Assessment of Salmonella spp . Attachment to Reusable Plastic Containers Based on Scanning Electron Microscopy and BAX ® PCR

Reusable Plastic Containers (RPC) coupons were used to determine the ability of Salmonella spp. to adhere and form potential biofilms on commercial RPCs. Attachment of Salmonella serovars Kentucky, Newport, Enteriditis, Heidelberg, and Typhimurium was evaluated. The RPC coupons served as a platform for generating biofilms of these microorganisms. Following biofilm formation on the RPC coupons, scanning electron microscopy (SEM) was performed to examine the coupons for bacterial presence. Additionally, the RPC coupons were subjected to a bacterial biofilm growth process and were subsequently sanitized using methods and sanitizing agents that are commonplace in commercial and industrial settings. Lastly, the RPC coupons were exposed to a bacterial biofilm growth process and swabbed using methods that closely mimic scrubbing actions performed during sanitation processes typically used in commercial and industrial settings. In all cases based on SEM assessment, bacteria not only attached to the RPC, but also could not be dislodged by the sanitizers or physical scrubbing that was applied.

Surface contamination of equipment and the potential risks associated with egg processing in general has been investigated (Suresh, Hatha, Sreenivasan, Sangeetha, & Lashmanaperumalsamy, 2006;Singh, Yadav, Singh, & Bharti, 2010;Utrarachkij et al., 2012).For example, in a study on Thailand egg farms and markets, Utrarachkij et al., (2012) concluded that reusable egg trays used for these eggs could serve as a potential source of horizontal Salmonella transmission.
Based on past research, the question arises as to whether Salmonella and other foodborne pathogens can attach to surfaces such as RPC materials that they might come in contact with during transportation of eggs, and once attached, if these microorganisms can be dislodged from such surfaces.Certainly, foodborne pathogens such as Salmonella are known to attach to a variety of surfaces, and can become part of communities encased in polymeric substances resulting in difficult to remove biofilms (Kalmokoff et al. 2001;de Oliveira, Brugnera, Alves, & Piccoli, 2010;Steenanckers, Hermans, Vanderleyden, & De Keersmaecker, 2012) Treatment 5 (200 ppm chlorine solution) was conducted as follows: the corner of the coupon was grasped, dipped in the chlorine solution, aggressively shaken for 5 seconds, and shaken to remove excess.The coupon was placed on a wire rack, dried for two min, and placed in a stomacher bag.Treatment 6 (untreated control) was conducted as follows: the corner of the coupon grasped but not exposed to treatment, and transferred directly to a sterile stomacher bag.The extra coupons needed for SEM imaging were removed from the treatment groups and held at 4 °C.
A PC1 Master Test Kit (Packers Chemical, Inc., Cuba City, WI) (titration kit to test concentration of quaternary ammonium and chlorine) was used to determine the actual level of quaternary ammonium and chlorine for the respective treatment.Once all treatments were performed and all coupons were in their corresponding stomacher bags, 20 mL of buffered peptone water (BPW) (Becton Dickinson and Company) was added, and they were shaken vigorously for 30 s.All samples were incubated at 35 ± 1 °C for 18 to 24 h.After incubation, the coupon samples were tested for the presence of Salmonella using the BAX ® PCR system according to the manufacturer's instruction (Dupont, 2014).Each coupon was examined using SEM for visual confirmation of attachment and potential biofilm formation.

Salmonella Biofilm Formation Process and Impact of Swabbing (Study III)
All five Salmonella serovars were prepared as a cocktail as described previously in biofilm study I.The RPC coupons were prepared by sanitizing each coupon with 70% isopropyl alcohol and were subsequently dried.
Each coupon was aseptically and thoroughly rinsed with sterile DI water to remove any sanitizer residue.Five 90 mL sterile specimen cups were labeled and RPC coupons were inserted into each cup.Aliquots (40 mL) of TSB were aseptically dispensed into each cup and 0.5 mL of inoculum was added into each cup containing the coupon and TSB.The cups were placed onto a platform shaker (110 rpm) and were incubated at 35 ± 1 °C for 18 h.The coupons were removed individually and aseptically from the respective cups, and the coupons were rinsed with sterile DI water to remove planktonic cells.The coupons were placed into a labeled sterile 90 mL specimen cup and rinsed as previously described.Once all coupons were rinsed and placed into the respective cups, 40 mL of the TSB was aseptically dispensed into the cup, the coupon was ensured to be submerged, and the cups were incubated on the platform shaker (110 rpm) at 35 ± 1 °C for 72 h.
After the final incubation step, each coupon was aseptically removed and transferred to a tray that had previously been covered with foil and sanitized with isopropyl alcohol.Using sanitized forceps, the corner of the coupon was grasped and sterile DI water was dispensed over the coupon to remove planktonic cells, and each coupon was placed into an individual sterile cup.
The entire surface of each coupon was swabbed using a PUR-Blue™ DUO™ swab (World Bioproducts) that was moistened with BPW.Swabbing was done aggressively and with pressure to remove as much of the Salmonella biofilm as possible.The swab was returned to its corresponding tube containing 9 mL of BPW.For each of the five coupons, the swabbing was repeated two more times (three swabs per coupon) changing swabs for each swab.Once all swabs were performed, the RPC coupons were placed into a sterile stomacher bag and 20 mL of sterile BPW was added.A negative control was prepared by pouring 20 mL of the BPW into a sterile stomacher bag.A positive control was prepared by pouring 20 mL of the BPW into a sterile stomacher bag, and one Salmonella Bioball ® was added to the BPW.All samples (swabs and coupons) were incubated at 35 ± 1 °C for 18 to 24 h.After incubation, treatment samples and controls were tested for the presence of Salmonella using the BAX ® PCR system.

Disposal Protocols for Samples and Chemicals
Samples and testing materials were disposed of at completion of analysis with the approval of the WBA project's team leader and reference to WI-A-011 (Laboratory Waste and Disposal) for disposal procedures.When chemicals were used in this study, they were held on site for future use, returned to the customer, or discarded.Handling, storage, and/or disposal of all chemicals were performed appropriately according to the material safety data sheets (MSDS) and the actions taken were noted in the Research Project Design Form.

Biofilm Formation for Multiple Salmonella Serovars (Study I)
Reusable Plastic Containers were used in this study to determine the ability of Salmonella spp. to adhere to and form biofilms on the RPCs being used in commercial settings.The Salmonella biofilm was comprised of serovars Kentucky, Newport, Heidelberg, Enteritidis, and Typhimurium.The RPC's were disassembled and cut into 1 in² pieces (referred to as coupons).Preliminary studies using SEM provided visual confirmation that S.
Enteritidis adhered to the RPC (Figure 3a), and an uninoculated RPC coupon was viewed using SEM to confirm

Conclusion
Microbial contamination on surfaces such as RPC materials will most likely consist of more than one bacterial species and will probably be fairly complex.How this microbial composition influences the before and after biofilm formation by organisms such as Salmonella may impact the extent of biofilm formation as well as the ability to clean and sanitize surfaces containing these biofilms.More comprehensive microbial studies need to be conducted to better identify the dynamics of microbial diversity and their potential interactions with foodborne pathogens such as Salmonella.Microbiome sequencing offers opportunities to more thoroughly characterize these microbial populations and detect patterns that may contribute to the more persistent contamination problems.Elucidating these microbial populations may allow for an assessment of the sequence of events that initiates biofilm formation as well as which non-Salmonella microorganisms are most likely to favor the establishment of Salmonella biofilms.
. The objectives in the