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Biofilm formation by Staphylococcus aureus on food contact surfaces: Relationship with temperature and cell surface hydrophobicity
Accepted Manuscript Biofilm formation by Staphylococcus aureus on food contact surfaces: Relationship with temperature and cell surface hydrophobicity P. Di Ciccio, A. Vergara, A.R. Festino, D. Paludi, E. Zanardi, S. Ghidini, A. Ianieri PII:
S0956-7135(14)00630-6
DOI:
10.1016/j.foodcont.2014.10.048
Reference:
JFCO 4148
To appear in:
Food Control
Received Date: 15 July 2014 Revised Date:
17 October 2014
Accepted Date: 25 October 2014
Please cite this article as: Di Ciccio P., Vergara A., Festino A.R., Paludi D., Zanardi E., Ghidini S. & Ianieri A., Biofilm formation by Staphylococcus aureus on food contact surfaces: Relationship with temperature and cell surface hydrophobicity, Food Control (2014), doi: 10.1016/j.foodcont.2014.10.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Biofilm formation by Staphylococcus aureus on food contact surfaces: relationship with
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temperature and cell surface hydrophobicity
3 P. Di Ciccioa*, A. Vergarab, A. R. Festinob, D. Paludib, E. Zanardia, S. Ghidinia, A. Ianieria a
Department of Food Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy
b
Faculty of Veterinary Medicine, University of Teramo, P.zza Aldo Moro 45, 64100, Teramo, Italy
*Corresponding author. Tel: (0039) 0521032753; Fax: (0039) 0521032752; e-mail: [email protected] (P. Di Ciccio)
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Department of Food Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy
11 ABSTRACT
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Staphylococcus aureus (S.aureus) is a pathogenic bacterium capable of developing biofilms on food
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processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study
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was to evaluate the ability of S. aureus to form biofilm on food processing surfaces (polystyrene
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and stainless steel) with regard to different temperatures (12 and 37°C) and cellular hydrophobicity.
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Biofilm assays were performed on n. 67 S.aureus isolates from food, food processing environments
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and food handlers and n. 3 reference strains (S.aureus ATCC 35556, S. aureus ATCC 12600 and
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S.epidermidis ATCC 12228). A strain-specific variation in biofilm formation within S.aureus
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strains tested was observed. At 37°C, n. 38/67 (56.7%) of strains were biofilm producer in at least
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one tested surface. A total of n. 25/38 (65.7%) of strains were biofilm producer on polystyrene
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whereas n. 24/38 (63.1%) were biofilm producer on stainless steel. Moreover, n. 11/38 (28.9%) of
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strains were biofilm producers on both selected surfaces. The majority of S.aureus strains which
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produced biofilms (n. 17/38 – 44.7%), were isolated from food environments. At 12°C, only one
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S.aureus strain from food handler (S.aureus 374) was biofilm producer. Cell surface hydrophobicity
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level increased with temperature. Additionally, a statistically significant difference (P < 0.001) was
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found between hydrophobicity at 37°C and 12°C. Finally, the architecture of biofilm formed by
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S.aureus strains on polystyrene and stainless steel surfaces at selected temperatures was observed
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by scanning electron microscopy. The appearance of thick extracellular products in strongly
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(S.aureus ATCC 35556 – positive control) and the absence of those products in the non-biofilm
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producer (S.epidermidis ATCC 12228 – negative control) is presented.
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Keywords: Staphylococcus aureus; Biofilm, Polystyrene; Stainless steel, Hydrophobicity
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ACCEPTED MANUSCRIPT 1. Introduction
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Bacteria most commonly live by adhering to surfaces and forming organized communities called
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biofilms (Malheiros et al., 2010). Biofilms are structured communities of bacterial cells enclosed in
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a self-produced polymeric matrix and adhered to inert or living surfaces (Costerton et al., 1999).
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The presence of biofilm on food contact surfaces is considered as an health hazard. Microbial
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biofilms, in fact, may contain a considerable number of both spoilage and pathogenic
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microorganisms (Giaouris et al., 2005). Exposure of pathogens to surfaces may take place either by
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direct contact with contaminated materials or indirectly through airborne microflora
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(Kusumaningrum et al., 2003). The direct contact with raw materials or food products may cause
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secondary contamination by which the product may become unsafe (Vlkova et al., 2008). Several
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bacteria are known to form biofilms on different materials (Costerton et al., 1978, Di Ciccio et al.,
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2012).However, biofilm formation is influenced by the nature of subtrata, cell surface charge,
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presence of flagella and microbial growth phase (Pagedar & Singh, 2012). The majority of surfaces
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in food processing plants are made of stainless steel that can be easily cleaned and is resistant
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against chemical agents (Mattila-Sandholm & Wirtanen, 1992). However, it was detected by
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microscopy that even smooth surfaces made from stainless steel can be damaged by mechanical
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cleaning. Small cracks and scratches are formed on their surfaces and bacteria and organic residues
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can stick to them (Wirtanen et al., 1996). S.aureus is a very adaptable organism and can live in a
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wide variety of environments as biofilm (Almeida & Oliver, 2001; Rode et al., 2007). Additionally,
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biofilm production is recognized as an important virulence factor for bacteria of the genus
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Staphylococcus (Cucarella et al., 2002; Vasudevan et al., 2003; Fox et al., 2005).S.aureus biofilm
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on food contact surfaces, in fact, poses a serious risk of food contamination (Gibson et al., 1999). It
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has been frequently found in surfaces of food processing plants being responsible for outbreaks
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related to the consumption of fresh and processed foods worldwide (Balaban & Rasooly, 2000;
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Nostro et al., 2004; Braga et al., 2005; Hamadi et al., 2005; Marques et al., 2007; Rode et al., 2007
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Oulahal, Brice, Martial, & Degraeve, 2008;). Humans are common asymptomatic carriers of
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enterotoxigenic S.aureus in nose, throat, and skin. Thus, food handlers may contaminate food
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(Gutierezz et al., 2012). S.aureus can produce a multilayered biofilm embedded within a glycocalyx
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or slime layer with heterogeneous protein expression throughout (Nathan et al., 2011). For the food
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industry it is important to identify the conditions, under which S.aureus is able to survive and
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multiply with regard to food processing. The majority of studies, in fact, have been addressed to
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clinical aspects related to the biofilm formation by Staphylococcus genera such as S. intermedius on
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catheters and/or medical devices (Silva Meira et al., 2012). To date, the literature about the biofilm
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formation by food-related S.aureus strains is still scarce and there is a lack of information about the
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ACCEPTED MANUSCRIPT capacity of S.aureus isolated from food, food environments and/or food handlers of forming biofilm
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when exposed to different environmental conditions simulating those in food processing plants. In
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order to control the S.aureus biofilm in the food industry, the greater understanding of the
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interactions between microorganisms and food processing equipment is required. Regarding these
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aspects, this study was carried out with the aim of evaluating the ability of S. aureus strains isolated
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from food, food environments and food handlers, to form biofilms on polystyrene and stainless steel
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surfaces under different temperatures: 12 and 37° C. Still, the possible correlation between biofilm
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formation ability and cell hydrophobicity was examined. Finally, the architecture of biofilm formed
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by S.aureus strains on polystyrene and stainless steel surfaces at selected temperatures was
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observed by scanning electron microscopy (SEM).
