Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens

Transcription

1 Letters in Applied Microbiology ISSN RIGINAL ARTICLE Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens C. Cueva 1, S. Mingo 1, I. Muñoz-González 1, I. Bustos 1, T. Requena 1, R. del Campo 2, P.J. Martín- Álvarez 1, B. Bartolomé 1 and M.V. Moreno-Arribas 1 1 Instituto de Investigación en Ciencias de la Alimentación (CIAL), CSIC-UAM, Campus de la Universidad Autónoma de Madrid, Madrid, Spain 2 Servicio de Microbiología, Hospital Ramón y Cajal, IRYCIS and CIBERESP, Madrid, Spain Keywords antimicrobial activity, phenolic compounds, phenolic extracts, respiratory pathogenic bacteria, wine. Correspondence M.V. Moreno-Arribas, Instituto de Investigación en Ciencias de la Alimentación (CIAL), CSIC-UAM, Nicolás Cabrera 9, Campus de la Universidad Autónoma de Madrid, Madrid, Spain. victoria.moreno@csic.es : received 20 December 2011, revised 20 February 2012 and accepted 21 March 2012 doi: /j x x Abstract Aims: To investigate the effect of seven wine phenolic compounds and six oenological phenolic extracts on the growth of pathogenic bacteria associated with respiratory diseases (Pseudomonas aeruginosa, Staphylococcus aureus, Moraxella catarrhalis, Enterococcus faecalis, sp Group F, agalactiae and pneumoniae). Methods and Results: Antimicrobial activity was determined using a microdilution method and quantified as IC 50. Mor. catarrhalis was the most susceptible specie to phenolic compounds and extracts. Gallic acid and ethyl gallate were the compounds that showed the greatest antimicrobial activity. Regarding phenolic extracts, GSE (grape seed extract) and GSE- (oligomeric-rich fraction from GSE) were the ones that displayed the strongest antimicrobial effects. Conclusions: Results highlight the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. The antimicrobial activity of wine phenolic compounds was influenced by the type of phenolic compounds. Gram-negative bacteria were more susceptible than Gram-positive bacteria to the action of phenolic compounds and extracts; however, the effect was species-dependent. Significance and Impact of Study: The ability to inhibit the growth of respiratory pathogenic bacteria as shown by several wine phenolic compounds and oenological extracts warrants further investigations to explore the use of grape and wine preparations in oral hygiene. Introduction The oral cavity harbours an abundant microbiota, usually dominated by anaerobic bacteria. The concentration of oral bacteria is about CFU g )1 in the dental plaque and bacteria per millilitre in saliva (Guarner and Malagelada 2003). Concerning to the microbial diversity, it is estimated that the human oral cavity harbours over 700 different bacterial species (Aas et al. 2005), which of the most belong to genera Gemella, Neisseria, Granulicatella, Porphyromonas, and Veillonella. In addition to the resident oral species, several studies have shown that the oral cavity may act as a reservoir of important respiratory pathogens such as pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa, which could lead to infection of lower respiratory tract (Paju and Scannapieco 2007; Zuanazzi et al. 2010) by means of aspiration of micro-organisms (van Uffelen et al. 1984). Poor oral health, significant amounts of dental plaque and or periodontal diseases could provide a favourable environment for the development of pathogen micro-organisms (Zuanazzi et al. 2010). For instance, patients with periodontitis showed greater prevalence of Corynebacterium diphtheriae, Escherichia coli, Enterococcus faecalis, Ps. aeruginosa and Staph. aureus (Souto et al. 2006). Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology 557

