Biofilm Development and Sanitizer Inactivation of Listeria monocytogenes and Salmonella typhimurium on Stainless Steel and Buna-n Rubber

Transcription

1 750 Journal of Food Protection, Vol. 56, No. 9, Pages (September 1993) Copyright, International Association ot Milk, Food and Environmental Sanitarians Biofilm Development and Sanitizer Inactivation of Listeria monocytogenes and Salmonella typhimurium on Stainless Steel and Rubber AMY B. RONNER and AMY C. L. WONG* Department of Food Microbiology and Toxicology, Food Research Institute, 1925 Willow Drive, University of Wisconsin, Madison, Wisconsi (Received for publication December 2, 1992) ABSTRACT Biofilm formation by seven strains of Listeria monocytogenes and one strain of Salmonella typhimurium on and rubber was examined under two nutrient conditions. The type of surface, nutrient level, and organism influenced biofilm development and production of extracellular materials. had a strong bacteriostatic effect on L. monocytogenes, and biofilm formation on under low nutrient conditions was reduced for four of the seven strains tested. was less bacteriostatic toward S. typhimurium. It inhibited the growth of several other pathogens to varying degrees. An ethylene propylene diamine monomer rubber was less inhibitory than, and Viton rubber had no effect. The effectiveness of sanitizers on biofilm bacteria was examined. Biofilms were challenged with four types of detergent and nondetergent sanitizers. Resistance to sanitizers was strongly influenced by the type of surface. Bacterial biofilm populations on were reduced 3-5 log by all the sanitizers, but those on were resistant to these sanitizers and were reduced less than 1-2 log. In contrast, planktonic (suspended) bacteria were reduced 7-8 log by these sanitizers. Chlorine and anionic acid sanitizers generally removed extracellular materials from biofilms better than iodine and quaternary ammonium detergent sanitizers. Scanning electron microscopy demonstrated that biofilm cells and extracellular matrices could remain on sanitized surfaces from which no viable cells were recovered. Cleaning-in-place procedures are the predominant method for cleaning and sanitizing food processing equipment. The application of a sanitizer is the final step in a cleaning-inplace procedure. Commonly used sanitizers have proven effective against bacteria in suspension (planktonic cells) (14) but are not routinely tested against cells adhering to food contact surfaces. Bacteria exposed to surfaces can attach readily and irreversibly, even within 1 min {12,16,21), and microcolonies several cells thick can develop over time. Such biofilms have been observed on glass, rubber,, cast iron, and many types of plastics (2,6,8,15,16,19,20,22). Biofilm bacteria appear to be less sensitive to antibiotics (1,9,19) and sanitizers (2,6-8,10-12,15,17,18). Many bacteria develop an extracellular matrix (ECM), believed to be polysaccharide in nature, that may help cells to adhere firmly to surfaces and may confer resistance to antibacterial agents. Other factors such as nutrient level of the growth medium, type of attachment surface, and species or strain differences have been shown to influence the amount of adherence to surfaces or resistance to sanitizers. LeChevallier et al. (10) found that biofilms of Klebsiella pneumoniae grown in a dilute mineral salts medium with a glucose concentration of 1 mg/l were significantly more resistant to free chlorine compared to biofilms grown in undiluted medium with a glucose concentration of 10 g/l. Best et al. (2) found that Listeria monocytogenes was slightly more resistant to various sanitizers than Listeria innocua, but two strains of L. monocytogenes were equally resistant. Krysinski et al. (8) tested a 3-strain cocktail of L. monocytogenes on three surfaces for bacterial attachment and sanitizer