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.Microorganisms in foods may be derived from the raw materials, ingredients, personnel, or the work environment. Epidemics of listeriosis, which is rare but serious when it affects the so-called at-risk population (43), have been traced back to environmental contamination of food processing areas (48). Listeria monocytogenes genotypes, absent from the raw materials, may be repeatedly found in the finished products and on work surfaces (3). It is therefore interesting to identify the factors that affect adhesion strength and hence transfer of L. monocytogenes from an inert surface to a food following contact. Such data can be useful for quantitative analysis of the risks associated with ready-to-eat foods that may be contaminated during manufacture (11,18). Most studies of detachment of microbial cells use biofilms formed under dynamic conditions in which detachment is induced by liquid flow. These studies have shown the influence of numerous factors on the detachme...

Positive Correlation Between the Expression of inlA and inlB Genes of Listeria monocytogenes and Its Attachment Strength on Glass Surface

“…The constant A is affected by the initial number of attached cells, but the k value is independent of A. 24,26,31,32 The k values are listed in Table 1 and most values fall in the range of 0.2∼0.5. An ATCC strain LM_7644 (serotype 1/2c) had the highest (p<0.05) k value (0.74), while LM_S20 (serotype 1/2b) and LM_S28 (serotype 4b), samples that were isolated from catfish plants, had the lowest k values (0.22) with the highest (p<0.05) attachment strength ( Table 1).…”

“…24,26,31,32 By plotting the logarithm of the number of transferred colonies as the function of the plate succession number, a one-phase curve for all L. monocytogenes strains was obtained, and the results of two representatives (LM_S3 and LM_27) were shown in Figure 1. Regression analysis showed that the data fit with the general equation CFU=A× e −kN , where CFU is the number of colonies transferred, A is the original ordinate, N is the plate succession number, and k is the value that reflects the ease of detachment of attached bacteria from the glass.…”

“…The attachment strength of L. monocytogenes strains was measured using a blot succession method 24,26,31,32 with some modifications. Incubation time and temperature were similar to processing plant conditions.…”

The effect of surface properties on the strength of attachment of fungal spores using AFM perpendicular force measurements

“…Whilst bacterial attachment has been extensively studied [26][27][28][29], considerably less attention has been given to fungal attachment with the exception of the opportunistic pathogen Candida albicans [30,31]. 0927-7765/$ -see front matter © 2010 Elsevier B.V. All rights reserved.…”

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

“…Work has been published regarding listerial transfer from either processing surfaces to foods or vice versa. [1][2][3][4][5][6][7] The atomic force microscopy (AFM) was invented in 1986 8 , and since then, it has been used to image stainless steel 9-13 as well as bacterial cells. [14][15][16][17][18] To generate images using AFM, a cantilever is rastered over a surface, and the change in location of a reflected laser focused on the top of the cantilever is used to monitor the surface topography of the sample.…”