Evaluation of Rhamnolipid and Surfactin to Reduce the Adhesion and Remove Biofilms of Individual and Mixed Cultures of Food Pathogenic Bacteria
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Food Control 25 (2012) 441e447 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria Milene Zezzi do Valle Gomes, Marcia Nitschke* Depto. Físico-Química, Instituto de Química de São Carlos (IQSC) e USP, Av. Trabalhador São Carlense, 400, Caixa Postal 780, São Carlos, SP, CEP 13560-970, Brazil article info abstract Article history: Biofilms represent a great concern for food industry, since they can be a source of persistent contami- Received 18 August 2011 nation leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and Received in revised form the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, 16 November 2011 microbial compounds that can modify the physicochemical properties of surfaces changing bacterial Accepted 22 November 2011 interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion Keywords: and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Bacterial adhesion Biofilms Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene Biosurfactants as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of Rhamnolipids L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% Surfactin adhesion of L. monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L. monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in View metadata, citation and similar papers at core.ac.uk brought to you by CORE concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids canprovided be explored by Elsevier to- Publisher control Connector the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens. Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license. 1. Introduction Biofilm formation can be controlled by cleaning strategies using chemical and physical methods however, bacteria living in biofilms The attachment of bacteria to surfaces and the consequent are more resistant to disinfectants and more difficult to remove biofilm formation has serious implications in food, environmental mechanically when compared to planktonic forms (Simões et al., and medical fields. The occurrence of biofilm in food processing 2010). Surfactants are the chemical products usually utilized for environments can lead to spoilage and transmission of diseases cleaning food contact surfaces. representing a risk for the health of consumers and a cause of In last years biosurfactants (BS), surface-active compounds of economical losses to industry (Simões, Simões, & Vieira, 2010). microbial origin, have been attracted attention due to their low Listeria monocytogenes, Salmonella Enteritidis and Staphylo- toxicity and high biodegradability when compared to synthetic coccus aureus are examples of food-borne pathogenic bacteria able surfactants (Banat et al., 2010; Nitschke et al., 2005). Bacterial to form biofilms in a variety of surfaces commonly used in food derived biosurfactants exhibit a variety of chemical structures and environment such as stainless steel, plastics, rubber and glass properties useful for industrial purposes. Surfactin, a lipopeptide (Marques et al., 2007; Meylheuc, van Oss, & Bellon-Fontaine, 2001; produced by Bacillus subtilis and rhamnolipid, a glycolipid from Oliveira, Oliveira, Teixeira, Azeredo, & Oliveira, 2007; Stepanovic, Pseudomonas aeruginosa are the best known biosurfactants. These Cirkovic, Ranin, & Svabic-Vlahovic, 2004). molecules present strong surface activity, emulsion forming ability and has shown antimicrobial properties (Nitschke, Costa, & Contiero, 2010; Ongena & Jacques, 2008). * Corresponding author. Tel.: þ55(0) 16 33738101; fax: þ55(0) 16 33739952. Previous studies has shown that adsorption of biosurfactants to E-mail address: [email protected] (M. Nitschke). a solid surface can modify its hydrophobicity affecting the adhesion 0956-7135 Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.foodcont.2011.11.025 442 M. Zezzi do Valle Gomes, M. Nitschke / Food Control 25 (2012) 441e447 process and consequently the biofilm formation. A 0.1% surfactin polystyrene surface: rhamnolipids 0.25%, 0.5% and 1.0% (w/v) solution was reported to reduce significantly the adhesion of aqueous solutions and surfactin 0.1%, 0.25% and 0.5% (w/v) aqueous L. monocytogenes and E. sakazakii to stainless steel and poly- solutions. A 96 well polystyrene microplate was filled with 200 mlof propylene (Nitschke et al., 2009). The pre-conditioning of poly- each concentration of biosurfactant. After 24 h at room tempera- styrene with 0.1% solution