Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida Paula Saá Ibusquiza * , Juan J.R. Herrera, Daniel Vázquez-Sánchez, Marta L. Cabo ** Instituto de Investigaciones Marinas (C.S.I.C.), Eduardo Cabello 6, 36208 Vigo, Pontevedra, Spain article info Article history: Received 13 May 2011 Received in revised form 23 September 2011 Accepted 3 October 2011 Keywords: Biofilm Disinfection Listeria monocytogenes Pseudomonas putida Benzalkonium chloride Electronic microscopy abstract Comparison between the resistance to BAC and the microscopic structure between mixed-species bio- films formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25  C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC- adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium implicated in numerous foodborne outbreaks (Denny & McLauchlin, 2008; Gilmour et al., 2010). It is widely recognized that one of the most frequent contamination routes of L. monocytogenes in foods is through cross contamination from the surfaces in food processing plants (Ammor et al., 2004; Norwood & Gilmour, 1999; Porsby, Vogel, Mohr, & Gram, 2008), where it is able to attach and form resistant biofilms (Aase, Sundheim, Langsrud, & Rørvik, 2000; Takahashi et al., 2009; To, Favrin, Romanova, & Griffiths, 2002). This resistance is related to the three- dimensional structure of the biofilm, which defines the interac- tions between the bacterial species (Wuertz, Okabe, & Hausner, 2004) and determines the degree to which chemicals can access the cells (Bourion & Cerf, 1996; Qu, Daley, Istivan, Rouch, & Deighton, 2010). Moreover, it has been demonstrated that biofilm cells are phenotypically and genotypically different from the cor- responding planktonic cells (Nadell, Xavier, & Foster, 2009). But the situation is even more complicated, as it is known that more than two species coexist in real biofilms and it has been demonstrated that this could increase their resistance to biocides (Kastbjerg & Gram, 2009; Sharma & Anand, 2002). Some studies on L. monocytogenes have analysed the attachment of multispecies biofilms involving this bacterium, with different results. It has been found that in some cases the association with other bacteria increases the number of adhered cells in the mixed biofilm: L. monocytogenes with Pseudomonas (Hassan, Birt, & Frank, 2004; Sasahara & Zottola, 1993) or with Flavobacterium (Bremer, Monk, & Osborne, 2001). However, other studies have found that the number of adhered cells in the mixed biofilm decreased: L. monocytogenes with Pseu- domonas fragi and Staphylococcus xylosus (Norwood & Gilmour, 2001); with Staphyloccocus sciuri (Leriche & Carpentier, 2000); and with Enterococcus faecium (Minei, Gomes, Regianne, D’Angelis, & De Martinis, 2008). Lastly, some studies found that the number of adhered cells did not vary due to the presence of other species in the biofilm: L. monocytogenes with Staphyloccocus aureus (Rieu, Lemaître, Guzzo, & Piveteau, 2008), except for one strain; and with Salmonella enterica and Pseudomonas putida (Chorianopoulos, Giaouris, Skandamis, Haroutounian, & Nychas, 2007). * Corresponding author. Instituto Investigaciones Marinas (C.S.I.C.), Microbiology and Sea Food Technology, Eduardo Cabello 6, 36208 Vigo, Pontevedra, Spain. Tel.: þ34 678068622; fax: þ34 986 292762. ** Corresponding author. E-mail addresses: psaa@iim.csic.es (P. Saá Ibusquiza), marta@iim.csic.es (M.L. Cabo). Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.002 Food Control 25 (2012) 202e210