Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

Abstract

Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.

Listeria monocytogenes-carrying consortia in food industry. Composition, subtyping and numerical characterisation of mono-species biofilm dynamics on stainless steel

“…Association capacity assays of dual-species biofilms demonstrated how L. monocytogenes isolates were able to form biofilms along with other microorganisms (Giaouris et al, 2013;Schirmer et al, 2013;Talon et al, 2007;Van der Veen and Abee, 2011). Outcomes obtained showed that Pseudomonas sp.…”

“…L. monocytogenes is able to form multispecies biofilms with both Gram positive and Gram negative species (Daneshvar Alavi and Truelstrup Hansen, 2013;Rieu et al, 2008;Van der Veen and Abee, 2011) and the interaction among species forming these consortia varies depending on the genera implicated and the environmental conditions (Van der Veen and Abee, 2011). Various studies involving L. monocytogenes multispecies biofilms have highlighted the complexity of such interactions and the different effects that associated bacteria could have on L. monocytogenes in terms of the number of adhered cells (Carpentier and Chassaing, 2004;Leriche and Carpentier, 2000;Rieu et al, 2008) or the resistance to a sanitisation treatment (Giaouris et al, 2013;Kostaki et al, 2012;Pan et al, 2006;Saá Ibusquiza et al, 2012;Van der Veen and Abee, 2011). Hence it seems to be clear that increasing the knowledge regarding multispecies biofilms could provide key information so as to improve control strategies and reduce food product contamination.…”

A 3-year hygiene and safety monitoring of a meat processing plant which uses raw materials of global origin

“…The agarose plugs were digested with 10 U XbaI (New England Biolands) for 16 h at 37°C and the restriction fragments were differentiated on a 1% w/v agarose gel, using 0.5 M Tris-Borate buffer at 14°C in CHEF-DR II (Bio-Rad Lab., Hercules, CA, USA) with the following parameters: electric field of 6 V/cm, switching times of 2.2-63.2 s, at 14°C for 20 h. The agarose gels were stained with ethidium bromide (0.5 μg/ml) for 1 h, destained for 2 h in water and the obtained fingerprints were captured using a GelDoc system (Bio-rad Lab). All isolates of L. monocytogenes that were identified by the molecular methods and phenotypic assays were subjected to DNA macrorestriction with PFGE for fingerprinting, according to Giaouris et al (2013). Plugs of the isolates were digested with 10 U of ApaI enzyme (New England Biolabs, Ipswich, MA, USA) at 25°C C for 16 h and the restriction fragments were differentiated on 1% w/v agarose gel with the following set of parameters: an initial switch of 4 s, a final switch time of 40 s, 6 V/cm, and a run time of 18 h.…”

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments

“…Nevertheless, it has been extensively demonstrated that biofilms exhibit higher resistance/tolerance to BAC compared to planktonic cells both in Gram-positives such as L. monocytogenes (Saá Ibusquiza et al 2011;Nakamura et al 2013) and in Gram-negatives such as Escherichia coli (Houari & Di Martino 2007) or Pseudomonas sp. (Giaouris et al 2013). …”