resistance on three different surfaces. They found that the type of surface (, polyester, or polyester backed with polyurethane) had little effect on the rate of cell attachment but affected the efficacy of various sanitizers and cleaners. The purpose of this study was to determine the ability of L. monocytogenes and Salmonella typhimurium to adhere to and form biofilms on and rubber, a gasket material commonly used in food processing environments, under two different nutrient conditions. In addition, we compared the ability of four chemically different commercial sanitizers to inactivate biofilm and planktonic bacteria. During this study, we found that had a strong influence on biofilm growth and resistance to sanitizers, which we have further attempted to define. MATERIALS AND METHODS Bacterial cultures All bacteria used in this study were from the culture collection of the Food Research Institute. Seven strains of L. monocytogenes were tested: Scott A, a clinical isolate; 01, (raw milk isolate); 02 and 03 (isolated from truck wash drains at a dairy plant); 04, 05, and 07 (isolated from floor drains in a dairy plant). All the strains were stored in glycerol at -80 C, and were subcultured twice in tryptose phosphate broth (, Difco, Detroit, MI), at room temperature (21-24 C) before use. 5. typhimurium strain 101 was a clinical isolate originally obtained from the Wisconsin State Laboratory of Hygiene. It was

2 LISTERIA AND SALMONELLA BIOFILMS 751 stored on nutrient agar slants at 4 C and grown in at 37 C for 18 h before use. In addition to L. monocytogenes and S. typhimurium, Staphylococcus epidermidis, Staphylococcus aureus, Yersinia enterocolitica, and Escherichia coli 0157:H7 were used in zone inhibition tests of and three rubber compounds. They were grown in tryptic soy broth (Difco) at 30 C overnight before use. Test surfaces Stainless steel (type 304, #4 finish) and rubber (70 Durometer, "food grade", Bardon Rubber Co., Union Grove, WI) were used for biofilm development and sanitizer challenge experiments. Biofilm development and zone inhibition tests were also performed on two rubber compounds with different formulations, EPDM (an ethylene propylene diamine monomer), and Viton (a steam-resistant compound) both from Bardon Rubber Co. All the materials were cut into 1 x 1 cm 2 chips, washed by soaking in warm alkaline cleaner (Micro, International Products Corp., Trenton, NJ) for 30 min, rinsed thoroughly with distilled water, and air-dried. Biofilm development Two growth media were used for biofilm development, and peptone glucose phosphate broth (, a lower nutrient medium containing 0.5% Bacto peptone, 0.1% glucose, 0.25% NaCl, and 0.25% Na 2 HP0 4 ). All ingredients for except glucose were mixed, adjusted to ph 7.0, and autoclaved. Filter-sterilized glucose solution was added after autoclaving, to obtain a final concentration of 0.1%. Each experiment consisted of three flasks, each containing five chips of either or, and 50 ml of either or. Flasks were inoculated with 10" CFU/ml of L. monocytogenes or S. typhimurium and incubated at room temperature (21-24 C) on an orbital shaker at 75 rpm. All culture flasks were incubated for 2 d, except L. monocytogenes cultures with in, which were incubated for 4 d. Enumeration of biofilm cells Biofilm bacteria were enumerated by rinsing the chips vigorously in sterile distilled water to remove nonadherent cells, then scraping and swabbing according to the method of Frank and Koffi (6). We amended this method slightly by adding several 4-mm glass beads to the tubes in which the swabs were vortexed, to facilitate dispersal of the swab fibers. The organisms were plated on nonselective and selective agar plates to determine numbers of injured cells. L monocytogenes was plated on tryptose phosphate agar ( with 1.5% Bacto agar, Difco) and lithium