Construction and analysis of fractional multifactorial designs to study attachment strength and transfer of Listeria monocytogenes from pure or mixed biofilms after contact with a solid model food

Abstract

The aim of this study was to establish which of seven factors influence the adhesion strength and hence bacterial transfer between biofilms containing Listeria monocytogenes (pure and two-species biofilms) and tryptone soya agar (TSA) as a solid organic surface. The two-species biofilms were made of L. monocytogenes and one of the following species of bacteria: the nonpathogenic organisms Kocuria varians, Pseudomonas fluorescens, and Staphylococcus sciuri and CCL 63, an unidentified gram-negative bacterium isolated from the processing plant environment. We used biofilms prepared under conditions simulating open surfaces in meat-processing sites. The biofilm's adhesion strength and population were evaluated by making 12 contacts on a given whole biofilm (4.5 cm(2)), using a new slice of a sterilized TSA cylinder for each contact, and plotting the logarithm CFU . cm(-2) detached by each contact against the contact number. Three types of detachment kinetics were observed: biphasic kinetics, where the first slope may be either positive or negative, and monophasic kinetics. The bacteria that resisted a chlorinated alkaline product and a glutaraldehyde- and quaternary ammonium-based disinfectant had greater adhesion strengths than those determined for untreated biofilms. One of the four non-Listeria strains studied, Kocuria varians CCL 56, favored both the attachment and detachment of L. monocytogenes. The stainless steel had smaller bacterial populations than polymer materials, and non-Listeria bacteria adhered to it less strongly. Our results helped to evaluate measures aimed at controlling the immediate risk, linked to the presence of a large number of CFU in a foodstuff, and the delayed risk, linked to the persistence of L. monocytogenes and the occurrence of slightly contaminated foods that may become dangerous if L. monocytogenes multiplies during storage. Cleaning and disinfection reduce the immediate risk, while reducing the delayed risk should be achieved by lowering the adhesion strength, which the sanitizers used here cannot do at low concentrations.

FIG. 1.

Slope k₂ as a function of slope k₁ in curves of the logarithm of CFU cm⁻² of L. monocytogenes detached by contact of a mixed biofilm with a solid food model versus the contact number.

FIG. 2.

Example of each of the three types of detachment kinetics observed. These curves plot the logarithm of the number of CFU cm⁻², detached from a biofilm through contact with a solid food model, against the contact order. (a) Biphasic curve, where the first slope k₁ is positive. This is a 1-day biofilm of K. varians grown at 25°C with added glucose on stainless steel and then subjected to a chemical shock with Galorox JH 3 (a chlorinated alkaline product). (b) Biphasic curve where the value for k₁ is negative and below the value for k₂. The results are from a 1-day biofilm of P. fluorescens grown at 15°C with added glucose and calcium on polyurethane (PU2). (c) Monophasic curve (k₁ = k₂). These results are from a 2-day biofilm of K. varians grown at 25°C with added glucose and calcium on polyurethane (PU1).

FIG. 3.

Logarithm of the number of contacts needed to reach 1 CFU cm⁻² of L. monocytogenes on the biofilm support as a function of the log (−k₂) (a) and the logarithm of the L. monocytogenes population in mixed biofilms (b).