader control strain). Remarkable degradations of AHLs were also observed when samples were treated with CHS. The obtained results thus revealed that the intensity of the AHL bands was reduced with increasing concentrations of

FIG 5 AHL degradation potential of CHS. The remaining AHL concentrations of samples were measured after the incubation of 2.7 ml of each CHS concentration 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 was obtained 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 of C4-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, AHL nondegrader strain (P. fluorescens P3/pME6000); Hs, hexasaccharide.

2174

aac.asm.org

Antimicrobial Agents and Chemotherapy

April 2015 Volume 59 Number 4

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

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 incubated statically at 37°C. The supernatant was used to examine hemolytic activity (A) and proteolytic activity (B). Plate assays were used to determine the inhibition of spreading ability (C) and lipolytic activity (D) of CHS, and results were expressed as the percent inhibition compared to that of the untreated controls. Inhibition values were directly plotted as the means from six independent experiments ⫾ SD against various concentrations of CHS. *, **, and *** indicate a significance at P values of ⬍0.05, ⬍0.01, and ⬍0.001.

Quorum-Sensing Blocker

CHS. At 5 mg/ml, CHS displayed very faint bands of both C4-AHL and C12-AHL, with mean values of 2 ␮M for both, compared with the controls by 10 and 7 ␮M, respectively. The rest of the low concentrations tested were also shown to be considerably effective in a dose-response picture. Antibiofilm activity of CHS. Direct microscopic observations of biofilm inhibition and biofilm eradication after exposure to CHS are known to provide valuable information of the action of antibiofilm compounds; therefore, light microscopy and confocal laser scanning microscopy (CLSM) analyses were performed. Inhibition of biofilm formation in S. aureus showed a clear dose response to increasing concentrations of CHS (Fig. 6A). A thick coating of biofilm was observed in the control, whereas a remarkable decrease in the number of microcolonies was observed in the CHS-treated biofilm. Application of 5 mg/ml CHS exhibited a visible biofilm formation inhibition compared with that of H2O. Moreover, CHS deteriorated the architecture of the biofilm too, as it was more evident from CLSM analysis (Fig. 6B). The CHS was also found to be very effective in eradicating the previously formed S. aureus biofilm. The CLSM microphotographs indicated the death of cells in the biofilm when treated with CHS (Fig. 6C). Biofilm inhibition and biofilm eradication were also quantified using 96-well-microtiter-plate assays. The attained results showed

April 2015 Volume 59 Number 4

a concentration-dependent antibiofilm activity of CHS in S. aureus. Concentrations of 5 mg/ml efficiently dislodged the biofilm biomass, inhibiting biofilm formation (P ⬍ 0.001) by 100% and also eradicating the preformed biofilm (P ⬍ 0.001) by 71.5% with respect to the untreated controls (Fig. 6D). The rest of the low concentrations, including 3 and 4 mg/ml, were also found to be statistically effective (P ⬍ 0.05). Moreover, at the lowest concentration tested (1 mg/ml), inhibition and eradication of biofilms were 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, linezolid, and erythromycin, was evaluated in the presence of CHS. The results revealed that CHS at 5 mg/ml with 15 ␮g/ml of each antibiotic enhanced the susceptibility of S. aureus to cell death, as examined by CLSM analysis (Fig. 7). DISCUSSION

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

Antimicrobial Agents and Chemotherapy

aac.asm.org

2175

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

FIG 6 Dose-dependent biofilm inhibitory potential of CHS. Microscopic images of S. aureus biofilms grown in the absence and presence of CHS and examined under light microscopy after crystal violet (CV) staining (A) and confocal laser scanning microscopy (CLSM) after staining with and SYTO-9 (B). (C) The eradication 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 of CLSM. (D) Quantification of biofilm inhibition and eradication by CHS by using microtiter plate assays. The tested concentrations of CHS were 1, 3, and 5 mg/ml. Saline water (0.85%) was included as a positive control with which to compare each treatment. Data are results from three independent assays in triplicate. 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.