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80 2. Materials and Methods
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2.1 Bacterial Strains
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The experiment was conducted on n.67 S.aureus strains: n. 19, n. 26 and n. 22 strains isolated from
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food, food environments and food handlers, respectively. The biofilm production was also
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examinated using three S.aureus reference strains. In particular, S.aureus ATCC 35556 is a reported
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biofilm producer that has been shown to form a strong biofilm (Cramton et al., 1999; Seidl et al.,
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2008), whereas S.epidermidis ATCC 12228 was used as negative biofilm producer (Atshan et al.,
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2012; Lee et al., 2013). Finally, a known additional S.aureus reference strain (ATCC 12600) was
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used to define into different categories the S.aureus isolates (n.67). Prior to the conduction of
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experiments, all strains were activated by culturing twice in 10 mL tryptic soy broth (TSB - Oxoid
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S.p.A., Milan, Italy) at 37°C for 24 h.
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2.2 Biofilm Production Assay
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A previously described method was used (Di Bonaventura et al., 2008). Polystyrene tissue culture
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plates (961mm2) and AISI 304 stainless steel chips (530 mm2) were used for biofilm formation
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assays at 12 and 37°C. These two temperatures were selected by their relevance to the food industry
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(12°C) and in infectious disease (37°C). Moreover, polystyrene and stainless steel were selected
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because they are the most widely used material in the construction of food processing equipment
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and they have different physicochemical characteristics: hydrophilic for stainless steel and
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hydrophobic for polystyrene. Briefly, Stainless steel chips were degreased before use by overnight
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immersion in ethanol, then rinsed thoroughly in distilled water and autoclaved for 15 min. at 121°
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C. Cultures of S.aureus were prepared, from overnight TSA-growth, in TSB by incubating at
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selected temperatures: 12 and 37°C. Cultures were then washed three times with phosphate-
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reach a concentration of about 108 CFU ml-1 by reading the optical density (OD) level at 550 nm
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(UV Mini-1240 - Shimadsu). Three milliliters (ml) of the standardized inocula were then added to
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polystyrene tissue culture plates (35mm diameter) and stainless steel chips. Samples were then
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incubated at 12°C and at 37°C. After 24h incubation, non-adherent cells were removed by dipping
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each sample three times in sterile PBS. Samples were fixed at 60°C for 1 h and stained with 3 ml of
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2% crystal violet solution in 95% ethanol for 15 min. After staining, samples were washed thrice
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with distilled water. Negative controls underwent the same treatment but without inoculation. The
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quantitative analysis of biofilm production was performed by adding 3 ml of 33% acetic acid to
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destain the samples. From each sample 200 µl were transferred to a microtiter plate and the OD
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level of the crystal violet present in destaining solution was measured at 492 nm (Varian SII Scan
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Cary 100). Considering different growth area of tested surfaces (polystyrene: 961 mm2 and stainless
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steel: 530 mm2), results were normalized by calculating the biofilm production indices (BPIs) as
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follows: BPI = [OD
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were performed in triplicate.
mean biofilm
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surface (mm2)−1] × 1000. Two independent sets of all experiments
119 2.3 Cell surface hydrophobicity assay
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S.aureus hydrophobicity was evaluated, at selected temperatures: 12 and 37°C, by microbial
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adherence to n-hexadecane (MATH) test according to Mattos-Guaraldi et al. (1999), with slight
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modification. Briefly, 4 ml of standardized inocula in PBS (OD550 = 0.8) were overlayed with 0.4
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ml of n-hexadecane (Sigma- Aldrich). After 1-min agitation by vortexing, the phases were allowed
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to separate for 15 min at room temperature. The results were expressed as the proportion of the cells
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which were excluded from the aqueous phase, determined by the equation as follows: [(A0-A) A0-1]
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x 100, where A0 and A are the initial and final optical densities of the aqueous phase, respectively.
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S.aureus strains were classified as: highly hydrophobic, for values >50%; moderately hydrophobic,
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for values ranging from 20 to 50% and hydrophilic, for values <20%. All experiments were carried
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out in triplicate and repeated in two independent sets of experiments. The data were analyzed by
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using one way ANOVA followed by Newman–Keuls multiple comparison test (set at 5%).
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2.4 SEM of S.aureus biofilms
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For visualization of S.aureus biofilm architecture, SEM images were taken. For SEM analysis, the
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reference strains (S.aureus ATCC 35556 and S.epidermidis ATCC 12228) were selected. Biofilms
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were prepared, as described above, at selected temperatures (12°C – 37°C), for 24 h on polystyrene
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tissue plates and stainless steel chips, then washed by dipping three times in sterile PBS to remove 4
ACCEPTED MANUSCRIPT non-adherent cells. Samples were dehydrated in ethanol–water mixtures, with increasing ethanol
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concentrations (65%, 75%, 85%, 95% and 100%) and finally overnight air-dried. Dehydrated
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specimens were coated with gold-palladium by Polaron E5100 II (Polaron Instruments Inc.,
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Hatfield, CA). After processing, samples were observed with a Philips XL30CP scanning electron
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microscope in the high-vacuum mode at 15 kV (Philips, Eindhoven, Netherlands). The images were
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processed for display using photoshop (Adobe Systems Inc., San Jose, CA, USA) software.
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144 3. Results
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3.1 Biofilm-forming ability of S.aureus strains
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Biofilm formation, expressed as BPI, was compared with reference strains: S.aureus ATCC 35556
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(positive control - BPIPC), S.aureus ATCC 12600 (reference strain – BPI12600) and S.epidermidis
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ATCC 12228 (negative control - BPINC) for each isolate. In particular, the BPI value of S.aureus
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ATCC 12600 (reference strain – BPI12600) was half the BPI value of positive control (ATCC 35556)
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on both surfaces tested at 37°C (Table 1). All isolates (n.67) were defined into different categories
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on the basis of their BPIs values. The cutoff point for the biofilm production was the BPI value
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obtained by negative control on polystyrene (BPINC = 0.294) and stainless steel (BPINC = 0.149).
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S.aureus strains showing ability to produce biofilms were classified as weak (BPINC ≤ S.aureus
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BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC).
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Considerable variations in biofilm-forming ability were observed between the n.67 S.aureus strains
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tested under different temperatures (12 and 37°C) and surfaces (polystyrene and stainless steel). At
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37°C, n. 38/67 (56.7%) of S.aureus strains were biofilm producer in at least one tested surface. A
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total of n. 25/38 (65.7%) of S.aureus strains were biofilm producer on polystyrene whereas n. 24/38
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(63.1%) were biofilm producer on stainless steel. Moreover, n. 11/38 (28.9%) of S.aureus strains
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were biofilm producers on both selected surfaces. Figure 1 shows the ability of the S.aureus strains
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to produce biofilm on polystyrene and stainless steel at 12 and 37°C whereas summarized results of
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S.aureus isolates from different sources are shown in Table 2 and Table 3. The majority of S.aureus
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strains which produced biofilms (n. 17/38 – 44.7%), were isolated from food environments. Among
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the S.aureus strains from food environments, n. 2/17 (11.7%) were classified as moderate biofilm
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producer (S.aureus 193 and S.aureus 194) on polystyrene whereas they were no biofilm producer
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on stainless steel. Anyway, at 37°C the highest BPI value (BPI=1.019), which was greater than
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BPIPC (BPI=0.758), was showed on polystyrene by food isolated strain (S.aureus 281), although this
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one was a weak biofilm producer on stainless steel. At 12°C, only one S.aureus strain isolated from
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food-handler (S.aureus 374) was biofilm producer and it was classified as weak biofilm producer on
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both selected surfaces. This strain at 37°C was classified as moderate biofilm producer on
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polystyrene and weak biofilm producer on stainless steel.
