Probiotic-mediated competition, exclusion and displacement in biofilm formation by food-borne pathogens

Letters in Applied MicrobiologyVolume 56, Issue 4 p. 307-313 Original ArticleFree Access Probiotic-mediated competition, exclusion and displacement in biofilm formation by food-borne pathogens J. Woo, J. Woo Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, KoreaSearch for more papers by this authorJ. Ahn, Corresponding Author J. Ahn Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Korea Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA Correspondence J. Ahn, Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 200-701, Korea. E-mail: juheeahn@kangwon.ac.krSearch for more papers by this author J. Woo, J. Woo Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, KoreaSearch for more papers by this authorJ. Ahn, Corresponding Author J. Ahn Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Korea Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA Correspondence J. Ahn, Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 200-701, Korea. E-mail: juheeahn@kangwon.ac.krSearch for more papers by this author First published: 30 January 2013 https://doi.org/10.1111/lam.12051Citations: 71AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The objective of this study was to examine the inhibitory effect of probiotic strains on pathogenic biofilm formation in terms of competition, exclusion and displacement. Probiotic strains (Lactobacillus acidophilus KACC 12419, Lact. casei KACC 12413, Lact. paracasei KACC 12427 and Lact. rhamnosus KACC 11953) and pathogens (Salmonella Typhimurium KCCM 40253 and Listeria monocytogenes KACC 12671) were used to evaluate the auto-aggregation, hydrophobicity and biofilm formation inhibition. The highest auto-aggregation abilities were observed in Lact. rhamnosus (17·5%), Lact. casei (17·2%) and Lact. acidophilus (15·1%). Salm. Typhimurium had the highest affinity to xylene, showing the hydrophobicity of 53·7%. The numbers of L. monocytogenes biofilm cells during the competition, exclusion and displacement assays were effectively reduced by more than 3 log when co-cultured with Lact. paracasei and Lact. rhamnosus. The results suggest that probiotic strains can be used as alternative way to effectively reduce the biofilm formation in pathogenic bacteria through competition, exclusion and displacement. Significance and Impact of the Study This study provides new insight into biofilm control strategy based on probiotic approach. Probiotic strains effectively inhibited the biofilm formation of Listeria monocytogenes through the mechanisms of competition, exclusion and displacement. These findings contribute to better understand the probiotic-mediated competition, exclusion and displacement in biofilm formation by pathogens. Introduction Most bacteria undergo a transition from planktonic mode to biofilm mode depending on bacterial community and environmental conditions (Costerton et al. 1999). Unlike the planktonic cells, biofilm cells have distinctive characteristics such as antibiotic resistance, preservative tolerance and enhanced virulence, leading to chronic infections (Parsek and Singh 2003; Anderson and O'Toole 2008; Brady et al. 2008; Hancock et al. 2010). Biofilms are widespread in nature, dwelling on the food contact surfaces, medical device and surgical implants (Costerton et al. 1995; Parsek and Singh 2003). The ability of pathogenic bacteria to form biofilms plays an important role in the pathogenesis of infectious diseases (Martinez-Medina et al. 2009; Hancock et al. 2010). Therefore, more attention has been paid to the prevention and control of biofilm formation in bacterial pathogens. In this study, probiotic strains were used as an alternative strategy for controlling biofilm formation by pathogens. The use of probiotic strains can be considered as an alternative approach for reducing biofilm formation that has received much research attention in food and clinical industries. Probiotic strains known as nonpathogenic, safe and health beneficial are defined as live supplements for improving the intestinal microbial balance (Fuller 1989). Lactobacilli are the dominant