Srivastava et al.

are 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 was measured at A600. Data are results from six independent assays. 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.

Moreover, CHS in combination with antibiotics enhanced the susceptibility of S. aureus to cell death. Colostrum is the first stage of breast milk that occurs during pregnancy and provides essential protection from microbial pathogens to very susceptible mammary glands through high antimicrobial content (26). Colostrum contains carbohydrates, proteins, vitamin A, and a low content of lipids. The most pertinent bioactive constituents in colostrum are growth factors and antimicrobial factors (12). A study conducted by Zivkovic and Barile has demonstrated that the acidic oligosaccharides of colostrum played an important role in the prevention of adhesion of pathogenic bacteria to the epithelial surface, but they were unable to describe which mechanism was responsible for this effect (27). In our study, a bioactive hexasaccharide component was identified in colostrum which was found to be accountable for regulating the QS system in S. aureus. Several QS-based VFs, such as lipase, protease, and swarming motility, have been found to be associated with staphylococcal pathogenesis (28). Several proteases have been reported to be produced by both health-associated and community-associated S. aureus strains, and some of them have been suggested to have a role in the degree of virulence (19). Moreover, production of lipase has been reported to be upregulated in the biofilm mode of bacterial growth (29). Protease and lipase have been reported to destroy or deactivate a wide range of biological tissues and immunological agents. In this regard, suppression of lipase and protease could block or manage S. aureus invasion to fully develop a functional biofilm. Previous studies have documented the possible involvement of oligosaccharide-based protease and lipase inhibitor in the inhibition of virulence behaviors of several pathogenic bacteria (30). A study by Alur at al. (31) reported how Hakea gibbosa gum containing an oligosaccharide inhibits a model protease, pyroglutamate aminopeptidase. In the present study, significant reduction in both protease and lipase activities was observed when treated with CHS, thereby confirming the reduction in pathogenicity of S. aureus. In order to make continued progress toward inhibiting dis-

2176

aac.asm.org

eases caused by S. aureus, novel therapeutic strategies should be developed, aimed at the VFs involved in the pathogenesis of this bacterium. The chronic biofilms regulated by QS are considered an important cause of chronic and recurrent infections by S. aureus, in particular because of their ability to form and persist on medical surfaces and indwelling devices (9). Moreover, biofilms are much more resistant to conventional antibiotics than their planktonic counterparts (5, 9, 16). By aiming to prevent or inhibit biofilm formation, therapeutic tactics for the prevention of biofilm development in S. aureus would be precise, and unlike at higher doses of antibiotics, the body bacterial flora would not necessarily be inhibited. However, most chemotherapeutic strategies are based on killing cells of pathogenic bacteria by antibiotics, although growing evidence suggests that the ability of these organisms to develop chronic biofilms might be more important than their level in targeted tissue. However, recent studies suggest naturally produced dietary compounds having a nonantibiotic nature, such as mannose-containing polysaccharides and others, could also inhibit biofilm development, controlling microbial interaction with interfaces (32). Colostrum is rich in oligosaccharides, and in particular neutral and acidic anti-infective oligosaccharides have been identified to date (33). It would therefore be plausible that the presence of CHS is related to the inhibition and destruction of S. aureus biofilms. The potential of CHS to inhibit the QS system and biofilm formation of bacteria is a very new idea, and it has not been previously reported by other authors. It has been reported that colostrum protects the mammary gland from pathogenic microorganisms (34). The mammary gland is very susceptible to microbial infection, because mammary secretions provide an excellent source of nutrients for pathogens, and the gland, through the teat opening, has a direct exposure to the external environment (26). On the other hand, a number of studies have been published on anti-QS and antibiofilm activities of dietary compounds (5, 13, 35, 36). AHLs are a class of QS signaling molecules, synthesized by LasI-RhlI synthease, which diffuse freely across the cell membrane

Antimicrobial Agents and Chemotherapy

April 2015 Volume 59 Number 4

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

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) which

Quorum-Sensing Blocker

ACKNOWLEDGMENTS We are thankful to C. S. Nautiyal, Director, CSIR-National Botanical Research 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 and OLP-0089). B.R.S. is also thankful to the CSIR, India, for awarding Pool Scientists Scheme [13(8595-A) 2012-Pool].