173 3.2 Cell surface hydrophobicity: effect of temperature and association with biofilm formation
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Cell surface hydrophobicity level increased with temperature. As a matter of fact, a statistically
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significant (P < 0.001) difference was found between hydrophobicity at 37°C and those produced at
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12°C (Fig. 2). In particular, among the 67 strains tested at 37°C, n. 43 (64.1%) strains resulted
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highly hydrophobic, n. 21 (31.3%) strains moderately hydrophobic and n. 3 (4.4%) strains
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hydrophilic. At 12°C, n. 21 (31.3%) strains resulted highly hydrophobic, n. 25 (37.3%) strains
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moderately hydrophobic and n.21 (31.3%) strains hydrophilic.
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181 3.3 SEM analysis of S.aureus biofilm
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Representative micrographs of biofilms produced by two reference strains (S.aureus ATCC 35556
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and S.epidermidis ATCC 12228) are shown in Figures 2. In particular, a scanning electron
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micrograph illustrating the appearance of thick extracellular products in strongly biofilm producer
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strain (S.aureus ATCC 35556) and the absence of those products in the non-biofilm producer strain
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(S. epidermidis ATCC 12228) is presented. Biofilm formation by S.aureus ATCC 35556 was
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clearly observed on both polystyrene and stainless steel after 24 h incubation period at 37°C, as
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shown in Figures 2 (a – b). On the contrary, at 37°C the negative biofilm producer (S.epidermidis
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ATCC 12228) showed an absence of extracellular products on selected surfaces as shown in Figure
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2 (c – d).
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4. Discussion
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Microbial adhesion and biofilms are of great importance for the food industry and occur on a high
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variety of food contact surfaces (Donlan & Costerton, 2002; Simões et al., 2010; Di Ciccio et al.,
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2012; Flemming et al., 2013). The biofilms enhance the ability of bacteria to survive stresses and
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consituite potential reservoirs for pathogens such as S.aureus (Abdallah et al., 2014). Several
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authors, in fact, have reported the ability of bacteria to form biofilms on materials commonly used
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in the food sector, such as the stainless steel, glass, rubber, polycarbonate, polyurethane,
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polystyrene, polypropylene, titanium, aluminum, and ceramic (Donlan 2001; Simões et al., 2010;
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Vazquez-Sanchez et al., 2013; Hamadi et al., 2014). The contaminated surfaces, in fact, in
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spreading pathogens to foods is already well established in food processing, catering and domestic
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environment (Silva-Meira et al., 2012; Giaouris et al., 2014). The environmental conditions
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encountered in this sector, such as temperature, nutrient availability, surface type, pH and humidity,
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ACCEPTED MANUSCRIPT provide for the bacterial growth and their biofilm formation. Moreover, some authors underlined the
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presence of biofilms on the food contact surfaces despite the use of disinfection procedures
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(Gounadaki et al., 2008; Gutierrez et al., 2012; de Jesus Pimetel-Filho et al., 2014). S.aureus and its
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biofilm formation are recognized as a serious clinical problem and little is known about the ability
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of wild S.aureus strains isolated from food, food-handlers and food environments to form biofilms
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when they are exposed to conditions simulating those in food processing plants (Leite de Souza et
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al., 2014). In addition, several studies have tested a limited number of food-related strains. In the
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present study, n.3 standard type strains and n.67 food-related strains were employed and the biofilm
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formation was compared at 12°C and 37°C. These two temperatures were selected by their
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relevance to the food industry (12°C), as stated by Regulation (EC) n.853/2004 of the European
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Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for on the
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hygiene of foodstuffs, and in infectious disease (optimum growth temperature: 37°C). Polystyrene
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and stainless steel were selected because they are the most widely used materials in the food sector.
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In particular, stainless steel is the most frequently used material in the construction of food
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processing equipments due to its mechanical strength, corrosion resistance and resistance to damage
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caused by the cleaning process, whereas polystyrene is one of the most commonly plastic used in
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the food industry, mainly for packaging (de Jesus Pimentel-Filho et al., 2014). Based on our results,
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considerable variations in the ability to form biofilms on polystyrene and stainless steel were shown
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among S. aureus isolates. At 37°C the formation of biofilms by S.aureus occurred preferentially on
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polystyrene (65.7%) compared to stainless steel (63.1%). These findings are in accordance with the
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results of Pagedar et al., (2010) who suggested that hydrophobicity is also a relevant factor in the
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formation of biofilms by S.aureus strains. Moreover, in the present study only one S.aureus strains
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isolated from food-handler showed the ability to form biofilm on polystyrene and stainless steel at
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12°C. The ability of S.aureus to colonize surfaces at low temperatures used in the food industry
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may contribute to the persistence of the bacterium in food processing environments, consequently
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increasing cross-contamination risks. Based on our results, at 37°C biofilm was produced at higher
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levels than at 12°C. In particular, at 37°C, the highest amount (BPI=1.019) of biofilm was formed
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on polystyrene by a food isolated strain (S.aureus 281). However, that S.aureus strain showed a BPI
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greater than BPIPC (BPI=0.758) on polystyrene, although it was a weak biofilm producer
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(BPI=0.198) on stainless steel. Several studies have shown that the temperature changes, which take
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place in both food and medical environments, affect biofilm formation (Cerca and Jefferson 2008;
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Nilsson et al. 2011). Anyway, the effect of temperature changes remains unclear on the biofilm
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formation of S.aureus. Results obtained by Vazquez-Sanchez et al. (2013) on n.26 S.aureus isolated
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from seafood and n.2 S.aureus reference strains showed that most of the strains had a higher biofilm
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However, Pagedar et al. (2010) reported a higher cell count of the S. aureus biofilm at 25 °C in
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contrast to that obtained at 37°C on stainless steel. Otherwise, Silva Meira et al. (2012) assessed the
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biofilm formation by n.3 S.aureus from food services on stainless steel and polypropylene surfaces
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at 7 and 28 °C. The isolates of S.aureus revealed high capability to adhere and form biofilm on the
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tested surfaces in both assayed incubation temperature after three days of cultivation. These authors
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showed that there is no clear effect of the incubation temperature on the biofilm formation of
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S.aureus. This discrepancy may reflect the difference in experimental conditions. In fact, Oulahal et
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al. (2008) found that the effect of the growth temperature on the formation of S.aureus biofilm is
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affected by several environmental factors such as nutrient availability and surface type. Biofilm
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formation depends on the characteristics of surface, the bacterial cell, the growth medium and other
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environmental factor (de Jesus Pimentel-Filho et al., 2014). On the other hand, in a study carried out
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by Rode et al. (2007), the biofilm formation on polystyrene was estimated for n.10 S.aureus strains
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incubated at various temperatures and the results indicated that biofilm production is higher at sub-
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optimal temperatures. In addition, the authors also found that the effect of temperature on biofilm
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formation was dependent on the presence of glucose and NaCl (Rode et al. 2007). Finally, in
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another investigation performed in 2014 by Vazquez-Sanchez et al. (2014), the biofilm-forming
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ability of S.aureus strains (n.26) isolated from fish products was assessed on stainless steel at 25°C.