2 Wine phenolics against respiratory pathogens C. Cueva et al. Grapes and wines are considered rich sources of polyphenolic compounds, including hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, flavan-3-ol monomers, oligomeric and polymeric procyanidins, flavonols, stilbenes and anthocyanins (only present in red varieties) (Monagas et al. 2003). Although there is substantial literature reporting antimicrobial properties of polyphenols against many bacteria species (Jayaprakasha et al. 2003; Özkan et al. 2004; Cueva et al. 2010), the information about their effect on oral pathogens is scarce (Requena et al. 2010). Studies carried out with tea and cranberry polyphenols have shown an inhibitory effect on biofilm formation by oral pathogens such as mutans, oralis, sobrinus and Porphyromonas gingivalis (Bodet et al. 2008). Recently, it has been found that wine and grape phenolic extracts, as well as pomace phenolic extracts, were able to inhibit the growth of different spp. strains associated with dental caries (Thimothe et al. 2007; Furiga et al. 2009). With the final aim of seeking natural products that could be used in oral hygiene, in this study, we have investigated the antimicrobial properties of pure phenolic compounds and oenological phenolic extracts against potential respiratory pathogens from the oral cavity. Materials and Methods Phenolic compounds (+)-Catechin, 3,4-dihydroxycinnamic acid (caffeic acid) and 3,4,5-trihidroxibenzoic acid (gallic acid) were obtained from Sigma (St Louis, M, USA); ())-epicatechin from Fluka AG (Buchs, Switzerland), 4-hydroxyphenethyl alcohol (tyrosol) and 3-(2-hydroxyethyl) indole (tryptophol) from Aldrich (Steinheim, Germany) and ethyl gallate from Extrasynthèse (Genay, France). Stock solutions (10 mg ml )1 ) were prepared by dissolving phenolic compounds in a mixture dimethyl sulfoxide (DMS) water (10 : 90, v v), except for caffeic acid, which was dissolved in a mixture DMS water (30 : 70, v v) to improve their solubility. Phenolic extracts A total of six commercial oenological phenolic extracts that are sold as ingredients for dietary supplements and cosmetics were tested. A grape seed extract (GSE), Vitaflavan Ò (50% flavan-3-ols monomers and 47% procyanidins), a GSE-derived monomeric-rich fraction (GSE-M, 70% flavan-3-ols monomers and 28% procyanidins) and an oligomeric-rich fraction (GSE-, 21% flavan-3-ols monomers and 78% procyanidins), both obtained from Vitaflavan Ò, were kindly provided by Dr. Piriou (Les Dérives Resiniques & Terpéniques, S.A., France). A wine extract, ProvinolsÔ, was kindly supplied by Safic-Alcan Especialidades S.A.U. (Barcelona, Spain). A pomace extract, Eminol Ò, was supplied by Central Lechera Asturiana (CAPSA, Spain). Also, a resveratrol-rich extract, Revidox Ò, obtained from grape seed skins and kindly provided by Dr Espín (CEBAS-CSIC, Spain) was included in this study. Table 1 reports the source, total polyphenols and antioxidant activity of all extracts used in this study. The concentration of total phenolic compounds in the extracts was determined by the Folin Ciocalteu colorimetric method (Singleton and Rossi 1965), whereas antioxidant activity was evaluated by the RAC (oxygen radical absorbance capacity) method using fluorescein as a fluorescence probe (Dávalos et al. 2004). Regarding phenolic composition of the extracts, previous studies have demonstrated that they contained some of the pure phenolic compounds tested (Tabasco et al. 2011; Sánchez- Patán et al. 2012). Bacterial strains and culture conditions A total of seven bacterial isolates were included in this study. Staphylococcus aureus ATCC was obtained from the Spanish Type Culture Collection (CECT), Moraxella catarrhalis, Pseudomonas aeruginosa PA1, Enterococcus faecalis V583, sp Group F, agalactiae and pneumoniae were clinical isolates obtained from human samples at the