chloride-phenylethanol-moxalactam agar (13) and incubated for 48 h at 30 C. S. typhimurium was plated on tryptose phosphate agar and MacConkey agar and incubated at 37 C for 48 h. Numbers of cells recovered were calculated as CFU/cm 2 chip and were the average of at least two trials. Sanitizers Four types of sanitizers from Diversey Wyandotte Corp., Wyandotte, MI, were tested: a quaternary ammonium detergent disinfectant (Quat-256); an iodine detergent sanitizer and germicide (Accord II); a liquid chlorinated sanitizer (Dibac); and an anionic acid sanitizer (Dividend). They were diluted according to label directions with autoclaved tap water, with an average hardness as CaC0 3 of 270 ppm (3). Sanitizer concentrations, contact times, and neutralizes are listed in Table 1. Sanitizer treatment Biofilm bacteria. For each sanitizer treatment, three chips were removed from the culture flasks with sterile forceps, rinsed vigorously with sterile distilled water, and drained on a sterile filter pad for about 1 min. Chips were submerged in the sanitizer, or in 0.01 M phosphate-buffered saline (PBS, ph 7.2) as a control, for the recommended contact time, then in the appropriate neutralizer for TABLE 1. Sanitizer concentrations, contact times, and neutralizers. Concentration" Contact time" Neutralizer PBS 0.01 M 2 min Tween-lecithin b quat 200 ppm 1 min Tween-lecithin (Quat-256) iodine 25 ppm 10 min 0.01 M sodium (Accord II) thiosulfate chlorine 100 ppm 2 min 0.01 M sodium (Dibac) thiosulfate anionic acid 200 ppm 2 min M NaOH (Dividend) ' Manufacturer's recommended concentration and contact time for nonporous surfaces. b Tween-lecithin: stock solution consisted of (per liter) 40 g L-alecithin (Sigma, St. Louis, MO), 0.5 ml Tween-80 (Sigma), 1.25 ml 0.1 M PBS. Working solution consisted of 25 ml of stock solution plus 6.25 ml of 0.1 M PBS diluted to 500 ml in distilled water. 30 s, andrinsedin sterile distilled water. Two of the treated chips were scraped, swabbed, and plated to enumerate viable biofilm cells. Scanning electron microscopy (SEM). A third chip was prepared for SEM by fixing the biofilms for 2 h in 0.01 M cacodylate buffer containing 5% glutaraldehyde (EM grade; Polysciences Inc., Warrington, PA) and 0.15% ruthenium red (Sigma Chemical Co., St. Louis, MO), rinsingfivetimes in distilled water, thenfixingin 1% osmium tetroxide (Polysciences) in cacodylate buffer containing 0.05% ruthenium red for 2 h, at room temperature. Chips were dehydrated in a cacodylate buffer-ethanol series, critical point dried in a Samdri-780A drier with C0 2 replacing ethanol, coated with gold, and viewed in a Hitachi S-570 scanning electron microscope at 20 or 25 kv. Planktonic bacteria. Planktonic cells from biofilm growth flasks were harvested by centrifugation at 3,000 x 9 in a Sorvall RC-5B centrifuge at 4 C for 10 min and resuspended in 0.01 M PBS to a final concentration of approximately 10 7 CFU/ml. Five milliliters of the cell suspension was added to a tube containing 5 ml of double-strength sanitizer for the recommended contact time. Five milliliters of this cell-sanitizer mixture was added to 5 ml of neutralizer for 30 s, then diluted as necessary, and plated. Numbers of recovered cells were calculated as CFU/ml. Results from all sanitizer experiments were the average of two trials. Zone inhibition tests Zone inhibition tests were performed to determine the bacteriostatic activity of the rubber materials. Tests were performed by adjusting overnight bacterial cultures to a turbidity of 0.5 McFarland Standard, dipping a sterile swab into each culture, and spreading the swab over the entire surface of a tryptic soy agar plate. Sterile chips of the test surfaces were placed on the plates, which were incubated at 30 C for h, until confluent bacterial lawns had formed. Clear zones were measured in millimeters from the edges of the chips. RESULTS Biofilm development Typical counts of planktonic and biofilm cells of two representative L. monocytogenes strains and S. typhimurium