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Most strains showed a biofilm-forming ability higher than the reference strain. Some studies have
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found that the biofilm formation on hydrophobic substrata occurred to a greater extent than that on
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hydrophilic ones (Cerca et al., 2005; Pagedar et al., 2010). Anyway, Da Silva Meira et al. (2012)
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have stated that stainless steel (hydrophilic) and polystyrene (hydrophobic) have no significant
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effect on the biofilm formation of S.aureus. Besides hydrophobicity and surface tension parameters,
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the surface roughness has been found as an essential factor affecting biofilm formation including
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those of P. aeruginosa and S.aureus (Arnold & Bailey 2000; Katsikogianni et al., 2006; Tang et al.,
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2011, Lee et al., 2013; de Jesus Pimentel-Filho et al., 2014, Giaouris et al., 2014). As for as the
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bacterial cell hydrophobicity is concerned, our results suggest that growth temperature may
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influence the hydrophobicity in S.aureus. In particular, the cell surface hydrophobicity level
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increases with temperature. A statistically significant difference, in fact, was found between
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hydrophobicity at 37°C and those showed at 12°C. In order to evaluate the architecture of the
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biofilms, the SEM analysis on S.aureus strains was carried out. The scanning electronic microscopy
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allows the observation of bacteria/surface interaction and may be used as a semi-quantitative
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technique. Thus, in this study to confirm the presence of an extracellular polysaccharide and
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glycoprotein network layer, the SEM analysis was used. Biofilm formation by S.aureus ATCC
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b. An important factor observed was the production of a considerable amount of exopolysaccharide
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matrix by positive reference strain (S.aureus ATCC 35556) on both polystyrene and stainless steel
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while the negative reference strain (S.epidermidis ATCC 12228) did not produce extracellular
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polysaccharides. Moreover, at 37°C, biofilm exhibited a complex organization, in term of cell
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number and extracellular polysaccharides produced. In particular, the polystyrene surface was
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totally colonized by the positive reference strains strain (ATCC 35556) and cells were embedded in
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a large thick layer while the stainless steel was partly colonized and cells were aggregated in
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clusters (Figure 2 a- b). Finally, At 37°C negative biofilm producer (ATCC 12228) showed an
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absence of network layers on both surfaces such as polystyrene and stainless steel (Figure 2 c- d).
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Our study attempted to investigate the biofilm formation, expressed as BPI, by wild isolates of
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S.aureus and to correlate the BPI values with the SEM images. It can be concluded that some
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assayed isolates of S.aureus presented highlighted capacity to form biofilm on polystyrene and/or
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stainless steel surfaces. Further studies focusing on the capability of S.aureus isolates from food
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services to form biofilm when they are exposed to conditions simulating those in food processing
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plants are needed to confirm these initial findings.
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289 4.1 Conclusions
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Microbial biofilms enhance the ability of bacteria to survive stress and can cause problems in the
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food industry. In fact, the persistence of biofilm on food contact surfaces, and equipment, may
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constitute a continuous source of contamination. Our results suggest that the biofilm formation of
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S.aureus is influenced by environmental conditions relevant for the food industry such as
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temperature and cell surface properties. In the present study, several S.aureus strains from food,
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food environments and food-handlers were biofilm producers in at least one assay. This fact is of
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public health concern because it indicates a potential source for persistence of S.aureus
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contamination in the food industry. Based on our results, in fact, the majority of S.aureus strains
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that showed the ability to form biofilm on the tested surfaces were isolated from food environments.
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The prevention and control of S.aureus biofilms in food processing environment should be based on
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an integrated efforts. A regular cleaning and disinfection of all equipment and food contact surfaces,
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also during processing, and an ambient temperature of not more than 12ºC in the food processing
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plants are essential to avoid or reduce the risk of the S.aureus biofilm formation in the food
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industry. In addition, the processing equipments should be designed with high standards of hygiene
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in mind. In conclusion, in order to reduce the microbiological risk related to the biofilm formation,
306
a better understanding of how S.aureus attaches and form biofilm when it is exposed to conditions
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simulating those in food processing plants is needed. Moreover, it is of importance to improve
308
hygienic conditions to control the emergence of biofilms in the food sector.
309 Acknowledgement
311
The present study falls within the Cost Action FA-1202: “A European Network for mitigating
312
bacterial colonisation and persistence on foods and food processing environments”. We thank Dr. L.
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Arizza, Microscopy Center, Faculty of Science - University of Aquila, Italy for his technical
314
assistance.
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formation. Veterinary Microbiology, 92, 179–185.
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factors on the adherence and biofilm formation of natural Staphylococcus aureus isolates. Current
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biofilms–a Review. Czech Journal of Food Science, 26, 309-323.
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Fig 1. Biofilm formation of S.aureus strains at 37°C. The results, expressed as BPI, are mean of
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three independent experiments.
Fig 2. Scanning electron microscopy (SEM) images of biofilm formed by reference strains at 37°C: S.aureus ATCC 35556 (positive control) on polystyrene (BPI= 0.758, a) and stainless steel (BPI= 0.801, b); S.epidermidis 12228 (negative control) on polystyrene (BPI= 0.294, c) and stainless steel
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(BPI= 0.149, d). Magnification: 20 000x.
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Reference Strains
OD mean biofilm Polystyrenea
BPI polystyrene
OD mean biofilm stainless steela
BPI stainless steel
S.aureus ATCC 35556 (positive control)
0.756 ± 0.15
0.758
0.489 ± 0.05
0.801
S.aureus ATCC 12600
0.450 ± 0.07
0.405
0.321 ± 0.02
0.486
S.epidermdis ATCC 12228 (negative control)
0.343 ± 0.05
0.294
0.133 ± 0.00
0.149
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values are expressed as OD mean ± standard deviation
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Table 1 Biofilm formation, expressed as BPIs, by reference S.aureus strains on polystyrene and stainless steel at 37° C.
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Table 2 Biofilm formation by S.aureus strains* on polystyrene at 37°C Source
Strains
Biofilm
Weak
Moderate
Strong
producer*
producer*
producer*
producer*
%
n
%
n
%
n
%
19
7a
36.8
2
28.5
4
57.1
1
14.3
Food-handlers
22
5b
22.7
3
60
2
40
0
Food-environments
26
13 c
50
6
46.1
5
38.4
2
15.4
Total
67
25
37.3
11
44
11
44
3
12
*
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n Food
0
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classified by comparison of each S.aureus strain BPI with the BPI values of the reference strains as follows: biofilm producers (S.aureus BPIs ≥ BPINC); weak (BPINC ≤ S.aureus BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC) biofilm producers. a n.3 S.aureus strains were biofilm producer on both selected surfaces b n.2 S.aureus strains were biofilm producer on both selected surfaces c n.6 S.aureus strains were biofilm producer on both selected surfaces
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Table 3 Biofilm formation by S.aureus strains on stainless steel at 37°C Strains
Biofilm
Weak *
Moderate *
producer
producer
n
%
n
a
36.8 31.8
Food
19
7
Food-handlers
22
7b c
Food-environments
26
10
Total
67
24
Strong
*
producer*
producer
%
n
%
0
0
0
0
2
28.5
0
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Source
%
n
7
100
5
71.4
38.4
9
90
1
10
0
35.8
21
87.5
3
12.5
0
0
0
0
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* classified by comparison of each S.aureus strain BPI with the BPI values of the reference strains as follows: biofilm producers (S.aureus BPIs ≥ BPINC); weak (BPINC ≤ S.aureus BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC) biofilm producers. a n.3 S.aureus strains were biofilm producer on both selected surfaces b n.2 S.aureus strains were biofilm producer on both selected surfaces c n.6 S.aureus strains were biofilm producer on both selected surfaces
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a S.aureus strain-specific variation in biofilm formation was observed at 37°C, 28.9% of strains were biofilm producers on polystyrene and stainless steel at 37°C, 56.7% of strains were biofilm producer in at least one tested surface
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cell surface hydrophobicity level increases with temperature
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at 12°C, only one S.aureus strain was biofilm producer on both tested surfaces
S0956-7135(14)00630-6
DOI:
10.1016/j.foodcont.2014.10.048
Reference:
JFCO 4148
To appear in:
Food Control
Received Date: 15 July 2014 Revised Date:
17 October 2014
Accepted Date: 25 October 2014
Please cite this article as: Di Ciccio P., Vergara A., Festino A.R., Paludi D., Zanardi E., Ghidini S. & Ianieri A., Biofilm formation by Staphylococcus aureus on food contact surfaces: Relationship with temperature and cell surface hydrophobicity, Food Control (2014), doi: 10.1016/j.foodcont.2014.10.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Biofilm formation by Staphylococcus aureus on food contact surfaces: relationship with
2
temperature and cell surface hydrophobicity
3 P. Di Ciccioa*, A. Vergarab, A. R. Festinob, D. Paludib, E. Zanardia, S. Ghidinia, A. Ianieria a
Department of Food Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy
b
Faculty of Veterinary Medicine, University of Teramo, P.zza Aldo Moro 45, 64100, Teramo, Italy
*Corresponding author. Tel: (0039) 0521032753; Fax: (0039) 0521032752; e-mail: [email protected] (P. Di Ciccio)
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4 5 6 7 8 9 10
Department of Food Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy
11 ABSTRACT
13
Staphylococcus aureus (S.aureus) is a pathogenic bacterium capable of developing biofilms on food
14
processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study
15
was to evaluate the ability of S. aureus to form biofilm on food processing surfaces (polystyrene
16
and stainless steel) with regard to different temperatures (12 and 37°C) and cellular hydrophobicity.