species in the intestinal microflora in human and animals (Collins and Gibson 1999; Collado et al. 2007). Although the exact mechanisms of Lactobacilli-induced beneficial effects still remain unclear, the proposed mechanisms for their antimicrobial activity include the competition for adhesion sites and nutrients and the production of growth-inhibiting compounds, leading to the modulation of intestinal communities, the competitive exclusion of pathogens and the stimulation of immune functions (Nurmi et al. 1992; Servin and Coconnier 2003). However, relatively few studies have focused on the control of pathogenic biofilm formation by using probiotic concept. Therefore, the objective of this study was to investigate the ability of probiotic strains to compete with, exclude and displace pathogens during biofilm formation. The food-borne pathogens, Salm. Typhimurium and L. monocytogenes, were used as representative Gram-negative and Gram-positive bacteria, respectively. Results and discussion As bacterial biofilms are highly resistant to antibiotics and preservatives, it is essential to develop alternative method to control biofilm formation. This study demonstrates the auto-aggregation and adhesion properties (hydrophobic, electron-donor and electron-acceptor characteristics) of probiotic and pathogenic bacteria in association with the probiotic-mediated effect on the inhibition of pathogenic biofilm formation. Auto-aggregation properties of probiotic and pathogenic bacteria The auto-aggregation properties of pathogenic bacteria (Salm. Typhimurium and L. monocytogenes) and probiotic strains (Lact. acidophilus, Lact. casei, Lact. paracasei and Lact. rhamnosus) are shown in Fig. 1a. The auto-aggregation abilities of bacterial cells varied with the types of solutions, PBS and TSB. The highest auto-aggregation abilities were observed in Lact. rhamnosus (29·4%) and L. monocytogenes (24·7%) tested in PBS, which were significantly decreased to 17·5 and 5·2%, respectively, when tested in TSB. When compared with the PBS, the auto-aggregation values in TSB were not significantly changed in Salm. Typhimurium, Lact. acidophilus, Lact. casei and Lact. paracasei, ranging between 10·8 and 17·2%. The auto-aggregation responsible for the binding capability between cells varies according to temperature, osmolality and acidic conditions (Bereksi et al. 2002; Chavant et al. 2002). The auto-aggregation values obtained from TSB were more acceptable than those obtained from PBS as the biofilm formation assay was conducted in TSB. This observation suggests that the solutions used for the auto-aggregation assay need to be taken into consideration in evaluating cell surface property. Most probiotic strains and Salm. Typhimurium tested in TSB showed relatively higher auto-aggregation than L. monocytogenes, which was highly correlated with the biofilm formation (Soderling et al. 2011). The auto-aggregation ability is directly associated with the initial attachment of bacteria to biotic and abiotic surfaces, which is required to biofilm formation and mainly involved in the virulence of infectious pathogens (Van Loosdrecht et al. 1990; Kim et al. 2009). Figure 1Open in figure viewerPowerPoint Auto-aggregation abilities (a; □, PBS; ■, TSB) and cell surface activities (b; □, hydrophobic; , electron donor; ■, electron acceptor) of pathogenic bacteria (Salm. Typhimurium, ST; Listeria monocytogenes, LM) and probiotic strains (Lactobacilli. acidophilus, LA; Lact. casei, LC; Lact. paracasei, LP; Lact. rhamnosus, LR). Different letters (A–D, a–c and x–z) on the bar are significantly different at P < 0·05. *Indicates significant difference between PBS and TSB at P < 0·05. Data are the mean values of three replicates, and the error bars indicate the standard deviation (n = 3). Adhesion properties of probiotic and pathogenic bacteria to solvents The cell surface characteristics of food-borne pathogens and probiotic strains are shown in Fig. 1b. The affinities to apolar xylene, polar acidic chloroform and polar basic ethyl acetate were used to evaluate the hydrophobic surface, electron-donor and electron-acceptor properties of bacterial cells, respectively (Bellon-Fontaine et al. 1996; Kos et al. 2003). The most hydrophobic strain was Salm. Typhimurium (53·7%), followed by L. monocytogenes (27·4%), whereas the least hydrophobic strain was Lact. paracasei (3·7%), showing the least affinity to xylene. The greatest affinity to chloroform was observed in Salm. Typhimurium (82·4%), followed by L. monocytogenes (65·2%), Lact. rhamnosus (43·6%) and Lact. casei (24·5%). However, Lact. paracasei showed the least affinity to chloroform (7·3%). The highest affinity to ethyl acetate was observed in Salm. Typhimurium (27·4%), while Lact. paracasei showed the least affinity to ethyl acetate (11·1%). Compared with the pathogenic bacteria, relatively low affinities to xylene, chloroform and ethyl acetate were observed in probiotic strains with the exception of Lact. rhamnosus. Salm. Typhimurium showed higher hydrophobicity than other strains used in this study. The hydrophobicity of cell surface plays an important role in bacterial attachment, colonization and biofilm formation (Chavant et al. 2002; Gorski et al. 2003; Di Bonaventura et al. 2008; Zou et al. 2012). The food-borne pathogens, Salm. Typhimurium and L. monocytogenes, showed the highest affinities to chloroform, indicating their acidic and electron-donor properties. The bacterial affinities to ethyl acetate were low in all probiotic strains when compared with those to xylene and chloroform. This is in good agreement with previous report that probiotic strains had basic and low electron-acceptor properties (Pelletier et al. 1997). Probiotic-mediated inhibition of biofilm formation by pathogens The inhibitory effects of probiotic strains on biofilm formation by food-borne pathogens were evaluated in (i) competition (probiotic planktonic cells with pathogenic planktonic cells) (ii) exclusion (pathogenic planktonic cells on probiotic biofilm cells) and (iii) displacement (probiotic planktonic cells on pathogenic biofilm cells) to simulate the conditions occurred in bacterial communities (Figs. 2-4). Figure 2Open in figure viewerPowerPoint Competition abilities of probiotic planktonic cells (Lact. acidophilus, LA; Lact. casei, LC; Lact. paracasei, LP; Lact. rhamnosus, LR) against Salm. Typhimurium (ST) (a) and L. monocytogenes (LM) (b) planktonic cells. Log reduction in pathogenic (□) and probiotic (■) biofilm cells was estimated by subtracting the biofilm cell counts in co-cultures from those at the controls after 24-h incubation. Different letters (A-B and a-c) on the bar are significantly different at P < 0·05. *Indicates significant difference in the reduction between pathogen and probiotic strains at P < 0·05. Data are the mean values of three replicates, and the error bars indicate the standard deviation (n = 3). Figure 3Open in figure viewerPowerPoint Exclusion abilities of probiotic biofilm cells (Lact. acidophilus, LA; Lact. casei, LC; Lact. paracasei, LP; Lact. rhamnosus, LR) against Salm. Typhimurium (ST) (a) and L. monocytogenes (LM) (b) planktonic cells. Log reduction in pathogenic (□) and probiotic (■) biofilm cells was estimated by subtracting the biofilm cell counts in co-cultures from those at the controls after 24-h incubation. Different letters (A–B and a–d) on the bar are significantly different at P < 0·05. *Indicates significant difference in the reduction between pathogen and probiotic strains at P < 0·05. Data are the mean values of three replicates, and the error bars indicate the standard deviation (n = 3). Figure 4Open in figure viewerPowerPoint Displacement abilities of probiotic planktonic cells (Lact. acidophilus, LA; Lact. casei, LC; Lact. paracasei, LP; Lact. rhamnosus, LR) against Salm. Typhimurium (ST) (a) and L. monocytogenes (LM) (b) biofilm cells. Log reduction in pathogenic (□) and probiotic (■) biofilm cells was estimated by subtracting the biofilm cell counts in co-cultures from those at the controls after 24-h incubation. Different letters (A–C and a–d) on the bar are significantly different at P < 0·05. *Indicates significant different in the reduction between pathogen and probiotic strains at P < 0·05. Data are the mean values of three replicates, and the error bars indicate the standard deviation (n = 3). Competition The abilities of probiotic planktonic cells to competitively inhibit the biofilm formation by food-borne pathogens are shown in Fig. 2. During the co-culture, the numbers of probiotic and Salm. Typhimurium biofilm cells were reduced when compared with the single-strain cultures (controls) of probiotic strains and Salm. Typhimurium with the exception of Lact. rhamnosus (Fig. 2a). No significant reductions were observed in the numbers of Salm. Typhimurium biofilm cells when co-cultured with probiotic strains. The number of Lact. acidophilus biofilm cells was significantly reduced by 2·5 log, while that of Lact. rhamnosus biofilm cells was increased by 0·7 log when compared with the control. The numbers of L. monocytogenes biofilm cells were reduced by more than 2 log when co-cultured with probiotic strains (Fig. 2b). Compared with Salm. Typhimurium, the biofilm formation by L. monocytogenes was highly susceptible to probiotic cultures. Exclusion The abilities of probiotic biofilm cells to exclude biofilm formation by food-borne pathogens are shown in Fig. 3. The biofilm formation by Salm. Typhimurium was inhibited by probiotic biofilm cells (Fig. 3a). Similar to the competition assay, L. monocytogenes biofilm cells were highly susceptible to probiotic cultures, showing more than 3 log reduction (Fig. 3b). Compared with Salm. Typhimurium, in the presence of probiotic strains, the considerable reductions were observed in L. monocytogenes biofilm cells. Probiotic strains produce various antimicrobial compounds including organic acids, hydrogen peroxide and bacteriocins, which can interfere with the pathogenic growth (Vesterlund et al. 2006). The production of growth-inhibiting factors by probiotic strains may result in the reduction of biofilm formation by pathogens. The enhanced biofilm formation was observed in Lact. rhamnosus during the competition and exclusion, indicating that Lact. rhamnosus has a potential growth advantage over Salm. Typhimurium and L. monocytogenes. This can be a beneficial factor to control the adhesion and colonization of pathogenic bacteria (Hancock et al. 2010). The exopolysaccharides released from probiotic strains can also inhibit the biofilm formation by pathogens (Kim et al. 2009). Displacement The abilities of probiotic planktonic cells to displace pathogenic biofilm cells are shown in Fig. 4. The displacement activity of probiotic planktonic cells was not effective against Salm. Typhimurium (Fig. 4a), while the numbers of L. monocytogenes biofilm cells were effectively reduced by Lact. acidophilus (1·5 log), Lact. casei (1·9 log), Lact. paracasei (3·9 log) or Lact. rhamnosus (3·6 log) (Fig. 4b). The biofilm formation by L. monocytogenes was less inhibited in displacement assay than competition and exclusion assays (Fig. 4b). This observation was consistent with the previous report that the probiotic-mediated effect was less in post-treatment than pretreatment (Tahmourespour and Kermanshahi 2011). The biofilm formation by pathogens was clearly inhibited in competition, exclusion and displacement, suggesting that probiotic planktonic cells may affect the inhibition of pathogenic biofilm formation. The probiotic planktonic cells can inhibit the pathogenic adhesion and colonization through the competition for limited sites and resources (Collado et al. 2007; Tahmourespour and Kermanshahi 2011). In conclusion, the adhesion reduction can be a potential approach to effectively control the pathogenic infections as the biofilm formation process is initiated by adhesion. The most significant finding in this study was that the competition, exclusion and displacement could be possible inhibitory mechanisms of probiotic strains on L. monocytogenes biofilm formation. The application of probiotic strains can be useful method to reduce the risk of biofilm infections. The mechanistic approach involved in biofilm formation would provide new insight into biofilm control strategies. Therefore, the probiotic-mediated regulatory pathways on biofilm formation are currently under investigation in our laboratory to elucidate the virulence mechanisms associated with biofilm formation. Materials