Abstract: This study investigated the ability of three different pathogens, Escherichia coli O157:H7, Listeria monocytogenes and Pseudomonas aeruginosa, to attach food soiled (salad dressing, cooking oil, milk and yogurt) polyvinyl chloride (PVC) plastic. The pre-soiled PVC coupons were incubated with 108 CFU/cm2 of each bacterium in 50% tryptic soy broth for 6 h to allow attachment. It was found that the effect of food products was not significant while the effect of bacterial strain was highly significant (p = 0.0005). Mean attachment of P. aeruginosa (5.5 log CFU/cm2) to pre-soiled coupons was the highest (p<0.05), compared to L. monocytogenes (5.0 log CFU/cm2) and E. coli O157:H7 (4.5 log CFU/cm2). This study indicates the capability of each bacterial pathogen, E. coli O157:H7, L. monocytogenes or P. aeruginosa, to equally attach to different pre-soiled PVC surfaces. P. aeruginosa showed the greatest potential to attach to PVC when compared to E. coli O157:H7 and L. monocytogenes.
INTRODUCTION
Biofilm formation can compromise the cleanliness of food contact surfaces and environmental surfaces by spreading detached organisms to food and other areas in processing plants. These detached organisms are different from normal microorganisms suspended in an aquatic environment because they can be more resistant to environmental and chemical stresses including some food preservation methods (de-Beer et al., 1994; Mosteller and Bishop, 1993; Ronner and Wong, 1993). Biofilm eradication is still an on-going research (Lebert et al., 2007). To effectively control biofilm in food processing plants, biofilm formation must be elucidated. Microbes can attach to various surfaces such as stainless steel, wood and rubber to form biofilms (Trachoo, 2003). When a surface is contacted with food residues, its physical and chemical properties can be modified (Chmielewski and Frank, 2003). Another word, the pre-soiled surface may be conditioned for bacterial attachment. This may desirably affect biofilm formation. Moreover food residues can also affect the effectiveness of chemical sanitizer treatment (Fatemi and Frank, 1999). Cleanability of stainless steel decreases in food soiled surfaces (Somers and Wong, 2004). Nowadays, polyvinyl chloride (PVC) is an important material used for drinking water pipe manufacture in tropical countries where hot water utilization in households is not common. Information on attachment of foodborne pathogens on PVC plastic surface soiled with different food products is limited. This study compared the attachment of Escherichia coli O157:H7, Listeria monocytogenes and Pseudomonas aeruginosa on pre-soiled (salad dressing, cooking oil, milk and yogurt) plastic surfaces.
MATERIALS AND METHODS
Culture preparation: Frozen stock cultures of Escherichia coli and Listeria monocytogenes were obtained from Foodborne Pathogens and Biofilm Laboratory, Non-aerobic Microniche Research Unit, Mahasarakham University. Pseudomonas aeruginosa (357 K. Komagata) was obtained from Thailand’s Microbiological Resources Center (Bangkok MIRCEN). The strains were recovered on tryptic soy agar slants (TSA, HiMedia Laboratories, Mumbai). The incubation temperature was 37°C for E. coli O157:H7 and 32°C for L. monocytogenes and P. aeruginosa. Before each use, the stock cultures were activated by a series of three transfers on tryptic soy agar (TSA, HiMedia).
Preparation of pre-soiled coupons: PVC (1x4x0.25 cm) coupons were cleaned by soaking in 2% HCl for 20 min, neutralized by rinsing with clean water and steriled in an autoclave at 121°C for 8 min. Pre-soiled PCV coupons were prepared by aseptically dipping the steriled PVC coupons in four different food products, salad dressing, cooking oil, milk and yogurt (available from a local market, Table 1) for 10 min and rinsing with sterile water.
Attachment comparison study: A fresh culture of each strain was grown in a tryptic soy broth (TSB) for 12 h. One-tenth milliliter of this broth culture was then added to 50% TSB (HTSB) and incubated for another 12 h to obtain a suspension of 109 CFU/cm2 of each strain (Table 2). Cell density was determined by measuring optical density of the cell suspension at 660 nm and by plate count on TSA. Pre-soiled PVC coupons were incubated with 108 CFU/cm2 of each strain in HTSB at 37°C for E. coli O157: H7 and 32°C for L. monocytogenes and P. aeruginosa for 6 h to allow cells to attach on coupons. Clean (unsoiled) PVC coupons were included as controls. After 6-h attachment, loosely attached cells were removed from each PVC coupons by rinsing with sterile water. Cells were then removed from the PVC coupons by vortexing (vortex model G-560E, Scientific Industries, NY) in 10 mL peptone water and 5 g glass beads (Sigma, St. Louis; 425-to 600 μm diameter) for 90 sec (Trachoo et al., 2002). Attached cell numbers were determined by spread plate technique on TSA.