17
Biofilm assays were performed on n. 67 S.aureus isolates from food, food processing environments
18
and food handlers and n. 3 reference strains (S.aureus ATCC 35556, S. aureus ATCC 12600 and
19
S.epidermidis ATCC 12228). A strain-specific variation in biofilm formation within S.aureus
20
strains tested was observed. At 37°C, n. 38/67 (56.7%) of strains were biofilm producer in at least
21
one tested surface. A total of n. 25/38 (65.7%) of strains were biofilm producer on polystyrene
22
whereas n. 24/38 (63.1%) were biofilm producer on stainless steel. Moreover, n. 11/38 (28.9%) of
23
strains were biofilm producers on both selected surfaces. The majority of S.aureus strains which
24
produced biofilms (n. 17/38 – 44.7%), were isolated from food environments. At 12°C, only one
25
S.aureus strain from food handler (S.aureus 374) was biofilm producer. Cell surface hydrophobicity
26
level increased with temperature. Additionally, a statistically significant difference (P < 0.001) was
27
found between hydrophobicity at 37°C and 12°C. Finally, the architecture of biofilm formed by
28
S.aureus strains on polystyrene and stainless steel surfaces at selected temperatures was observed
29
by scanning electron microscopy. The appearance of thick extracellular products in strongly
30
(S.aureus ATCC 35556 – positive control) and the absence of those products in the non-biofilm
31
producer (S.epidermidis ATCC 12228 – negative control) is presented.
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Keywords: Staphylococcus aureus; Biofilm, Polystyrene; Stainless steel, Hydrophobicity
34 35 1
ACCEPTED MANUSCRIPT 1. Introduction
37
Bacteria most commonly live by adhering to surfaces and forming organized communities called
38
biofilms (Malheiros et al., 2010). Biofilms are structured communities of bacterial cells enclosed in
39
a self-produced polymeric matrix and adhered to inert or living surfaces (Costerton et al., 1999).
40
The presence of biofilm on food contact surfaces is considered as an health hazard. Microbial
41
biofilms, in fact, may contain a considerable number of both spoilage and pathogenic
42
microorganisms (Giaouris et al., 2005). Exposure of pathogens to surfaces may take place either by
43
direct contact with contaminated materials or indirectly through airborne microflora
44
(Kusumaningrum et al., 2003). The direct contact with raw materials or food products may cause
45
secondary contamination by which the product may become unsafe (Vlkova et al., 2008). Several
46
bacteria are known to form biofilms on different materials (Costerton et al., 1978, Di Ciccio et al.,
47
2012).However, biofilm formation is influenced by the nature of subtrata, cell surface charge,
48
presence of flagella and microbial growth phase (Pagedar & Singh, 2012). The majority of surfaces
49
in food processing plants are made of stainless steel that can be easily cleaned and is resistant
50
against chemical agents (Mattila-Sandholm & Wirtanen, 1992). However, it was detected by
51
microscopy that even smooth surfaces made from stainless steel can be damaged by mechanical
52
cleaning. Small cracks and scratches are formed on their surfaces and bacteria and organic residues
53
can stick to them (Wirtanen et al., 1996). S.aureus is a very adaptable organism and can live in a
54
wide variety of environments as biofilm (Almeida & Oliver, 2001; Rode et al., 2007). Additionally,
55
biofilm production is recognized as an important virulence factor for bacteria of the genus
56
Staphylococcus (Cucarella et al., 2002; Vasudevan et al., 2003; Fox et al., 2005).S.aureus biofilm
57
on food contact surfaces, in fact, poses a serious risk of food contamination (Gibson et al., 1999). It
58
has been frequently found in surfaces of food processing plants being responsible for outbreaks
59
related to the consumption of fresh and processed foods worldwide (Balaban & Rasooly, 2000;
60
Nostro et al., 2004; Braga et al., 2005; Hamadi et al., 2005; Marques et al., 2007; Rode et al., 2007
61
Oulahal, Brice, Martial, & Degraeve, 2008;). Humans are common asymptomatic carriers of
62
enterotoxigenic S.aureus in nose, throat, and skin. Thus, food handlers may contaminate food
63
(Gutierezz et al., 2012). S.aureus can produce a multilayered biofilm embedded within a glycocalyx
64
or slime layer with heterogeneous protein expression throughout (Nathan et al., 2011). For the food
65
industry it is important to identify the conditions, under which S.aureus is able to survive and
66
multiply with regard to food processing. The majority of studies, in fact, have been addressed to
67
clinical aspects related to the biofilm formation by Staphylococcus genera such as S. intermedius on
68
catheters and/or medical devices (Silva Meira et al., 2012). To date, the literature about the biofilm
69
formation by food-related S.aureus strains is still scarce and there is a lack of information about the
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when exposed to different environmental conditions simulating those in food processing plants. In
72
order to control the S.aureus biofilm in the food industry, the greater understanding of the
73
interactions between microorganisms and food processing equipment is required. Regarding these
74
aspects, this study was carried out with the aim of evaluating the ability of S. aureus strains isolated
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from food, food environments and food handlers, to form biofilms on polystyrene and stainless steel
76
surfaces under different temperatures: 12 and 37° C. Still, the possible correlation between biofilm
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formation ability and cell hydrophobicity was examined. Finally, the architecture of biofilm formed
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by S.aureus strains on polystyrene and stainless steel surfaces at selected temperatures was
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observed by scanning electron microscopy (SEM).