3 752 RONNER AND WONG 101 are shown in Table 2. All the L. monocytogenes strains and S. typhimurium grew well and developed biofilms on in both and. These organisms became turbid within 24 h of growth and attained populations of 10 8 to 10 9 CFU/ml after 48 h at room temperature. After 2 d, the corresponding biofilms contained 10 5 to 10 6 CFU/ cm 2. The appearance of biofilms under SEM did not necessarily correspond with the quantitative results of the growth experiments, but observations on biofilm structure and the presence of ECM could be made. Electron photomicrographs indicated that biofilm development and ECM production by L. monocytogenes (Fig. 1) were higher on than on for all strains tested. Generally, fewer cells were seen on chips in than in, although at higher magnifications, there was no visible difference in appearance between cells (not shown). had a slight bacteriostatic effect on L. monocytogenes and S. typhimurium grown in. There was a 24-h lag in development of turbidity in these cultures; however, by 48 h planktonic cells grew to 10 9 CFU/ml, and final biofilm cell counts reached 10 5 to 10 6 CFU/cm 2. Biofilm formation and ECM production by L. monocytogenes in appeared lower on than on stainless steel and may have been strain dependent. Strains 01, 04, and 05 produced extensive biofilms and ECM on (Fig. 2a), but few cells of strain 03 remained on the chips after SEM processing. TABLE 2. Growth of planktonic and biofilm cells. Planktonic cells Biofilm cells log CFU/ml log CFU/cm 2 L. monocytogenes 01 a " L. monocytogenes 02 c S. typhimurium had a pronounced bacteriostatic effect on L. monocytogenes in. These cultures showed a lag of up to 72 h in development of turbidity, and in some trials the culture media did not become turbid after 4 d. Counts of the planktonic cells in these cultures ranged from 10 4 to 10 8 CFU/ ml. L, monocytogenes exhibited two different patterns of growth and biofilm formation on in which are represented by strains 01 and 02 in Table 2. Biofilms of L. monocytogenes strains 01, 03, 04, and 05 on contained lower numbers of cells in than the other strains. After 4 d, these biofilms contained <10 2 to 10 3 CFU/cm 2. A representative photomicrograph of strain 01 is shown in Fig. 2b. L. monocytogenes strains 02, 07, and Scott A grew somewhat better on in, even though a lag time of up to 72 h was observed until turbidity developed. Planktonic and biofilm cell counts for these strains were 10 8 CFU/ ml and 10 5 CFU/cm 2, respectively. Using SEM, few L. monocytogenes cells of any strain were seen on in, even when viable counts were as high as 10 5 CFU/cm 2. The type of surface and growth medium appeared to have less influence on S. typhimurium, except for an initial 24-h lag time in growth in the presence of with both media. An example of a S. typhimurium biofilm is shown in Fig. 3. This organism produced ECM under all growth conditions. Most notably, S. typhimurium adhered better to than L. monocytogenes, and biofilms could be seen on chips incubated in both and. Sanitizer treatments Effectiveness of the sanitizers against biofilm bacteria depended on the type of organism and surface (Table 3). L. monocytogenes strains 01 and 02 represent the groups that adhered in lower and in higher numbers, respectively, to in. The type of medium appeared to have no effect on the resistance of biofilm cells developing on. In a Similar results were obtained for strains 03, 04, and 05. b Incubated for 4 d; all other treatments incubated for 2 d. c Similar results were obtained for strains 07 and Scott A. Figure 1. L. monocytogenes strain <_>!..'.. \: :. '... ^ >'...' I'I'B, showing extensive biofilm formed after 48 h. Bar = 60 \im.