REFERENCES 1. Gonzalez-Ortiz G, Quarles Van Ufford HC, Halkes SB, Cerda-Cuellar M, Beukelman CJ, Pieters RJ, Liskamp RM, Perez JF, Martin-Orue SM. 2014. New properties of wheat bran: anti-biofilm activity and interference with bacteria quorum-sensing systems. Environ Microbiol 16:1346 –1353. http://dx.doi.org/10.1111/1462-2920.12441. 2. Cimolai N. 2008. MRSA and the environment: implications for comprehensive control measures. Eur J Clin Microbiol Infect Dis 27:481– 493. http://dx.doi.org/10.1007/s10096-008-0471-0. 3. Campoccia D, Montanaro L, Baldassarri L, An YH, Arciola CR. 2005. Antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates from implant orthopedic infections. Int J Artif Organs 28:1186 –1191. 4. Hastings JW. 2004. Bacterial quorum-sensing signals are inactivated by mammalian cells. Proc Natl Acad Sci U S A 101:3993–3994. http://dx.doi .org/10.1073/pnas.0400874101. 5. Singh BN, Singh HB, Singh A, Singh BR, Mishra A, Nautiyal CS. 2012. Lagerstroemia speciosa fruit extract modulates quorum sensing-controlled virulence factor production and biofilm formation in Pseudomonas aeruginosa. Microbiology 158:529 –538. http://dx.doi.org/10.1099/mic.0 .052985-0.