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80 2. Materials and Methods
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2.1 Bacterial Strains
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The experiment was conducted on n.67 S.aureus strains: n. 19, n. 26 and n. 22 strains isolated from
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food, food environments and food handlers, respectively. The biofilm production was also
85
examinated using three S.aureus reference strains. In particular, S.aureus ATCC 35556 is a reported
86
biofilm producer that has been shown to form a strong biofilm (Cramton et al., 1999; Seidl et al.,
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2008), whereas S.epidermidis ATCC 12228 was used as negative biofilm producer (Atshan et al.,
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2012; Lee et al., 2013). Finally, a known additional S.aureus reference strain (ATCC 12600) was
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used to define into different categories the S.aureus isolates (n.67). Prior to the conduction of
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experiments, all strains were activated by culturing twice in 10 mL tryptic soy broth (TSB - Oxoid
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S.p.A., Milan, Italy) at 37°C for 24 h.
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2.2 Biofilm Production Assay
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A previously described method was used (Di Bonaventura et al., 2008). Polystyrene tissue culture
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plates (961mm2) and AISI 304 stainless steel chips (530 mm2) were used for biofilm formation
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assays at 12 and 37°C. These two temperatures were selected by their relevance to the food industry
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(12°C) and in infectious disease (37°C). Moreover, polystyrene and stainless steel were selected
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because they are the most widely used material in the construction of food processing equipment
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and they have different physicochemical characteristics: hydrophilic for stainless steel and
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hydrophobic for polystyrene. Briefly, Stainless steel chips were degreased before use by overnight
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immersion in ethanol, then rinsed thoroughly in distilled water and autoclaved for 15 min. at 121°
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C. Cultures of S.aureus were prepared, from overnight TSA-growth, in TSB by incubating at
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selected temperatures: 12 and 37°C. Cultures were then washed three times with phosphate-
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reach a concentration of about 108 CFU ml-1 by reading the optical density (OD) level at 550 nm
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(UV Mini-1240 - Shimadsu). Three milliliters (ml) of the standardized inocula were then added to
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polystyrene tissue culture plates (35mm diameter) and stainless steel chips. Samples were then
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incubated at 12°C and at 37°C. After 24h incubation, non-adherent cells were removed by dipping
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each sample three times in sterile PBS. Samples were fixed at 60°C for 1 h and stained with 3 ml of
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2% crystal violet solution in 95% ethanol for 15 min. After staining, samples were washed thrice
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with distilled water. Negative controls underwent the same treatment but without inoculation. The
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quantitative analysis of biofilm production was performed by adding 3 ml of 33% acetic acid to
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destain the samples. From each sample 200 µl were transferred to a microtiter plate and the OD
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level of the crystal violet present in destaining solution was measured at 492 nm (Varian SII Scan
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Cary 100). Considering different growth area of tested surfaces (polystyrene: 961 mm2 and stainless
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steel: 530 mm2), results were normalized by calculating the biofilm production indices (BPIs) as
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follows: BPI = [OD
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were performed in triplicate.
mean biofilm
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surface (mm2)−1] × 1000. Two independent sets of all experiments
119 2.3 Cell surface hydrophobicity assay
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S.aureus hydrophobicity was evaluated, at selected temperatures: 12 and 37°C, by microbial
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adherence to n-hexadecane (MATH) test according to Mattos-Guaraldi et al. (1999), with slight
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modification. Briefly, 4 ml of standardized inocula in PBS (OD550 = 0.8) were overlayed with 0.4
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ml of n-hexadecane (Sigma- Aldrich). After 1-min agitation by vortexing, the phases were allowed
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to separate for 15 min at room temperature. The results were expressed as the proportion of the cells
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which were excluded from the aqueous phase, determined by the equation as follows: [(A0-A) A0-1]
127
x 100, where A0 and A are the initial and final optical densities of the aqueous phase, respectively.
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S.aureus strains were classified as: highly hydrophobic, for values >50%; moderately hydrophobic,
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for values ranging from 20 to 50% and hydrophilic, for values <20%. All experiments were carried
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out in triplicate and repeated in two independent sets of experiments. The data were analyzed by
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using one way ANOVA followed by Newman–Keuls multiple comparison test (set at 5%).
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132 133
2.4 SEM of S.aureus biofilms
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For visualization of S.aureus biofilm architecture, SEM images were taken. For SEM analysis, the
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reference strains (S.aureus ATCC 35556 and S.epidermidis ATCC 12228) were selected. Biofilms
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were prepared, as described above, at selected temperatures (12°C – 37°C), for 24 h on polystyrene
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tissue plates and stainless steel chips, then washed by dipping three times in sterile PBS to remove 4
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concentrations (65%, 75%, 85%, 95% and 100%) and finally overnight air-dried. Dehydrated
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specimens were coated with gold-palladium by Polaron E5100 II (Polaron Instruments Inc.,
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Hatfield, CA). After processing, samples were observed with a Philips XL30CP scanning electron
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microscope in the high-vacuum mode at 15 kV (Philips, Eindhoven, Netherlands). The images were
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processed for display using photoshop (Adobe Systems Inc., San Jose, CA, USA) software.
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144 3. Results
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3.1 Biofilm-forming ability of S.aureus strains
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Biofilm formation, expressed as BPI, was compared with reference strains: S.aureus ATCC 35556
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(positive control - BPIPC), S.aureus ATCC 12600 (reference strain – BPI12600) and S.epidermidis
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ATCC 12228 (negative control - BPINC) for each isolate. In particular, the BPI value of S.aureus
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ATCC 12600 (reference strain – BPI12600) was half the BPI value of positive control (ATCC 35556)
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on both surfaces tested at 37°C (Table 1). All isolates (n.67) were defined into different categories
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on the basis of their BPIs values. The cutoff point for the biofilm production was the BPI value
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obtained by negative control on polystyrene (BPINC = 0.294) and stainless steel (BPINC = 0.149).
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S.aureus strains showing ability to produce biofilms were classified as weak (BPINC ≤ S.aureus
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BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC).
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Considerable variations in biofilm-forming ability were observed between the n.67 S.aureus strains
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tested under different temperatures (12 and 37°C) and surfaces (polystyrene and stainless steel). At
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37°C, n. 38/67 (56.7%) of S.aureus strains were biofilm producer in at least one tested surface. A
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total of n. 25/38 (65.7%) of S.aureus strains were biofilm producer on polystyrene whereas n. 24/38
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(63.1%) were biofilm producer on stainless steel. Moreover, n. 11/38 (28.9%) of S.aureus strains
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were biofilm producers on both selected surfaces. Figure 1 shows the ability of the S.aureus strains
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to produce biofilm on polystyrene and stainless steel at 12 and 37°C whereas summarized results of
163
S.aureus isolates from different sources are shown in Table 2 and Table 3. The majority of S.aureus
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strains which produced biofilms (n. 17/38 – 44.7%), were isolated from food environments. Among
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the S.aureus strains from food environments, n. 2/17 (11.7%) were classified as moderate biofilm
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producer (S.aureus 193 and S.aureus 194) on polystyrene whereas they were no biofilm producer
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on stainless steel. Anyway, at 37°C the highest BPI value (BPI=1.019), which was greater than
168
BPIPC (BPI=0.758), was showed on polystyrene by food isolated strain (S.aureus 281), although this
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one was a weak biofilm producer on stainless steel. At 12°C, only one S.aureus strain isolated from
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food-handler (S.aureus 374) was biofilm producer and it was classified as weak biofilm producer on
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both selected surfaces. This strain at 37°C was classified as moderate biofilm producer on
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polystyrene and weak biofilm producer on stainless steel.