Ramón y Cajal Hospital (Madrid, Spain). Tryptic Soy Broth (TSB) was employed for Staph. aureus ATCC 25923, Ent. faecalis V583, Mor. catarrhalis and Strep. pneumoniae, Luria-Bertani (LB) broth for Ps. aeruginosa PA1 and Brain Heart Infusion (BHI) broth for sp. Group F and Strep. agalactiae. The strains Staph. aureus ATCC 25923, Table 1 Characteristic of the phenolic extracts used in this study Phenolic extracts rigin Total polyphenols (mg gallic acid equivalent per gram) Vitaflavan Ò Grape seeds Æ4 (GSE ) GSE-Mà Grape seeds Æ6 GSE- Grape seeds Æ8 Eminol Ò Red grape 34 1Æ4 pomace ProvinolsÔ Red wine Æ5 Revidox Ò Red grape skins Æ0 *RAC: oxygen radical absorbance capacity. GSE: grape seed extract. àgse-m: monomeric-rich fraction from Vitaflavan Ò. GSE-: oligomeric-rich fraction from Vitaflavan Ò. Antioxidant activity (RAC*, mmol Trolox per gram) 558 Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology

3 C. Cueva et al. Wine phenolics against respiratory pathogens Ent. faecalis V583 and Ps. aeruginosa PA1 were cultured aerobically at 37 C for h, whereas Mor. catarrhalis, Strep. pneumoniae, sp. Group F and Strep. agalactiae strains were cultured with 5% C 2. The strains were kept frozen at )70 C in a sterilized mixture of culture medium and glycerol (50 : 50, v v). Antibacterial activity assay Antibacterial assays were performed using a microdilution method according to Cueva et al. (2010). First, 20 ll of the phenolic solutions (78Æ lg ml )1 ), solvent (10 or 30% DMS) (growth control) or antibiotic solution (positive control) were added to the wells of sterile 96-well microplate. In a second step, 180 ll of broth medium was added to the blank wells. Finally, 180 ll of the diluted strain (inoculum of cfu ml )1 ) was added to the remaining microplate wells, including the controls. Bacterial growth was determined by reading the absorbance at 550 nm. The inhibition percentage of test microorganisms were calculated by comparing the control growth with those obtained from cultures with phenolic compounds or oenological extracts. The experiments were carried out in duplicate. For the active phenolic compounds and oenological extracts, the survival parameter IC 50 value was defined as the concentration required to obtain 50% inhibition of growth after the time of incubation and was estimated by nonlinear regression using the following sigmoidal dose response (with variable slope) equation: Top-Bottom Y ¼ Bottom þ ð1 þ 10 ððlogic 50 XÞSlopeÞ Þ where X represents the logarithm of concentration, Y is the response variable (%Inhibition) that starts at Bottom and goes to Top with a sigmoid shape, LogIC50 is the logarithmic of IC 50 and Slope represents the slope parameter. Prism for Windows release 4.03 (GraphPad Software, Inc., 2005, was used for the estimation of the four parameters. For each data set, the PRISM program also allows comparison of the fit to the previous sigmoidal dose response model (with four parameters) and the fit to the same model with the Bottom and Top parameters constrained to 0 and 100%, respectively. Results Antibacterial activity of phenolic compounds The phenolic compounds selected for this study represent components of different chemical structures present in wine and in oenological products. Table 2 reports the IC 50 values of all these compounds against the seven bacteria used in this study. Susceptibility to the phenolic compounds was found to differ considerably among the micro-organisms tested. Mor. catarrhalis was the most susceptible strain to all phenolic compounds and, in general, Gram-negative bacteria were more susceptible to these compounds than Grampositive bacteria (Table 2). Among the different compounds tested, gallic acid showed the most extensive effect, inhibiting the growth of six out of the seven