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Biofilm formation
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  International Journal of ELSEVIER Food Microbiology 26 (1995) 147-164 Intemational Journal of Food Microbiology Influence of culture conditions on biofilm formation by Escherichia coli 0157:H7 Ratih Dewanti, Amy c L Wong ã Food Research Institute Department of Food Microbiology and Toxicology 1925 Willow Drive University of WISconsin-Madison Madison WI 53705 USA Received 22 February 1994; revision received 27 June 1994; accepted 14 July 1994 Abstract Biofilms of Escherichia coli 0157:H7 were developed on stainless steel chips in trypticase soy broth (TSB), 1 5 dilution of TSB, 0.1 Bacto peptone (BP) and a minimal salts medium (MSM) supplemented with 0.04% of one of the following carbon sources: glucose, glycerol, lactose, mannose, succinic acid, sodium pyruvate or lactic acid. t was found that biofilms developed faster and a higher number of adherent cells (ca. 10 6 CFU cm 2) were recovered when the organisms were grown in the low nutrient media. Regardless of the carbon source, biofilms developed in MSM consisted of shorter bacterial cells and thicker extracellular matrix (ECM), with glucose as the best substrate for stable biofilm formation. Fewer bacteria in initial attachment, non-hydrophobicity of bacterial cells, lack of ECM formation and easy detachment of the biofilm bacteria may contribute to poor biofilm formation in TSB. ECM is probably important for the stability of biofilms; however, at 10°C and under anaerobic conditions, ECM seems to be unnecessary. Keywords Biofilm; Escherichia coli 0157:H7; Culture conditions; Extracellular matrix 1. Introduction Microbial attachment to surfaces and the development of biofilms are known to occur in many environments. Biofilms have been studied most extensively in marine and aquatic environments and medical areas (Characklis and Marshall, 1990 . Often biofilms in these situations create economic and health problems. For example, they cause fouling of industrial equipment such as heat exchangers (Bott, ã Corresponding author. Tel. (608)-263-1168. Fax: (608)-263-1114. 0168-1605/95/ 09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1605 94)00103-0  148 R Dewanti A C L Wong / Int. 1. Food Microbiology 26 1995) 147-164 1992) and ship hulls Cooksey and Wigglesworth-Cooksey, 1991), which results in reduced heat transfer, energy loss, increased fluid frictional resistance and accelerated corrosion. Biofilm formation in water distribution systems decreases water quality and increases health risks Block, 1992). Biofilm accumulation on teeth and gum, urinary tract, and implanted medical devices such as catheters Sheretz et aI., 1990) may lead to infections. Only recently has biofilm formation gained attention in food environments. The attachment of microorganisms and subsequent development of biofilms in food processing environments are potential sources of contamination and may lead to food spoilage or transmission of diseases. t has been shown that even with cleaning and sanitation procedures consistent with good manufacturing practices, microorganisms can remain on equipment surfaces Maxcy, 1964; Czechowski, 1990; Mattila et aI., 1990). These organisms may survive for prolonged periods depending on the environmental conditions Maxcy, 1971). Listeria monocytogenes and other Listeria spp. can be isolated frequently from various surfaces in dairy and meat processing environments Anonymous, 1988; Charlton et aI., 1990; Nelson, 1990). Biofilm development is a dynamic process. Bacteria exposed to surfaces attach readily. Under suitable conditions, organisms that remain irreversibly attached grow and develop into biofilms, usually embedded in a polymer matrix of microbial srcin Characklis and Marshall, 1990). This matrix is generally assumed to be polysaccharide in nature and is often referred to as glycocalyx Costerton et aI., 1987). Portions of biofilms may eventually detach and colonize other parts of the system. Cells in biofilms are generally hardier than their planktonic free-living) counterparts, and exhibit increased resistance to adverse conditions such as desiccation Costerton et aI., 1987), extreme temperatures Frank and Koffi, 1990) and the presence of antibiotics Nickel et aI., 1985) or sanitizers Marrie and Costerton, 1981; Stickler at aI., 1989). Several food spoilage and pathogenic bacteria have been reported to attach and form biofilms in vitro on food contact surfaces such as stainless steel, polystyrene or rubber Speers et aI., 1984; Czechowski, 1990). These biofilm bacteria are also known to be more resistant to cleaners and sanitizers Krysinski et aI., 1992 . Ronner and Wong 1993) found that attachment surface affected biofilm formation by L. monocytogenes and Salmonella typhimurium and also their relative resistance to sanitizers. Escherichia coli 0157:H7 was first identified as a pathogen in 1982 and is now recognized as an important cause of foodborne disease Doyle, 1991). The illnesses caused by this organism can be manifested as hemorrhagic colitis, hemolytic uremic syndrome HUS) and thrombotic thrombocytopenic purpura TIP). Hemorrhagic colitis is the most common syndrome, and is typified by severe abdominal pain and grossly bloody diarrhea. HUS is a leading cause of renal failure in children and patients often require dialysis and blood transfusions. Symptoms of TIP are similar but more severe than HUS. Death may result from HUS or TIP. Outbreaks due to E. coli 0157:H7 have been associated primarily with consumption of undercooked ground beef. The most recent outbreak involved over 500  R. Dewanti A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164 