173 3.2 Cell surface hydrophobicity: effect of temperature and association with biofilm formation
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Cell surface hydrophobicity level increased with temperature. As a matter of fact, a statistically
176
significant (P < 0.001) difference was found between hydrophobicity at 37°C and those produced at
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12°C (Fig. 2). In particular, among the 67 strains tested at 37°C, n. 43 (64.1%) strains resulted
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highly hydrophobic, n. 21 (31.3%) strains moderately hydrophobic and n. 3 (4.4%) strains
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hydrophilic. At 12°C, n. 21 (31.3%) strains resulted highly hydrophobic, n. 25 (37.3%) strains
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moderately hydrophobic and n.21 (31.3%) strains hydrophilic.
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181 3.3 SEM analysis of S.aureus biofilm
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Representative micrographs of biofilms produced by two reference strains (S.aureus ATCC 35556
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and S.epidermidis ATCC 12228) are shown in Figures 2. In particular, a scanning electron
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micrograph illustrating the appearance of thick extracellular products in strongly biofilm producer
186
strain (S.aureus ATCC 35556) and the absence of those products in the non-biofilm producer strain
187
(S. epidermidis ATCC 12228) is presented. Biofilm formation by S.aureus ATCC 35556 was
188
clearly observed on both polystyrene and stainless steel after 24 h incubation period at 37°C, as
189
shown in Figures 2 (a – b). On the contrary, at 37°C the negative biofilm producer (S.epidermidis
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ATCC 12228) showed an absence of extracellular products on selected surfaces as shown in Figure
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2 (c – d).
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4. Discussion
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Microbial adhesion and biofilms are of great importance for the food industry and occur on a high
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variety of food contact surfaces (Donlan & Costerton, 2002; Simões et al., 2010; Di Ciccio et al.,
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2012; Flemming et al., 2013). The biofilms enhance the ability of bacteria to survive stresses and
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consituite potential reservoirs for pathogens such as S.aureus (Abdallah et al., 2014). Several
198
authors, in fact, have reported the ability of bacteria to form biofilms on materials commonly used
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in the food sector, such as the stainless steel, glass, rubber, polycarbonate, polyurethane,
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polystyrene, polypropylene, titanium, aluminum, and ceramic (Donlan 2001; Simões et al., 2010;
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Vazquez-Sanchez et al., 2013; Hamadi et al., 2014). The contaminated surfaces, in fact, in
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spreading pathogens to foods is already well established in food processing, catering and domestic
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environment (Silva-Meira et al., 2012; Giaouris et al., 2014). The environmental conditions
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encountered in this sector, such as temperature, nutrient availability, surface type, pH and humidity,
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presence of biofilms on the food contact surfaces despite the use of disinfection procedures
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(Gounadaki et al., 2008; Gutierrez et al., 2012; de Jesus Pimetel-Filho et al., 2014). S.aureus and its
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biofilm formation are recognized as a serious clinical problem and little is known about the ability
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of wild S.aureus strains isolated from food, food-handlers and food environments to form biofilms
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when they are exposed to conditions simulating those in food processing plants (Leite de Souza et
211
al., 2014). In addition, several studies have tested a limited number of food-related strains. In the
212
present study, n.3 standard type strains and n.67 food-related strains were employed and the biofilm
213
formation was compared at 12°C and 37°C. These two temperatures were selected by their
214
relevance to the food industry (12°C), as stated by Regulation (EC) n.853/2004 of the European
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Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for on the
216
hygiene of foodstuffs, and in infectious disease (optimum growth temperature: 37°C). Polystyrene
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and stainless steel were selected because they are the most widely used materials in the food sector.
218
In particular, stainless steel is the most frequently used material in the construction of food
219
processing equipments due to its mechanical strength, corrosion resistance and resistance to damage
220
caused by the cleaning process, whereas polystyrene is one of the most commonly plastic used in
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the food industry, mainly for packaging (de Jesus Pimentel-Filho et al., 2014). Based on our results,
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considerable variations in the ability to form biofilms on polystyrene and stainless steel were shown
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among S. aureus isolates. At 37°C the formation of biofilms by S.aureus occurred preferentially on
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polystyrene (65.7%) compared to stainless steel (63.1%). These findings are in accordance with the
225
results of Pagedar et al., (2010) who suggested that hydrophobicity is also a relevant factor in the
226
formation of biofilms by S.aureus strains. Moreover, in the present study only one S.aureus strains
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isolated from food-handler showed the ability to form biofilm on polystyrene and stainless steel at
228
12°C. The ability of S.aureus to colonize surfaces at low temperatures used in the food industry
229
may contribute to the persistence of the bacterium in food processing environments, consequently
230
increasing cross-contamination risks. Based on our results, at 37°C biofilm was produced at higher
231
levels than at 12°C. In particular, at 37°C, the highest amount (BPI=1.019) of biofilm was formed
232
on polystyrene by a food isolated strain (S.aureus 281). However, that S.aureus strain showed a BPI
233
greater than BPIPC (BPI=0.758) on polystyrene, although it was a weak biofilm producer
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(BPI=0.198) on stainless steel. Several studies have shown that the temperature changes, which take
235
place in both food and medical environments, affect biofilm formation (Cerca and Jefferson 2008;
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Nilsson et al. 2011). Anyway, the effect of temperature changes remains unclear on the biofilm
237
formation of S.aureus. Results obtained by Vazquez-Sanchez et al. (2013) on n.26 S.aureus isolated
238
from seafood and n.2 S.aureus reference strains showed that most of the strains had a higher biofilm
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240
However, Pagedar et al. (2010) reported a higher cell count of the S. aureus biofilm at 25 °C in
241
contrast to that obtained at 37°C on stainless steel. Otherwise, Silva Meira et al. (2012) assessed the
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biofilm formation by n.3 S.aureus from food services on stainless steel and polypropylene surfaces
243
at 7 and 28 °C. The isolates of S.aureus revealed high capability to adhere and form biofilm on the
244
tested surfaces in both assayed incubation temperature after three days of cultivation. These authors
245
showed that there is no clear effect of the incubation temperature on the biofilm formation of
246
S.aureus. This discrepancy may reflect the difference in experimental conditions. In fact, Oulahal et
247
al. (2008) found that the effect of the growth temperature on the formation of S.aureus biofilm is
248
affected by several environmental factors such as nutrient availability and surface type. Biofilm
249
formation depends on the characteristics of surface, the bacterial cell, the growth medium and other
250
environmental factor (de Jesus Pimentel-Filho et al., 2014). On the other hand, in a study carried out
251
by Rode et al. (2007), the biofilm formation on polystyrene was estimated for n.10 S.aureus strains
252
incubated at various temperatures and the results indicated that biofilm production is higher at sub-
253
optimal temperatures. In addition, the authors also found that the effect of temperature on biofilm
254
formation was dependent on the presence of glucose and NaCl (Rode et al. 2007). Finally, in
255
another investigation performed in 2014 by Vazquez-Sanchez et al. (2014), the biofilm-forming
256
ability of S.aureus strains (n.26) isolated from fish products was assessed on stainless steel at 25°C.
