Elsevier

Food Control

Volume 35, Issue 1, January 2014, Pages 386-391
Food Control

Production of biofilm by Listeria monocytogenes in different materials and temperatures

https://doi.org/10.1016/j.foodcont.2013.07.023Get rights and content

Highlights

  • luxS and inlA genes may be involved in biofilm formation.

  • After 48 h, there was a decreased in biofilm formation.

  • L. monocytogenes adhered better to hydrophilic surfaces than to hydrophobic one.

Abstract

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L. monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L. monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30 °C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20 °C for 24 h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35 °C for 48 h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (p = 0.3847). We conclude that L. monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm.

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,

References (43)

  • B.L. Bassler et al.

    Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway

    Molecular Microbiology

    (1994)
  • C.S. Belval et al.

    Assessment of the roles of LuxS, S-ribosyl homocysteine, and autoinducer 2 in cell attachment during biofilm formation by Listeria monocytogenes EGD-e

    Applied and Environmental Microbiology

    (2006)
  • L.C. Blackman et al.

    Growth of Listeria monocytogenes as a biofilm on various food-processing surfaces

    Journal of Food Protection

    (1996)
  • M.K. Borucki et al.

    Variation in biofilm formation among strains of Listeria monocytogenes

    Applied and Environmental Microbiology

    (2003)
  • J. Chandra et al.

    In vitro growth and analysis of Candida biofilms

    Nature Protocols

    (2008)
  • P. Chavant et al.

    Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

    Applied and Environmental Microbiology

    (2002)
  • B.Y. Chen et al.

    Attachment strength of Listeria monocytogenes and its internalin negative mutants

    Food Biophysics

    (2008)
  • B.Y. Chen et al.

    Positive correlation between the expression of inlA and inlB genes of Listeria monocytogenes and its attachment strength on glass surface

    Food Biophysics

    (2009)
  • D.A. Daines et al.

    Haemophilus influenzae luxS mutants form a biofilm and have increased virulence

    Microbial Pathogenesis

    (2005)
  • G. DiBonaventura et al.

    Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity

    Journal of Applied Microbiology

    (2008)
  • A.F. Gonzalez Barrios et al.

    Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022)

    Journal of Bacteriology

    (2006)
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