April 2015 Volume 59 Number 4

6. de Kievit TR. 2009. Quorum sensing in Pseudomonas aeruginosa biofilms. Environ Microbiol 11:279 –288. http://dx.doi.org/10.1111/j.1462-2920 .2008.01792.x. 7. Gorske BC, Blackwell HE. 2006. Interception of quorum sensing in Staphylococcus aureus: a new niche for peptidomimetics. Org Biomol Chem 4:1441–1445. http://dx.doi.org/10.1039/b517681f. 8. Defoirdt T, Miyamoto CM, Wood TK, Meighen EA, Sorgeloos P, Verstraete W, Bossier P. 2007. The natural furanone (5Z)-4-bromo-5-(bromomethylene)3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR. Environ Microbiol 9:2486 –2495. http://dx .doi.org/10.1111/j.1462-2920.2007.01367.x. 9. Kalia VC. 2013. Quorum sensing inhibitors: an overview. Biotechnol Adv 31:224 –245. http://dx.doi.org/10.1016/j.biotechadv.2012.10.004. 10. Gordon CP, Williams P, Chan WC. 2013. Attenuating Staphylococcus aureus virulence gene regulation: a medicinal chemistry perspective. J Med Chem 56:1389 –1404. http://dx.doi.org/10.1021/jm3014635. 11. Packiavathy IA, Priya S, Pandian SK, Ravi AV. 2014. Inhibition of biofilm development of uropathogens by curcumin—an anti-quorum sensing agent from Curcuma longa. Food Chem 148:453– 460. http://dx .doi.org/10.1016/j.foodchem.2012.08.002. 12. Reiter B, Brock JH, Steel ED. 1975. Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Immunology 28:83–95. 13. Singh BN, Singh BR, Singh RL, Prakash D, Sarma BK, Singh HB. 2009. Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L. Food Chem Toxicol 47:778 –786. http://dx.doi.org/10.1016/j.fct .2009.01.009. 14. Saksena R, Deepak D, Khare A, Sahai R, Tripathi LM, Srivastava VM. 1999. A novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk. Biochim Biophys Acta 1428:433– 445. http://dx .doi.org/10.1016/S0304-4165(99)00089-6. 15. Glycoconjugate Journal. 1995. XIIIth International Symposium on Glycoconjugates. Seattle, USA, August 20-26, 1995. Abstracts. Glycoconj J 12:391–590. 16. Varaldo PE. 2002. Antimicrobial resistance and susceptibility testing: an evergreen topic. J Antimicrob Chemother 50:1– 4. http://dx.doi.org/10 .1093/jac/dkf093. 17. Singh BN, Singh BR, Singh RL, Prakash D, Dhakarey R, Upadhyay G, Singh HB. 2009. Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera. Food Chem Toxicol 47:1109 –1116. http://dx.doi.org/10.1016/j.fct.2009.01.034. 18. Nilsson IM, Hartford O, Foster T, Tarkowski A. 1999. Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis. Infect Immun 67:1045–1049. 19. McDonald CE, Chen LL. 1965. The Lowry modification of the Folin reagent for determination of proteinase activity. Anal Biochem 10:175– 177. http://dx.doi.org/10.1016/0003-2697(65)90255-1. 20. Smeltzer MS, Hart ME, Iandolo JJ. 1992. Quantitative spectrophotometric assay for staphylococcal lipase. Appl Environ Microbiol 58:2815–2819. 21. Medina-Martinez MS, Uyttendaele M, Meireman S, Debevere J. 2007. Relevance of N-acyl-L-homoserine lactone production by Yersinia enterocolitica in fresh foods. J Appl Microbiol 102:1150 –1158. http://dx.doi.org /10.1111/j.1365-2672.2006.03143.x. 22. Shaw PD, Ping G, Daly SL, Cha C, Cronan JE, Jr, Rinehart KL, Farrand SK. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci U S A 94: 6036 – 6041. http://dx.doi.org/10.1073/pnas.94.12.6036. 23. Melchior MB, Fink-Gremmels J, Gaastra W. 2006. Comparative assessment of the antimicrobial susceptibility of Staphylococcus aureus isolates from bovine mastitis in biofilm versus planktonic culture. J Vet Med B Infect Dis Vet Public Health 53:326 –332. http://dx.doi.org/10.1111/j.1439 -0450.2006.00962.x. 24. Pragman AA, Schlievert PM. 2004. Virulence regulation in Staphylococcus aureus: the need for in vivo analysis of virulence factor regulation. FEMS Immunol Med Microbiol 42:147–154. http://dx.doi.org/10.1016/j .femsim.2004.05.005. 25. Nakamura T, Hirasawa Y, Takai T, Mitsuishi K, Okuda M, Kato T, Okumura K, Ikeda S, Ogawa H. 2006. Reduction of skin barrier function by proteolytic activity of a recombinant house dust mite major allergen Der f 1. J Investig Dermatol 126:2719 –2723. http://dx.doi.org/10.1038/sj .jid.5700584.

Antimicrobial Agents and Chemotherapy

aac.asm.org

2177

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

upon reaching a putative threshold concentration. The transcriptional regulator, an LasR-RhlR receptor protein, binds to the diffusing AHLs, thus activating transcription of the target genes (4, 35). Many genes encoding virulence factors have been described to be regulated by AHLs (5, 9, 37). In our study, it was really interesting to see that the CHS was able to degrade both C4-AHLs and C12-AHLs. AHLs are a class of QS signaling molecules, synthesized by LasI-RhlI synthetase (36). Numerous studies have shown that inhibiting the production of AHLs of a pathogen can result in a significant inhibition of the expression of QS-controlled genes (37). The bacterial QS circuit can be interfered with by inhibition of AHL molecule biosynthesis, degradation of AHL molecules by bacterial lactonases, and using small molecules to block the activation of the AHL receptor protein (9). In our investigation, the AHL degradation potential of CHS was observed, suggesting that CHS has anti-QS activity against Gram-negative bacteria. Furthermore, CHS has been shown to have synergistic action with conventional antibiotics at low concentrations to enhance cell death of S. aureus, possibly due to interference with the QS process. These data thus provide a lead that CHS can be an effective anti-QS and antibiofilm agent against S. aureus. However, exhaustive studies exploring the exact molecular mechanisms pertaining to the antibacterial nature of CHS will certainly make the picture clearer. Conclusions. This study reports on dietary hexasaccharide from mare colostrum and its newly determined anti-QS activity. CHS attenuated production of virulence factors and biofilm development, and it also enhanced the susceptibility of S. aureus to antibiotics. Thus, CHS may play an effective role as a novel antipathogenic agent or could serve as the lead compound. Moreover, this approach is highly attractive, because it does not impose harsh selective pressure for the emergence of antibiotic resistance, opening the possibility of a new nonantibiotic-based antibacterial therapy by deactivating the QS of bacteria.