149 laboratory-confirmed cases and four deaths (CDC, 1993 . Other infection vehicles include unpasteurized milk, roast beef, apple cider and person-to-person transmission. A one-year, prospective, population-based study (MacDonald et aI., 1988 indicated that the incidence rate for the organism was 8/100000 person-years, compared to 21/100000 person-years for Salmonella and 7/100000 person-years for Shigella. Hence infection by E coli 0157:H7 is quite common. An understanding on how E coli 0157:H7 can establish and survive in the processing environment is essential to finding ways to prevent contamination. This study is a first step in delineating conditions under which this organism can attach and form biofilms, and in characterizing the process. Effects of nutrients, low temperature and anaerobic conditions on biofilm formation by these organisms were examined. 2 Materials and methods 2.1. Bacteria Escherichia coli 0157:H7 strain 932, a clinical isolate from a ground beef associated hemorrhagic colitis outbreak in 1982, was obtained from the Centers for Disease Control. The strain was maintained in 80 glycerol at -20°C and grown in trypticase soy broth (TSB, Becton Dickinson, Cockeysville, MD) for 16-18 h at room temperature prior to use. 2.2. Stainless steel chips Stainless steel (SS) type 304 with a 4 finish, commonly used in food processing equipment and contact surfaces, was cut into 1 1 cm chips. The chips were washed in a hot detergent solution 1 Micro; International Products, Corp., Trenton, NJ» for 1 h, rinsed in distilled water twice and air dried. Cleaned SS chips were dry autoclaved at 121°C for 20 min prior to use. 2.3. Biofilm development Escherichia coli 0157:H7 (approximately 10 7 CFU) was inoculated into 50 ml of growth medium in 125-ml Erlenmeyer flasks and four SS chips were placed in each flask. Incubation was at room temperature (22-25°C) with mild agitation (70-90 rpm) on a rotary shaker (Labline, Melrose Park, IL). At specified times, duplicate SS chips were removed and rinsed in sterile distilled water. Adherent cells on the SS surfaces were removed by scraping with a Teflon spatula, followed by swabbing with a calcium alginate swab (Frank and Koffi, 1990). The cells were dispersed and serially diluted in 0.01 M phosphate buffered saline (PBS), then surface plated on trypticase soy agar (TSA, Becton Dickinson) for enumeration. The other two SS chips were processed for scanning electron microscopy (SEM). Planktonic cells, i.e.  150 R Dewanti, A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164 bacteria suspended in the culture medium, were enumerated as above. Planktonic cells from certain growth conditions were also observed under SEM. Media used in this study were: TSB, li TSB, 0.1 Bacto peptone (BP, Difco) and a minimal salts medium (MSM) containing 7 g KH 2 P0 4, 3 g K 2 HP0 4, 1 g (NH4 )2 S04 0.1 g MgS0 4 and 1 mg of yeast extract per liter (Camper et al., 1991). The MSM was supplemented with the following carbon sources: 0.01-1.0 D-glu cose, 0.04-1.0 D-mannose, 0.04 D-lactose, 0.04 glycerol, 0.04 sodium pyruvate, 0.04 succinic acid or 0.04 lactic acid. The initial pH of all media ranged from 6.8 to 7.1. Carbon sources were filter sterilized using a 0.2 JLm cellulose acetate filter unit (Coming Inc., NY) before addition to the MSM. All media were sterilized at 121°C, 15 psi for 20 min. Biofilm formation in MSM-0.04 glucose was also examined at 10°C and under anaerobic conditions. For anaerobic incubation, 125-ml Erlenmeyer flasks containing bacterial cells and the SS chips were degassed with a vacuum pump. Air was replaced with a mixture of 80 nitrogen, 10 carbon dioxide and 10 hydrogen and the flasks were incubated at 22-25°C. 2 4 Scanning electron microscopy SEM) Biofilms from all growth conditions were prepared for SEM using a fixation method described by Birdsell et al. (1975) with minor modifications. The chips were rinsed twice in sterile distilled water and placed in 0.1 w Iv concanavalin A (con A; Sigma Co., St. Louis, MO) in 0.1 M phosphate buffered saline containing 0.1 CaCl 2 and 0.1 MgCl 2 (PBS-CM, pH 7.2) for 20 min. The SS chips were then washed in PBS-CM twice and fixed with 1 glutaraldehyde (Sigma) in 0.2 M cacodylate buffer (Sigma) overnight at 4°C. A second method of fixation (Fassel et al., 1992) employing ruthenium red (RR, Sigma) was also used for biofilms developed in TSB and MSM-0.04 glucose. Ruthenium red is a stain specific for acidic polysaccharide (Luft, 1971). Briefly, rinsed SS chips were placed in a pre-fixation solution containing 0.15 RR in 0.1 M cacodylate buffer for 1 h. The chips were rinsed in a wash buffer 0.1 M cacodylate buffer, pH 7.0-7.3), then fixed in 2 glutaraldehyde in 0.1 M cacodylate buffer containing 0.05 RR. After 2 h of fixation, SS chips were rinsed in wash buffer and placed in a post-fixation solution containing 2 osmium tetroxide in 0.2 M cacodylate buffer for 2.5 h. The chips were then rinsed five times in wash buffer. Stainless steel chips fixed with either method were dehydrated twice 5 min each time) in a graded ethanol series of 35, 50, 70, 85, 95 and 100 ethanol. Dehydration was completed in a Tousimis Sam Dri 7808 critical point dryer using carbon dioxide as the transition medium. The chips were mounted on SEM specimen stubs with silver paint and coated with gold-palladium alloy using a Polaron E-5000M vacuum evaporator (Bio Rad, Richmond, CA). Biofilms were viewed with a Hitachi S 570 scanning electron microscope. Planktonic cells from selected growth conditions were also viewed under SEM. Approximately 10 ml of culture was filtered using a 0.2 JLm cellulose acetate
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