and methods Bacterial strains and culture conditions Strains of Lactobacillus acidophilus KACC 12419, Lactobacillus casei KACC 12413, Lactobacillus paracasei KACC 12427 and Lactobacillus rhamnosus KACC 11953 were kindly provided by the Korean Agricultural Culture Collection (KACC; Suwon, Korea). Probiotic strains were cultured anaerobically in de Man–Rogosa–Sharpe (MRS; Difco, BD Diagnostic Systems, Sparks, MD, USA) broth at 37°C for 24 h using an AnaeroGen GasPak system (BBL, Cockeysville, MD, USA). Pathogenic strains, Salmonella Typhimurium KCCM 40253 and Listeria monocytogenes KACC 12671, were obtained from the KCCM and Korean Culture Center of Microorganisms (KCCM; Seoul, Korea), respectively, and cultured in trypticase soy broth (TSB; BD, Becton, Dickinson and Co.,) at 37°C for 20 h. All cultures were collected by centrifugation at 3000 g for 20 min at 4°C. Auto-aggregation assay The cell-to-cell interactions were evaluated according to the auto-aggregation assay (Del Re et al. 2000) with slight modifications. The probiotic and pathogenic bacterial cells grown in TSB at 37°C for 20 h were resuspended in phosphate-buffered saline (PBS; pH 7·2) and TSB to OD600 of 0·5 (A0). Each bacterial suspension (3 ml) was vortexed and incubated at 37°C for 2 h. The absorbance was measured at 600 nm (At). The auto-aggregation ability was estimated as follows: Bacterial adhesion to solvent assay The cell surface properties were evaluated using the bacterial affinity to solvent (BATS) assay (Kos et al. 2003) with minor modifications. The cultured bacterial cells were resuspended in PBS to OD600 of 0·5 (H0). The cell suspensions (3 ml each) were vortexed with 1 ml of xylene, chloroform or ethylene acetate for 1 min and allowed to stand for 5 min to separate the aqueous phase. The absorbance was measured at 600 nm (Ht). The affinities to solvents were estimated as follows: Preparation of biofilm cells The probiotic strains (Lact. acidophilus, Lact. casei, Lact. paracasei and Lact. rhamnosus) and pathogenic bacteria (Salm. Typhimurium and L. monocytogenes) (106 CFU ml−1 each) were incubated anaerobically in TSB at 37°C until biofilm cell density reached 106 CFU ml−1 on 12-well flat-bottomed polystyrene microtitre plate (BD Falcon; San Jose, CA, USA). After cultivation, each well was rinsed twice with PBS to remove nonadherent cells and then used for exclusion and displacement assays. Inhibition of biofilm formation The ability of probiotic strains to inhibit pathogenic biofilms was evaluated by three different assays, including competition, exclusion and displacement assays. For the competition assay, probiotic strain (Lact. acidophilus, Lact. casei, Lact. paracasei or Lact. rhamnosus) and pathogenic bacterium (Salm. Typhimurium or L. monocytogenes) were co-cultured at approximately 107 CFU ml−1 in 12-well plates for 24 h at 37°C. For the exclusion assay, pathogenic bacterial cells (107 CFU ml−1 each) were seeded in the preprepared probiotic biofilm cells on 12-well plates and incubated for 24 h at 37°C. For the displacement assay, probiotic cells (107 CFU ml−1 each) were seeded in the preprepared pathogenic biofilm cells on 12-well plates and incubated for 24 h at 37°C. After 24-h incubation, each well was rinsed twice to remove nonadherent cells. The monospecies cultures of probiotic strains and pathogenic bacteria were used as the controls. Enumeration of biofilm cells The biofilm cells were collected by using a cell scraper and serially (1 : 10) diluted with PBS. The proper dilutions were plated on MRS agar for probiotic strains, xylose lysine deoxycholate (XLD) agar for Salm.Typhimurium and modified oxford (MOX) agar for L. monocytogenes. The plates were incubated at 37°C for 24–48 h to enumerate bacterial counts. Statistical analysis All analyses were conducted in duplicate for three replicates. The Statistical Analysis System software (SAS) was used to evaluate auto-aggregation, hydrophobicity and biofilm formation data. The general linear model (GLM) and Fisher's least significant difference (LSD) procedures were used to determine significant differences at P < 0·05. 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