Food Protection Trends - March 2010 - 169

INTRODUCTION Listeria monocytogenes is a human pathogen that can cause foodborne disease. Meat and poultry have been implicated in foodborne outbreaks of listeriosis (4, 9). This organism is ubiquitous in nature and able to grow at relatively low temperatures. Therefore, L. monocytogenes is of particular concern in the poultry processing arena because after chilling, most of the plant and all products are maintained at a cool temperature. L. monocytogenes has been isolated from raw poultry products and in plants in which poultry undergoes further processing (2). L. monocytogenes can enter a poultry cooking and further processing plant with raw meat (2). Once in the facility, this organism can become a longterm resident, showing particular ability to persist for months and even years in floor drains (2). The inner surface of floor drains is an environment for complex biofilm communities, including multiple bacterial genera. Spoilage organisms such as Pseudomonas putida can also form biofilms and may be present in tandem with pathogenic bacteria. L. monocytogenes has been shown to attach espcially well to moist low-nutrient surfaces previously colonized with P. putida (5). Bacteria present as long-term residents in a further processing plant floor drain have the potential to escape this niche and cross contaminate other surfaces, including those that contact product. L. monocytogenes can attach and form biofilms on a variety of processing plant surfaces (1); plant persistent subtypes are especially capable biofilm formers (7). Once on a product contact surface, L. monocytogenes can be transferred to meat (10, 12). Plastic composite cutting boards, counters, trays and conveyors are common product contact surfaces in many poultry further processing plants. L. monocytogenes can attach to and survive on such plastic surfaces (11). Once established as an attached community, L. monocytogenes is more difficult to eradicate with heat or surface sanitizing treatments (3). Sanitizers have been shown to lessen but not eliminate L. monocytogenes when it is attached to plastic cutting board surfaces (13). Silver ions have an antibacterial affect that is well reviewed by Kampmann et al. (6). Silver ions can be immobilized in plastics and have been reported to have utility to lessen bacterial contamination associated with treated surfaces (6). The objective of this study was to test the ability of L. monocytogenes and P. putida to attach, grow and form stable biofilms on plastic cutting boards with and without silver ions added to the formulation. MATERIALS AND METHODS Cutting boards Plastic (polyethylene) cutting boards with and without silver ion treatment were purchased from the manufacturer (Bio-guard Plastics, Mendota, MN 55120). Control cutting boards were identified by a different color, but were otherwise identical to the treated boards. Each cutting board was cut into squares 2 cm by 2 cm. Squares were disinfected prior to inoculation by rinsing with 70% ethanol and then being air dried under germicidal ultra-violet light on sterile paper towels in a biological safety cabinet. Listeria monocytogenes culture and inoculation The L. monocytogenes culture used in this study was originally recovered from a poultry further processing plant and found to be capable of forming a biofilm. An overnight broth culture of L. monocytogenes was plated onto ten plates of BHI agar (Oxoid Ltd., Basingstoke, Hampshire, England), using a cotton tipped applicator to result in a lawn of growth; plates were incubated for 18–24 h at 37°C. Culture was removed from each of ten plates and suspended in 300 ml of PBS (pH 7). Serial dilutions and subsequent plating on BHI agar (18–24 h, 37oC) revealed a cellular density ranging from 2 to 7 × 108 cells L. monocytogenes per ml PBS. Ten 50 ml conical tubes (Tyco Healthcare Group LP, Mansfield, MA 02048) were filled with 25 ml aliquots of the cell suspension. One square of treated cutting board was placed into each of five tubes, and one square of control board was placed in each of the remaining five tubes. An uninoculated control tube of PBS was included for each type of cutting board to assure there was no contamination present on the cutting board squares prior to inoculation. Squares remained in the PBS cell suspension at 25oC for 2 h. Following incubation, each square was aseptically removed from the cell suspension and placed into a fresh tube with 25 ml sterile PBS. Each tube was inverted 4 times to remove unattached cells. Each square was then removed from PBS and placed into another fresh tube with sterile 1:10 strength BHI broth (Oxoid Ltd., Basingstoke, Hampshire, England). Dilute BHI broth was used to simulate the limited nutrients expected on a rinsed food contact surface. Squares were allowed to incubate in the dilute BHI broth for 24 h at 25oC. In the first set of experiments, squares were removed from the growth phase in dilute BHI, rinsed in a tube of PBS by inversion 4 times as previously described, and analyzed immediately. In the second set, the squares were removed from the dilute BHI, rinsed, placed on sterile paper towels and allowed to air dry for one hour under a biological safety cabinet before being transferred to a dry sterile 50 ml tube and subjected to 24 h of dry incubation at 25oC. Thus attached cells were exposed to the silver ion treatment for an additional 24 h. Prior to sampling, the dried squares were subjected to another rinse procedure as already described. Pseudomonas putida culture and inoculation The P. putida culture used was selected because it is known to be an excellent biofilm producer (5). The growth conditions and inoculum preparation methods for P. putida were similar to those previously described for L. monocytogenes with a few differences. The inoculum cell suspension ranged from 1.4 to 1.5 × 108 cells P. putida per ml PBS. P. putida was used only in the second set of experiments, with a 24-h post growth dry incubation period to allow attached cells to remain in contact with the antimicrobial plastic cutting boards. No P. putida biofilms were analyzed immediately after the growth phase. Detection of biofilm cells To remove biofilm cells from the cutting board squares, one cotton tipped MARCH 2010 | FOOD PROTECTION TRENDS

Food Protection Trends - March 2010

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