4 LISTERIA AND SALMONELLA BIOFILMS -70 * ^ s < - * v - " <. v, *'** " >** ** " 'Sri. "'Mi Figure 3. S. typhimurium biofilm and ECM on in. Bar = 1 \xm. and, S. typhimurium and L. monocytogenes populations were generally reduced 3-5 log by all the sanitizers. However, S. typhimurium was reduced slightly less than 3 log by the quaternary ammonium detergent sanitizer. There were no differences in numbers of cells recovered from selective and nonselective media, indicating that treated cells were inactivated and not injured. All the sanitizers were much less effective against biofilms on. Biofilms of both organisms grown on in both media were reduced less than 1-2 log by all the sanitizers. Some differences in the ability of sanitizers to remove cells and ECM from and were noted with SEM. Large numbers of L. monocytogenes cells often remained on chips in after sanitizer treatment, even though most of these cells were not viable. The anionic acid and chlorine sanitizers usually removed more of the ECM from these chips than the iodine and quaternary ammonium detergent sanitizers (Fig. 4). L. monocytogenes cells and ECM adhering to chips in and in tended to be removed by all the sanitizers, even though viable cells could be recovered as indicated by plate counts. Cells of S. typhimurium remained on the and chips after all sanitizer treatments, even where no viable cells were recovered. The anionic acid and chlorine sanitizers removed ECM from the chips in both media, while the iodine and quaternary ammonium detergent sanitizers allowed much of the ECM to remain (Fig. 5a,b). Biofilms on differed from this trend, and none of the sanitizers removed the ECM (Fig. 5c). Planktonic cells grown in the presence of and in both media were much more sensitive to sanitizers than biofilm cells (Table 4). L. monocytogenes strains 01, 02, 04, and 05 were reduced 7-8 log by all the sanitizers, and no viable cells were recovered (data not shown). Strains 03, 07, and Scott A were also reduced by 7-8 log, but in at least one of the duplicate trials, some viable cells (1 log or fewer) were recovered from the iodine or quaternary ammonium detergent sanitizer treatments. Planktonic S. typhimurium cells grown in the presence of and in both media were reduced 7-8 log by the iodine and chlorine sanitizers. The quaternary ammonium detergent and anionic acid sanitizers inactivated only 4- JOURNAL OF FOOD PROTECTION. VOL. 56, SEPTEMBER 1993

5 754 RONNER AND WONG TABLE 3. Survival of biofilm L. monocytogenes and S. typhimurium on and after sanitizer treatments. log 10 CFU/cm 2 after treatment with PBS Iodine (Accord II) Chlorine (Dibac) Quat (Quat-256) Anionic acid (Dividend) L. monocytogenes 01" b L. monocytogenes 02 c typhimurium a Similar results were obtained for strains 03, 04, and 05. b None recovered (detection limit 5 CFU/cm 2 ). c Similar results were obtained for strains 07 and Scott A. TABLE 4. Survival of planktonic L. monocytogenes and S. typhimurium after sanitizer treatments. log CFU/ml after treatment with PBS Iodine (Accord II) Chlorine (Dibac) Quat (Quat-256) Anionic acid (Dividend) L. monocytogenes 03 c " S. typhimurium a Similar results were obtained for strains 07 and Scott A. Cells of strains 01, 02, 04, and 05 were not recovered from these treatments. b None recovered (detection limit 1 CFU/ml).

6 LISTERIA AND SALMONELLA BIOFILMS 755 f * V Bacteriostatic activity of To determine if the bacteriostatic effect of could be neutralized by organic matter, an experiment was permi,*ht*r»-'>^ ^ *. Figure 4a. L. monocytogenes sfram 04 on in. Biofilm with ECM on PBS control chip. Bar = I \\m. Figure 4c. L. monocytogenes strain 04 on in. ECM remained after treatment with Quat-256. Bar = 1 \xm../.ut\ i I 3i: ^ formed in which washed chips were soaked for 30 min in, skim milk, or distilled water. Four chips from each soaking treatment were then incubated in flasks of with L. monocytogenes strain 03. None of the culture flasks showed growth at 4 d, indicating that these treatments had no effect on bacteriostatic activity. The bacteriostatic component was quite potent since a single chip in 50 ml of prevented growth of L. monocytogenes after 4 d. A trial was conducted in which a - flask was inoculated with 10 7 CFU/ml of L. monocytogenes strain 01, instead of the usual inoculum of 10 4 CFU/ml. After 4 d, biofilm and planktonic cell counts were 10 3 CFU/cm 2 and 10 7 CFU/ml, respectively, with the culture medium remaining clear. TABLE 5. Growth inhibition of various organisms by rubber compounds. ^ " Organism L. monocytogenes strain Scott A Inhibition zones (mm') from edge of chip EPDM Viton 3 b Figure 4b. L. monocytogenes strain 04 on in. ECM was removed after treatment with Dividend. Bar = 1 uni. S. typhimurium S. aureus log in most cases; the anionic acid sanitizer inactivated more than 6 log of cells grown in containing stainless steel chips. S. epidermidis E. coli 0157:H Y. enterocolitica Zone measured in mm from edge of chip. 1 No zone of inhibition. 4 ~ JOURNAL OF FOOD PROTECTION. VOL. 56, SEPTEMBER 1993