257
Most strains showed a biofilm-forming ability higher than the reference strain. Some studies have
258
found that the biofilm formation on hydrophobic substrata occurred to a greater extent than that on
259
hydrophilic ones (Cerca et al., 2005; Pagedar et al., 2010). Anyway, Da Silva Meira et al. (2012)
260
have stated that stainless steel (hydrophilic) and polystyrene (hydrophobic) have no significant
261
effect on the biofilm formation of S.aureus. Besides hydrophobicity and surface tension parameters,
262
the surface roughness has been found as an essential factor affecting biofilm formation including
263
those of P. aeruginosa and S.aureus (Arnold & Bailey 2000; Katsikogianni et al., 2006; Tang et al.,
264
2011, Lee et al., 2013; de Jesus Pimentel-Filho et al., 2014, Giaouris et al., 2014). As for as the
265
bacterial cell hydrophobicity is concerned, our results suggest that growth temperature may
266
influence the hydrophobicity in S.aureus. In particular, the cell surface hydrophobicity level
267
increases with temperature. A statistically significant difference, in fact, was found between
268
hydrophobicity at 37°C and those showed at 12°C. In order to evaluate the architecture of the
269
biofilms, the SEM analysis on S.aureus strains was carried out. The scanning electronic microscopy
270
allows the observation of bacteria/surface interaction and may be used as a semi-quantitative
271
technique. Thus, in this study to confirm the presence of an extracellular polysaccharide and
272
glycoprotein network layer, the SEM analysis was used. Biofilm formation by S.aureus ATCC
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274
b. An important factor observed was the production of a considerable amount of exopolysaccharide
275
matrix by positive reference strain (S.aureus ATCC 35556) on both polystyrene and stainless steel
276
while the negative reference strain (S.epidermidis ATCC 12228) did not produce extracellular
277
polysaccharides. Moreover, at 37°C, biofilm exhibited a complex organization, in term of cell
278
number and extracellular polysaccharides produced. In particular, the polystyrene surface was
279
totally colonized by the positive reference strains strain (ATCC 35556) and cells were embedded in
280
a large thick layer while the stainless steel was partly colonized and cells were aggregated in
281
clusters (Figure 2 a- b). Finally, At 37°C negative biofilm producer (ATCC 12228) showed an
282
absence of network layers on both surfaces such as polystyrene and stainless steel (Figure 2 c- d).
283
Our study attempted to investigate the biofilm formation, expressed as BPI, by wild isolates of
284
S.aureus and to correlate the BPI values with the SEM images. It can be concluded that some
285
assayed isolates of S.aureus presented highlighted capacity to form biofilm on polystyrene and/or
286
stainless steel surfaces. Further studies focusing on the capability of S.aureus isolates from food
287
services to form biofilm when they are exposed to conditions simulating those in food processing
288
plants are needed to confirm these initial findings.
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289 4.1 Conclusions
291
Microbial biofilms enhance the ability of bacteria to survive stress and can cause problems in the
292
food industry. In fact, the persistence of biofilm on food contact surfaces, and equipment, may
293
constitute a continuous source of contamination. Our results suggest that the biofilm formation of
294
S.aureus is influenced by environmental conditions relevant for the food industry such as
295
temperature and cell surface properties. In the present study, several S.aureus strains from food,
296
food environments and food-handlers were biofilm producers in at least one assay. This fact is of
297
public health concern because it indicates a potential source for persistence of S.aureus
298
contamination in the food industry. Based on our results, in fact, the majority of S.aureus strains
299
that showed the ability to form biofilm on the tested surfaces were isolated from food environments.
300
The prevention and control of S.aureus biofilms in food processing environment should be based on
301
an integrated efforts. A regular cleaning and disinfection of all equipment and food contact surfaces,
302
also during processing, and an ambient temperature of not more than 12ºC in the food processing
303
plants are essential to avoid or reduce the risk of the S.aureus biofilm formation in the food
304
industry. In addition, the processing equipments should be designed with high standards of hygiene
305
in mind. In conclusion, in order to reduce the microbiological risk related to the biofilm formation,
306
a better understanding of how S.aureus attaches and form biofilm when it is exposed to conditions
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simulating those in food processing plants is needed. Moreover, it is of importance to improve
308
hygienic conditions to control the emergence of biofilms in the food sector.
309 Acknowledgement
311
The present study falls within the Cost Action FA-1202: “A European Network for mitigating
312
bacterial colonisation and persistence on foods and food processing environments”. We thank Dr. L.
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Arizza, Microscopy Center, Faculty of Science - University of Aquila, Italy for his technical
314
assistance.
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Fig 1. Biofilm formation of S.aureus strains at 37°C. The results, expressed as BPI, are mean of
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three independent experiments.
Fig 2. Scanning electron microscopy (SEM) images of biofilm formed by reference strains at 37°C: S.aureus ATCC 35556 (positive control) on polystyrene (BPI= 0.758, a) and stainless steel (BPI= 0.801, b); S.epidermidis 12228 (negative control) on polystyrene (BPI= 0.294, c) and stainless steel
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(BPI= 0.149, d). Magnification: 20 000x.
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Reference Strains
OD mean biofilm Polystyrenea
BPI polystyrene
OD mean biofilm stainless steela
BPI stainless steel
S.aureus ATCC 35556 (positive control)
0.756 ± 0.15
0.758
0.489 ± 0.05
0.801
S.aureus ATCC 12600
0.450 ± 0.07
0.405
0.321 ± 0.02
0.486
S.epidermdis ATCC 12228 (negative control)
0.343 ± 0.05
0.294
0.133 ± 0.00
0.149
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values are expressed as OD mean ± standard deviation
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Table 1 Biofilm formation, expressed as BPIs, by reference S.aureus strains on polystyrene and stainless steel at 37° C.
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Table 2 Biofilm formation by S.aureus strains* on polystyrene at 37°C Source
Strains
Biofilm
Weak
Moderate
Strong
producer*
producer*
producer*
producer*
%
n
%
n
%
n
%
19
7a
36.8
2
28.5
4
57.1
1
14.3
Food-handlers
22
5b
22.7
3
60
2
40
0
Food-environments
26
13 c
50
6
46.1
5
38.4
2
15.4
Total
67
25
37.3
11
44
11
44
3
12
*
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n Food
0
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classified by comparison of each S.aureus strain BPI with the BPI values of the reference strains as follows: biofilm producers (S.aureus BPIs ≥ BPINC); weak (BPINC ≤ S.aureus BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC) biofilm producers. a n.3 S.aureus strains were biofilm producer on both selected surfaces b n.2 S.aureus strains were biofilm producer on both selected surfaces c n.6 S.aureus strains were biofilm producer on both selected surfaces
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Table 3 Biofilm formation by S.aureus strains on stainless steel at 37°C Strains
Biofilm
Weak *
Moderate *
producer
producer
n
%
n
a
36.8 31.8
Food
19
7
Food-handlers
22
7b c
Food-environments
26
10
Total
67
24
Strong
*
producer*
producer
%
n
%
0
0
0
0
2
28.5
0
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Source
%
n
7
100
5
71.4
38.4
9
90
1
10
0
35.8
21
87.5
3
12.5
0
0
0
0
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* classified by comparison of each S.aureus strain BPI with the BPI values of the reference strains as follows: biofilm producers (S.aureus BPIs ≥ BPINC); weak (BPINC ≤ S.aureus BPIs < BPI12600), moderate (BPI12600 ≤ S.aureus BPIs < BPIPC ) or strong (S.aureus BPIs ≥ BPIPC) biofilm producers. a n.3 S.aureus strains were biofilm producer on both selected surfaces b n.2 S.aureus strains were biofilm producer on both selected surfaces c n.6 S.aureus strains were biofilm producer on both selected surfaces
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a S.aureus strain-specific variation in biofilm formation was observed at 37°C, 28.9% of strains were biofilm producers on polystyrene and stainless steel at 37°C, 56.7% of strains were biofilm producer in at least one tested surface
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cell surface hydrophobicity level increases with temperature
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at 12°C, only one S.aureus strain was biofilm producer on both tested surfaces