Srivastava et al.

2178

aac.asm.org

33.

34. 35.

36.

37.

Vacca-Smith AM, Bowen WH. 2003. Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and ttfarnesol. J Antimicrob Chemother 52:782–789. http://dx.doi.org/10.1093 /jac/dkg449. Lane JA, Marino K, Naughton J, Kavanaugh D, Clyne M, Carrington SD, Hickey RM. 2012. Anti-infective bovine colostrum oligosaccharides: Campylobacter jejuni as a case study. Int J Food Microbiol 157:182–188. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.04.027. Vorbach C, Capecchi MR, Penninger JM. 2006. Evolution of the mammary gland from the innate immune system? Bioessays 28:606 – 616. http: //dx.doi.org/10.1002/bies.20423. Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, Manefield M, Costerton JW, Molin S, Eberl L, Steinberg P, Kjelleberg S, Hoiby N, Givskov M. 2003. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22:3803–3815. http://dx.doi.org/10.1093/emboj/cdg366. Rasmussen TB, Bjarnsholt T, Skindersoe ME, Hentzer M, Kristoffersen P, Kote M, Nielsen J, Eberl L, Givskov M. 2005. Screening for quorumsensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. J Bacteriol 187:1799 –1814. http://dx.doi.org/10.1128/JB.187.5.1799-1814 .2005. Hentzer M, Givskov M. 2003. Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J Clin Invest 112:1300 –1307. http://dx.doi.org/10.1172/JCI20074.

Antimicrobial Agents and Chemotherapy

April 2015 Volume 59 Number 4

Downloaded from http://aac.asm.org/ on May 23, 2015 by FLORIDA ATLANTIC UNIV

26. Stelwagen K, Carpenter E, Haigh B, Hodgkinson A, Wheeler TT. 2009. Immune components of bovine colostrum and milk. J Anim Sci 87:3–9. http://dx.doi.org/10.2527/jas.2008-1377. 27. Zivkovic AM, Barile D. 2011. Bovine milk as a source of functional oligosaccharides for improving human health. Adv Nut Int Rev J 2:284 – 289. http://dx.doi.org/10.3945/an.111.000455. 28. Saising J, Singdam S, Ongsakul M, Voravuthikunchai SP. 2012. Lipase, protease, and biofilm as the major virulence factors in staphylococci isolated from acne lesions. Biosci Trends 6:160 –164. http://dx.doi.org/10 .5582/bst.2012.v6.4.160. 29. Coenye T, Peeters E, Nelis HJ. 2007. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Microbiol 158: 386 –392. http://dx.doi.org/10.1016/j.resmic.2007.02.001. 30. Grishin A, Karyagina AS, Tiganova IG, Dobrynina OY, Bolshakova TN, Boksha IS, Alexeyeva NV, Stepanova TV, Lunin VG, Chuchalin AG, Ginzburg AL. 2013. Inhibition of Pseudomonas aeruginosa biofilm formation by LecA-binding polysaccharides. Int J Antimicrob Agents 42:471– 472. http://dx.doi.org/10.1016/j.ijantimicag.2013.07.003. 31. Alur HH, Desai RP, Mitra AK, Johnston TP. 2001. Inhibition of a model protease—pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa. Int J Pharm 212:171–176. http://dx.doi.org/10 .1016/S0378-5173(00)00609-8. 32. Koo H, Hayacibara MF, Schobel BD, Cury JA, Rosalen PL, Park YK,