bacteria tested. Both gallic acid and ethyl gallate were the compounds that showed the lowest IC 50 values for all bacteria, with the exception of sp Group F. Caffeic acid exhibited inhibitory effect against Gram-negative bacteria, Mor. catarrhalis and Ps. aeruginosa PA1 and Gram-positive bacteria, Ent. faecalis V583 and Staph. aureus ATCC (Table 2). Phenolic alcohols, tyrosol and tryptophol only showed inhibitory effect against Mor. catarrhalis and Ps. aeruginosa PA1. Among flavan-3-ols, (+)-catechin only showed inhibitory effect in the growth of Mor. catarrhalis and sp Group F, whereas ())-epicatechin was active against these two latter strains and against the other two streptococci, although at lower extent (Table 2). Antibacterial activity of phenolic extracts The antimicrobial activity of the six oenological phenolic extracts was also evaluated against the seven respiratory pathogens (Table 3). Mor. catarrhalis, Ps. aeruginosa PA1 and Strep. pneumoniae were susceptible to all phenolic extracts in different extents, whereas Strep. agalactiae was resistance to all of them. The lowest IC 50 values of the phenolic extracts were obtained for Mor. catarrhalis, with the exception of Eminol Ò (Table 3). Extracts from grape seeds were the most active inhibitors (lowest IC 50 ) against all the susceptible strains: Vitaflavan Ò was the most active for Ent. faecalis V583, Staph. aureus ATCC and Strep. pneumoniae; GSE- was for Mor. catarrhalis and Ps. aeruginosa PA1; and GSE-M was for sp. Group F. n the other hand, the wine extract ProvinolsÔ together with GSE-M and GSE- were found to show the most extensive antimicrobial effect, being active against all the strains tested, except Strep. agalactiae. Eminol Ò, a red grape pomace extract, only showed inhibitory effect against Mor. catarrhalis, Ps. aeruginosa PA1 and Strep. pneumoniae at higher concentrations. Finally, Revidox Ò, a resveratrol-rich extract, showed inhibitory effect against Mor. catarrhalis > Staph. aureus ATCC > Strep. pneumoniae > Ps. aeruginosa PA1 > Ent. faecalis V583 (Table 3). Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology 559

4 Wine phenolics against respiratory pathogens C. Cueva et al. Table 2 IC 50 (lg ml )1 ) values of the phenolic compounds studied against pathogenic bacteria Gram-negative bacteria Gram-positive bacteria Compounds Structure Moraxella catarrhalis Pseudomonas aeruginosa PA1 Enterococcus faecalis V583 Staphylococcus aureus ATCC sp Group F. agalactiae pneumoniae (+)-Catechin 129 n.e. n.e. n.e. 674 n.e. n.e. ())-Epicatechin 139 n.e. n.e. n.e Hydroxyphenethyl alcohol (Tyrosol) 15Æ n.e. n.e. n.e. n.e. n.e. 3-(2-Hydroxyethyl)indole (Tryptophol) N H n.e. n.e. n.e. n.e. n.e. Gallic acid 7Æ Æ9 n.e Ethyl gallate CH 3 1Æ n.e. n.e. 60Æ4 3,4-Dihydroxycinnamic acid (Caffeic acid) n.e. n.e. n.e. n.e.: no effect. Table 3 IC 50 (lg ml )1 ) values of the oenological phenolic extracts studied against pathogenic bacteria Gram-negative bacteria Gram-positive bacteria Extracts Moraxella catarrhalis Pseudomonas aeruginosa PA1 Enterococcus faecalis V583 Staphylococcus aureus ATCC sp Group F. agalactiae pneumoniae Vitaflavan Ò (GSE) 11Æ Æ5 n.e n.e. 195 GSE-M 8Æ n.e. 661 GSE- 7Æ n.e. 384 Eminol Ò n.e n.e n.e n.e ProvinolsÔ 30Æ n.e. 498 Revidox Ò n.e n.e. 295 n.e.: no effect. Discussion Previous studies have demonstrated that oral cavity is a potential reservoir for bacterial respiratory pathogens, particularly in people with poor oral health (Zuanazzi et al. 2010). This study reports valuable information about the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. Experiments with pure phenolic compounds would allow us to determine the influence of 560 Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology

5 C. Cueva et al. Wine phenolics against respiratory pathogens the phenolic compound type on their antimicrobial properties, whereas experiments with oenological extracts would assess the feasibility of their use in oral hygiene. In relation to the influence of pure phenolic compounds on the inhibition of the growth of potential respiratory pathogens, our results have shown that nonflavonoid compounds such as gallic acid, ethyl gallate and caffeic acid, tyrosol, tryptophol, were more active than flavonoids such as (+)-catechin and ())-epicatechin. These results are in accordance with Cueva et al. (2010), who reported greater inhibitory effect of phenolic acids in the growth of intestinal bacteria and pathogens in comparison with (+)-catechin and ())-epicatechin. Gallic acid and ethyl gallate were found to have the widest spectrum of antimicrobial activity, being effective against all tested bacteria, with the exception of the clinical strain sp Group F. The highest antimicrobial activity of gallic acid and ethyl gallate could be associated with the presence of the 3,4,5- trihydroxyphenyl group (the pyrogallol group), as suggested by Taguri et al. (2006). Besides, ethylation of gallic acid influences its antimicrobial properties depending on the strain studied (García-Ruiz et al. 2011). Among the scarce bibliography related to this topic, Rodríguez- Vaquero et al. (2007) also found that gallic acid and caffeic acid inhibited the growth of Ps. aeruginosa and Staph. aureus, which is in agreement with our results. The wine phenolic compounds with lower antimicrobial effect against the potential respiratory pathogens under study were tyrosol and tryptophol. It has been reported that Ps. aeruginosa was able to metabolize tyrosol giving hydroxytyrosol (Bouallagui and Sayadi 2006), which might explain, at least partially, the high IC 50 value obtained for this compound (Table 2). In relation to (+)- catechin and ())-epicatechin, it has been reported that flavan-3-ols significantly inhibited the growth of Strep. mutans and Strep. sobrinus (oral streptococci); however, they did not inhibit the growth of pathogenic bacteria such as Staph. aureus and Ps. aeruginosa strains (Rauha et al. 2000; Shan et al. 2007; Cueva et al. 2010), which agreed with our results. In contrast, other authors reported that certain strains of Staph. aureus and Ps. aeruginosa were inhibited by (+)-catechin and ())-epicatechin (Lee et al. 2006; Taguri et al. 2006; Rodríguez-Vaquero et al. 2007). It is remarkable that the susceptibility of bacteria tested to wine phenolic compounds may be physiologically relevant in the oral cavity, because the IC 50 values of the compounds tested are lower than the estimation of concentration present in red wine (García-Ruiz et al. 2008). The antimicrobial activities of wine and grape extracts in the prevention of periodontal oral diseases have been reported recently (Smullen et al. 2007; Furiga et al. 2009); however, there is not enough information about their ability to exert antimicrobial effects on potential respiratory pathogens. Testing different potential respiratory pathogens, our results have shown that extracts from grape seed, especially Vitaflavan Ò and GSE-, exerted higher antimicrobial activity than the rest of extracts obtained from red wine, grape pomace and grape skins (Xia et al. 2010). In this line, it has been reported that grape seed extracts showed antimicrobial activity against Staph. aureus, Ent. faecalis and Ps. aeruginosa strains, which is in agreement with our results (Baydar et al. 2004; Al-Habib et al. 2010); this effect may be associated with their phenolic content, as recently has been suggested by Bubonja-Sonje et al. (2011) for olive oil, cocoa and rosemary extract polyphenols. Additionally, the composition and structure of phenolic compounds from extracts