Data analysis: This study was conducted in the Foodborne Pathogens and Biofilm Research Laboratory (FBRL), department of food technology and nutrition, mahasarakham university, Thailand in summer 2005. A 3x5 factorial design in a randomized completed block design with four replications was used for data obtained from the attachment comparison study. The same frozen stock cultures and equipment were used in all replicates. The main effects were bacterial strains (E. coli O157:H7, L. monocytogenes and P. aeruginosa and food products (salad dressing, cooking oil, milk, yogurt and control). Data was analyzed with SAS software (SAS Institute, Cary, NC) using PROC ANOVA and GLM. Significant differences between means were determined using Scheffe’s method. Significance was determined by least square means at p = 0.05.
RESULTS AND DISCUSSION
Food products used in this study were purchased from a local market. Their chemical compositions are shown in Table 1. Cooking oil and salad dressing contained 99.9 and 21.0% fat, respectively, much greater than milk and yogurt. After rinsing with sterile water, only cooking oil-soiled PVC coupons left oil marks on paper (Fig. 1). Salad dressing, milk and yogurt contained proteins and carbohydrates at various concentrations. These nutrients have been reported to modify physical and chemical properties of stainless steel surface (Frank and Chmielewski, 2001; Somers and Wong, 2004). pH of food products were in acidic range except for milk which had the pH of 6.8, nearly neutral. To control the initial bacteria counts, 109 CFU/cm2 cell suspensions were prepared for all experiments. The microbial loads of cell suspension for attachment study of each strain were not significantly different (p>0.05) as shown in Table 2. Mean attachment of P. aeruginosa (5.8 log CFU/cm2) to unsoiled coupons was the highest (p<0.05) while that of E. coli O157:H7 (4.6 log CFU/cm2) was the lowest (p<0.05).
| Table 1: | Fat, proteins and carbohydrates contents1 of salad dressing, palm oil, milk and yogurt used in the experiment |
| Calculated from data on labels | |
| Fig. 1: | Oil marks (drawn in rectangular shapes) left on paper after pressing with polyvinyl chloride plastic (PVC) soiled with water (control), salad dressing and cooking oil |
| Table 2: | Microbial load1 before attachment |
| 1Means of three replicates, nsNot significantly different | |
| Table 3: | Attachment1 of E. coli O157:H7, L. monocytogenes and P. aeruginosa on polyvinyl chloride plastic preconditioned with salad dressing, cooking oil, milk and yogurt |
| A,B,CMean of four replicates with no common superscript letter differ (p<0.05 ); nsNot significantly different; 1Bacterial cells were removed by vortexing with glass beads for 90 sec and plated on tryptic soy agar media | |
Similarly, mean attachment of P. aeruginosa (5.5 log CFU/cm2) to pre-soiled coupons was the highest (p<0.05), compared to L. monocytogenes (5.0 log CFU/cm2) and E. coli O157:H7 (4.5 logCFU/cm2). The numbers of attached pathogens on pre-soiled coupons were high enough to potentially harm human subjects (Table 3). There were no differences among means of attachment of each strain attached to different foods. The effect of food products was not significant while the effect of bacterial strain was highly significant (p = 0.0005) (Table 3). This indicates the ability of all strains to attach on different food-soiled surfaces. Pseudomonas sp. was known for its ability to attach and form biofilm on a variety of surfaces (Cowell et al., 1999; Wirtanen et al., 2001). P. aeruginosa is an opportunistic pathogen causing respiratory infections in hospital worldwide due to its ability to produce slime and form biofilm which is extraordinarily resistant to antibiotics (Korber et al., 1994). Scientists at the University of Georgia reported that Pseudomonas sp. could produce the most biofilm on PVC compared to the other three biofilm producers (Trachoo et al., 2002). Temperature may affect bacterial metabolism, motility and thus biofilm formation (Costerton et al., 1995). C. jejuni could form biofilm at 42°C or its optimal growth temperature but not 12°C and 23°C (Trachoo and Brooks, 2005). In this study, the strains were allowed to attach to PVC plastic coupons at their optimal growth temperature to allow maximum attachment.
This study indicates the capability of each bacterial pathogen, E. coli O157:H7, L. monocytogenes or P. aeruginosa, to equally attach to different pre-soiled PVC surfaces. P. aeruginosa as a worldwide antibiotic resistant and biofilm forming bacteria, showed the greatest potential to attach to PVC when compared to E. coli O157:H7 and L. monocytogenes.
ACKNOWLEDGMENTS
This research was financially supported by the Division of Research Affairs, Mahasarakham University, Thailand.