Production of biofilm by Listeria monocytogenes in different materials and temperatures
Introduction
Listeria monocytogenes is a Gram-positive, mesophilic pathogen with psychrotrophic characteristics, involved in several outbreaks of foodborne illness (Gandhi & Chikindas, 2007; Wilks, Michels, & Keevil, 2006). L. monocytogenes cells can be fixed onto various surfaces, especially in inaccessible parts of industrial equipment due to biofilm formation (Pan, Breidt, & Kathariou, 2006). This can occur rapidly and the substrate, once attached, is difficult to remove (Frank & Koffi, 1990). L. monocytogenes has been found in biofilms on plastic surfaces, polypropylene, rubber, stainless steel and glass (Jeong & Frank, 1994). So, sanitization plays a very important role in improving food safety. Yang, Dier-Gonzalez, and Feirtag (2013) observed that electrochemically activated water (ECAW) presented good results, because 100 mg/L ECAW decreased at least 5 log CFU/ml on liquid culture and more than 4 log CFU in stainless steel coupon for Escherichia coli O157:H7, L. monocytogenes and Salmonella.
The luxS gene encodes S-ribosylhomocysteinase, which catalyzes the hydrolysis of S-ribosylhomocysteine to homocysteine and 4,5-dihydroxy-2,3-pentadione (DPD), and works as precursor of an autoinducer molecule (AI-2). AI-2 is involved in the quorum-sensing response in Vibrio harveyi (Bassler, Wright, & Silverman, 1994) and is also found in many Gram-positive and negative bacteria (Vendeville, Winzer, Heurlier, Tang, & Hardie, 2005) and has been considered essential in communication between species. Belval et al. (2006) concluded that L. monocytogenes also produces AI-2 molecules, and the luxS gene may be involved in the regulation of biofilm formation, including in other bacteria (Daines et al., 2005; Xu et al., 2006).
The gene inlaA, besides encoding internalin (InlA), seems to play a role in surface attachment (Chen, Kim, Jung, & Silva, 2008) and the adherence of bacteria to surfaces is affected by the physicochemical properties of the environment (pH and temperature), surface (hydrophobicity) and characteristics such as the motility of the bacteria (Moltz & Martin, 2005).
Environmental factors, such as temperature, may regulate genes related to virulence, or with structures that result in changes in the cell surface, which may affect compliance with the formation of flagella in L. monocytogenes (Liu, Graham, Bigelow, Morse, & Wilkinson, 2002).
The objective of this study was to evaluate the ability of 30 strains of L. monocytogenes isolated from the environment and different foods (vegetables and milk) and two standard strains (ATCC 7644 and ATCC 16313), to form biofilms on hydrophobic (polystyrene) and hydrophilic surfaces (glass and stainless steel) at different temperatures (4, 20 and 30 °C).
Section snippets
Samples
We used 30 strains previously isolated from milk, vegetables and food processing environments, in the Food Microbiology Laboratory, UNESP/Botucatu. In addition, two standard strains were used (ATCC 7644 and 16313).
PCR for the inlA and luxS genes
Each strain of L. monocytogenes was inoculated into brain heart infusion broth (Difco, Sparks, NV, USA) at 37 °C/24 h, and 300 μl was transferred to microfuge tubes, for DNA extraction and purification, using the commercial kit “MiniSpin” (GE Healthcare, Little Chalfont, UK),
PCR of the inlA and luxS genes
All samples previously identified as L. monocytogenes were confirmed by PCR for the inlA gene (Fig. 1). We also performed PCR reactions for the luxS gene, and 25 (78.1%) out of 32 strains were positive (Fig. 2).
Production of biofilm on hydrophilic materials (stainless steel and glass)
According to Table 1, biofilm production occurred on stainless steel at 4 °C for 24 h in 62.5% (20/32) of the strains. After 48 h, there was an increase to 87.5% (28/32) and by 72 h there was a decrease to 84.3% (27/32). At the same temperature, using glass slides, in 24 h of incubation,
Discussion
L. monocytogenes has been a serious problem in the food industry due to biofilm production, which leads to its prevalence in food processing environments. All strains possessed the inlA gene, specific to L. monocytogenes (Poyart, TrieuCuot, & Berche, 1996) and encode internalin (InlA), a surface protein that facilitates the entry of the microorganism into epithelial cells expressing the E-cadherin receptor; this is an important step in the pathogenesis of listeriosis (Nightingale, Windham,
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