may influence their antimicrobial properties. In particular, the degree of polymerization of the compounds might be pivotal for antimicrobial activity (Taguri et al. 2006; Xia et al. 2010). Concerning ProvinolsÔ and Revidox Ò, they exhibited antimicrobial activity against all bacteria tested with the exception of Strep. agalactiae and sp. Group F for ProvinolsÔ and Strep. agalactiae in the case of Revidox Ò. It has been previously reported that a red wine extract inhibited the growth of two oral streptococci, Strep. mutans and Strep. sobrinus (Furiga et al. 2009); however, their IC 50 values were higher than those obtained in our study, which could be due to differences in the strains as well as in the phenolic composition of the extracts. The resveratrol content of Revidox Ò might explain, at least partially, the antimicrobial activity exerted by this extract. In this sense, Chan (2002) found that resveratrol inhibited the growth of Staph. aureus, Ent. faecalis and Ps. aeruginosa, which is in accordance with our results. Finally, Eminol Ò only showed inhibitory effects against Mor. catarrhalis, Ps. aeruginosa PA1 and Strep. pneumoniae at high concentrations. This weak inhibitory effect may be related with its low phenolic content (Baydar et al. 2004). In general, phenolic extracts exhibited higher antimicrobial activity than pure phenolic compounds, being influenced by their individual phenolic composition. For instance, the extracts with higher content in gallic acid, such as Vitaflavan Ò and GSE-M (Tabasco et al. 2011), were those that showed greater antimicrobial activity. These results are in line with those of Shoko et al. (1999) who reported that in grapes, gallic acid was the most active compound for the inhibition of bacteria. Therefore, these results suggested that the antimicrobial activity of the extracts is related to the antimicrobial effects of their phenolic compounds. In addition, a synergistic effect of the main components of extracts cannot be ruled out (Rodríguez-Vaquero et al. 2010). Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology 561

6 Wine phenolics against respiratory pathogens C. Cueva et al. Results from both, phenolic compounds and oenological extracts, indicate that Gram-negative bacteria were more susceptible than Gram-positive bacteria, which may reflect differences in cell surface structures (Helander et al. 1998; Shan et al. 2007). This is a promising finding because many antibiotics are less active against Gram-negative than Gram-positive bacteria, probably due to a more complex cell wall structure of Gram-negative bacteria and the presence of additional lipopolysaccharides on their outer surface. Recently, Saavedra et al. (2010) have reported a synergistic effect between the antibiotic streptomycin and some phenolic compounds, such as gallic acid, against Gram-negative bacteria. From a clinical point of view, this finding could be interesting to decrease the antibiotic concentration necessary to treat oral bacterial infections. n the other hand, Mor. catarrhalis, which is known to be a major respiratory pathogen in paediatric and adult patient populations (Sarubbi et al. 1990), was also found to be the most susceptible strain to all phenolic standards and phenolic extracts tested. Similar results were obtained by Ayaz et al. (2008) who found that Mor. catarrhalis was the most susceptible Gram-negative strain against phenolic fractions (free, ester, glycoside and ester bound) of kale leaves and seeds. In conclusion, results of our study highlight the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. The antimicrobial activity of wine phenolic compounds was mainly conditioned by both, type of phenolic compound and bacteria species. Because their higher antibacterial activity, these extracts