of surfactin reduces by 84% the adhesion ture, solution was removed and the wells were gently washed 3 of L. monocytogenes ATCC 7644 whereas rhamnolipids at 0.75% times with 200 ml of sterile distilled water. Control wells were filled reduced the adhesion of L. monocytogenes ATCC 15313 by 82% with 200 ml of water. (Araújo, Lins, Santa Anna, Nitschke, & Freire, 2011). The anti- adhesive properties of biosurfactants shows to be dependant on 2.6. Adhesion test the surface and microorganism involved as well as the temperature, and the type and concentration of the surfactant (Shakerifard, The wells of the polystyrene microplate pre-conditioned with Gancel, Jacques, & Faille, 2009; Zeraik & Nitschke, 2010). Most biosurfactants were filled with 180 ml of TSYE broth, inoculated studies regarding anti-adhesive properties of biosurfactants were with 20 ml of bacterial suspension (of pure or mixed cultures) and conducted using pure cultures of microorganisms and in the incubated at 35 C for different time intervals. Cell suspension was absence of culture medium however, it is known that mixed removed, and wells washed with water, fixed with 200 mlof fi cultures are predominantly found in bio lms and that the presence methanol for 15 min and stained for 15 min with crystal violet 1.0% ’ of nutrients can affect the adhesion. Irie, O Toole, and Yuk (2005) (w/v). The wells were washed and filled with 200 ml of acetic acid fi reported that rhamnolipids can disperse bio lms of the respira- 33% (v/v). The adhesion kinetics of adhered cells was estimated by tory pathogen Bordetella bronchiseptica, suggesting another reading the optical density at 630 nm (Mireles, Toguchi, & Harshey, potential use for biosurfactants. The present work evaluates the 2001) using a microplate reader (TP Reader e Thermoplate). effect of the pre-conditioning of polystyrene surfaces with surfactin and rhamnolipid on the adhesion of pure and mixed growing 2.7. Disruption of pre-formed biofilms cultures of food pathogenic bacteria. The potential of the bio- surfactants to disrupt pre-formed biofilms of such bacteria was also Biofilm formation by L. monocytogenes, S. aureus and Salmonella investigated. Enteritidis was performed as individual cultures and mixed cultures. The 96 well polystyrene microplate was filled with 180 ml 2. Materials and methods of TSYE broth, inoculated with 20 ml of individual or mixed bacterial suspension and incubated at 35 C for 24 h. Cell suspension was 2.1. Bacterial strains removed and 180 ml of fresh TSYE broth and inoculum were added and incubated for more 24 h. After 48 h of growth the cell L. monocytogenes ATCC 19112, S. aureus ATCC 25923 and suspension was removed and the formed biofilm were added of Salmonella enterica Enteritidis PNCQ0301 were stored at - 20 Con 200 ml of surfactin (0.1% and 0.5% concentrations) or rhamnoli- trypticase soy broth (TSB) supplemented with 20% glycerol (v/v). pids(0.25% and 1.0% concentrations) solutions. The effectiveness of biosurfactants was verified after 2 h, 6 h and 12 h of contact with 2.2. Medium and culture conditions the biofilms. The wells were washed three times with 200 mlof distillated water and the adhered cells were quantified as described Bacterial strains were cultivated in trypticase soy broth (TSB) or À above. agar (TSA) supplemented with 6 g l 1 (w/v) of yeast extract (TSYE) and incubated at 35 C for 24 h. 2.8. Contact angle measurements 2.3. Preparation of bacterial suspension Polystyrene samples of 3 cm2 were cleaned as described by Bacteria from stock medium were inoculated on a TSYE agar Zeraik and Nitschke (2010). The samples were conditioned with the plate at 35 C for 24 h. Nine milliliters of sterile distilled water were biosurfactants for 24 h at room temperature, washed with 10 ml of added to the culture and then scraped from the plate. The dense water and left to dry in a desiccator for 24 h. As control, samples of À bacterial suspension obtained was adjusted to have 109 CFU ml 1 polystyrene were immersed in distillated water for 24 h. The and used to perform the adhesion tests. To prepared mixed contact angle of water was assessed by the sessile drop technique at m cultures, 1 ml of each standardized individual cultures were pooled 20 C using a drop volume of 4 l on a Contact Angle System (CAM in a sterile glass tube. 200- KSV). 2.4. Biosurfactants 2.9. Physicochemical characterization of cell surfaces Rhamnolipids were produced by P. aeruginosa LBI using soybean To evaluate hydrophobic/hydrophilic nature of bacterial cells oil as carbon source as described by Nitschke et al.