can serve as potential active components in formulations for oral hygiene. Acknowledgements This research was supported by the Spanish Ministry for Science and Innovation (AGL C02-00, AGL , AGL and CSD Consolider Ingenio 2010 FUN-C-FD Projects) and the Comunidad de Madrid (ALIBIRD P2009 AGR-1469 Project). C.C. is the recipient of fellowships from the FPI-MICINN programme. R.d.C. has a Miguel Servet contract (CB05 137) from the Instituto Nacional de Salud Carlos III-FIS. References Aas, J.A., Paster, B.J., Stokes, L.N., lsen, I. and Dewhirst, F.E. (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43, Al-Habib, A., Al-Saleh, E., Safer, A.M. and Afzal, M. (2010) Bactericidal effect of grape seed extract on methicillinresistant Staphylococcus aureus (MRSA). J Toxicol Sci 35, Ayaz, F.A., Hayirlioglu-Ayaz, S., Alpay-Karaoglu, S., Grúz, J., Valentová, K., Ulrichová, J. and Strnad, M. (2008) Phenolic acid contents of kale (Brassica oleraceae L. var. acephala DC.) extracts and their antioxidant and antibacterial activities. Food Chem 107, Baydar, N.G., Özkan, G. and Sadiç,. (2004) Total phenolic contents and antibacterial activities of grape (Vitis vinifera L.) extracts. Food Control 15, Bodet, C., Grenier, D., Chandad, F., fek, I., Steinberg, D. and Weiss, E.I. (2008) Potential oral health benefits of cranberry. Cri Rev Food Sci Nutr 48, Bouallagui, Z. and Sayadi, S. (2006) Production of high hydroxytyrosol yields via tyrosol conversion by Pseudomonas aeruginosa immobilized resting cells. J Agric Food Chem 54, Bubonja-Sonje, M., Giacometti, J. and Abram, M. (2011) Antioxidant and antilisterial activity of olive oil, cocoa and rosemary extract polyphenols. Food Chem 127, Chan, M.M.Y. (2002) Antimicrobial effect of resveratrol on dermatophytes and bacterial pathogens of the skin. Biochem Pharmacol 63, Cueva, C., Moreno-Arribas, M.V., Requena, T., Rodríguez, J.M., Vicente, F., Basilio, A., Bills, G.F., Martín-Álvarez, P.J. et al. (2010) Antimicrobial activity of phenolic acids against commensal, probiotic and pathogenic bacteria. Res Microbiol 161, Dávalos, A., Gómez-Cordovés, C. and Bartolomé, B. (2004) Extending applicability of the oxygen radical absorbance capacity (RAC-Fluorescein) assay. J Agric Food Chem 52, Furiga, A., Lonvaud, A. and Badet, C. (2009) In vitro study of antioxidant capacity and antibacterial activity on oral anaerobes of a grape seed extract. Food Chem 113, García-Ruiz, A., Bartolomé, B., Martínez-Rodríguez, A., Pueyo, E., Martín-Álvarez, P.J. and Moreno-Arribas, M.V. (2008) Potential of phenolic compounds for controlling lactic acid bacteria growth in wine. Food Control 19, García-Ruiz, A., Moreno-Arribas, M.V., Martín-Álvarez, P.J. and Bartolomé, B. (2011) Comparative study of the inhibitory effects of wine polyphenols on the growth of oenological lactic acid bacteria. Int J Food Microbiol 145, Guarner, F. and Malagelada, J.R. (2003) Gut flora in health and disease. Lancet 360, Helander, I.M., Alakomi, H.L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E.J., Gorris, L.G.M. and Von Wright, A. (1998) Characterization of the action of selected essential oil components on Gram-negative bacteria. J Agric Food Chem 46, Jayaprakasha, G.K., Selvi, T. and Sakariah, K.K. (2003) Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Res Int 36, Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology

7 C. Cueva et al. Wine phenolics against respiratory pathogens Lee, H.C., Jenner, A.M., Low, C.S. and Lee, Y.K. (2006) Effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota. Res Microbiol 157, Monagas, M., Gómez-Cordovés, C., Bartolomé, B., Laureano,. and Ricardo Da Silva, J.M. (2003) Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L Cv. Graciano, Tempranillo, and Cabernet Sauvignon. J Agric Food Chem 51, Özkan, G., Sagdiç,., Baydar, N.G. and Kurumahmutoglu, Z. (2004) Antibacterial activities and total phenolic contents of grape pomace extracts. J Sci Food Agric 84, Paju, S. and Scannapieco, F.A. (2007) ral biofilms, periodontitis, and pulmonary infections. ral Dis 13, Rauha, J.P., Remes, S., Heinonen, M., Hopia, A., Kähkönen, M., Kujala, T., Pihlaja, T. and Vuorela, P. (2000) Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. Int J Food Microbiol 56, Requena, T., Monagas, M., Pozo-Bayón, M.A., Martín-Álvarez, P.J., Bartolomé, B., del Campo, R., Ávila, M., Martínez- Cuesta, M.C. et al. (2010) Perspectives of the potential implications of wine polyphenols on human oral and gut microbiota. Trends Food Sci Technol 21, Rodríguez-Vaquero, M.J., Alberto, M.R. and Manca de Nadra, M.C. (2007) Antibacterial effect of phenolic compounds from different wines. Food Control 18, Rodríguez-Vaquero, M.J., Aredes Fernández, P.A., Manca de Nadra, M.C. and Strasser de Saad, A.M. (2010) Phenolic compound combinations on Escherichia coli viability in a meat system. J Agric Food Chem 58, Saavedra, M.J., Borges, A., Dias, C., Aires, A., Bennett, R.N., Rosa, E.S. and Simoes, M. (2010) Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria. Med Chem 6, Sánchez-Patán, F., Cueva, C., Monagas, M., Walton, G.E., Gibson, G.R., Quintanilla-López, J.E., Lebrón-Aguilar, R., Martín-Álvarez, P.J. et al. (2012) In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites. J Agric Food Chem 60, Sarubbi, F.A., Myers, J.W., Williams, J.J. and Shell, C.G. (1990) Respiratory infections caused by Branhamella catarrhalis: selected epidemiologic features. Am J Medicine 88 (Suppl. 1) S9 S14. Shan, B., Cai, Y.Z., Brooks, J.D. and Corke, H. (2007) Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomun burmannii): activity against foodborne pathogenic bacteria. J Agric Food Chem 55, Singleton, V.L. and Rossi, J.A. (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16, Shoko, T., Soichi, T., Megumi, M.M., Eri, F., Jun, K. and Michiko, K. (1999) Isolation and identification of an antibacterial compound from grape and its application to foods. Nippon Nogei Kagaku Kaishi 73, Smullen, J., Koutsou, G.A., Foster, H.A., Zumbé, A. and Storey, D.M. (2007) The antibacterial activity of plant extracts containing polyphenols against mutans. Caries Res 41, Souto, R., de Andrade, A.F.B., Uzeda, M. and Colombo, A.P.V. (2006) Prevalence of non oral bacteria in subgingival biofilm of subjects with chronic periodontitis. Braz J Microbiol 37, Tabasco, R., Sánchez-Patán, F., Monagas, M., Bartolomé, B., Moreno-Arribas, M.V., Peláez, C. and Requena, T. (2011) Effect of grape polyphenols on lactic acid bacteria and bifidobacteria growth: resistance and metabolism. Food Microbiol 28, Taguri, T., Tanaka, T. and Kouno, I. (2006) Antibacterial spectrum of plant polyphenols and extracts depending upon hydroxyphenyl structure. Biol Pharm Bull 29, Thimothe, J., Bonsi, J.A., Padilla-Zakour,.I. and Koo, H. (2007) Chemical characterization of red wine grape (Vitis vinifera and Vitis Interspecific Hybrids) and pomace phenolic extracts and their biological activity against mutans. J Agric Food Chem 55, van Uffelen, R., Van Saene, H.K.F., Fidler, V. and Lowenberg, A. (1984) ropharyngeal flora as a source of bacteria colonizing the lower airways in patients on artificial ventilation. Int Care Med 10, Xia, E.Q., Deng, G.F., Guo, Y.J. and Li, H.B. (2010) Biological activities of polyphenols from grapes. Int J Mol Sci 11, Zuanazzi, D., Souto, R., Mattos, B.A.M., Zuanazzi, M.R., Tura, B.G., Sansone, C. and Colombo, A.P.V. (2010) Prevalence of potential bacterial respiratory pathogens in the oral cavity of hospitalised individuals. Arch ral Biol 55, Letters in Applied Microbiology 54, ª 2012 The Society for Applied Microbiology 563