7 756 RONNER AND WONG Figure 5a. S. typhimurium on in. ECM removed after treatment with Dibac. Bar = 1 um.. Figure 5c. S. typhimurium on in. ECM remained after treatment with Dibac. Bar = 1 \im..' ft the clear zones underneath were touched with a sterile swab and streaked onto fresh agar. After incubation overnight at 30 C, moderate to heavy growth occurred, indicating that was bacteriostatic and not bactericidal. We also noted that had a bacteriostatic effect on several other organisms including S. epidermidis, S. aureus, Y. enterocolitica, and E. coli 0157:H7 (Table 5). Stainless steel chips did not inhibit any of these cultures. X "C0-- +r,. Figure 5b. S. typhimurium on in. ECM remained after treatment with Accord II. Bar = I \lm. We attempted to quantify the inhibitory effect of by zone inhibition tests. and chips were placed on plates seeded with L. monocytogenes or S. typhimurium. Overnight incubation produced clear zones of 3 and 2 mm on confluent lawns of L. monocytogenes and S. typhimurium, respectively. The chips were removed and Bacteriostatic activity of other rubber compounds Two other rubber compounds, EPDM and Viton, were tested by the zone inhibition test. EPDM inhibited S. epidermidis and S. aureus, but not L. monocytogenes or S. typhimurium. Viton did not inhibit any of the organisms tested (Table 5). The inhibitory effect of, Viton, and EPDM chips on L. monocytogenes strain 01 in was determined. While the flasks containing or EPDM chips showed a 24-h lag in development of turbidity, the flask containing Viton was turbid at 22 h. After 2 d, the Viton and EPDM chips both had counts of approximately 10 5 CFU/cm 2, while the chips had <5 CFU/cm 2. After 4 d, counts on EPDM and Viton chips remained at 10 5 CFU/cm 2, and the chips had 6 x 10 4 CFU/cm 2. Hence, had a strong bacteriostatic effect on L. monocytogenes, whereas EPDM had a lesser effect, and Viton had no effect. DISCUSSION Results of this study support previous reports of bacterial adherence to many types of surfaces found in food processing environments, and the notion that biofilm bacteria are more resistant to sanitizers than planktonic bacteria. We have shown that L. monocytogenes and S. typhimurium can form

8 LISTERIA AND SALMONELLA BIOFILMS 757 extensive biofilms and produce ECM on and at room temperature within 2 d. The behavior of biofilm cells was greatly influenced by the type of surface. rubber, a gasket material that is commonly used in the food processing industry, had a bacteriostatic effect on a number of bacteria including L. monocytogenes. The bacteriostatic effect of was most pronounced under lower nutrient conditions and was not mitigated by the presence of organic matter. EPDM was slightly less bacteriostatic. Despite this bacteriostatic effect, biofilms did form on. In two other studies concerning biofilm formation in this material, Czechowski (4,5) reported that bacterial attachment to gaskets in dairy processing lines increased with length of time in the line and the physical deterioration of the gasket surfaces. We found large numbers of cells attached to unused. Biofilms of L. monocytogenes and S. typhimurium grown on were more resistant to the four types of sanitizers that we tested than biofilms grown on. Mafu et al. (75) referred to nitrile rubber as a porous surface and showed that sanitizer efficiency was greater on nonporous surfaces. Except for the chlorine sanitizer, none of the sanitizers we tested had different manufacturer's instructions for use on porous surfaces. The lag in development of culture turbidity in the presence of, and the results of zone inhibition tests, suggest that some constituent of affects the rate of growth. Slow growth might enhance the resistance of cells to antimicrobial agents (/), which would partially explain why cells grown on are more resistant to sanitizers. Using a mixture of three L. monocytogenes strains, Krysinski et al. (8) found that cleaners were generally more effective than sanitizers in eliminating this pathogen from and plastic surfaces. However, in our study the detergent-sanitizer formulations (Accord II and Quat-256) we tested were generally less effective than the nondetergent sanitizer formulations (Dibac and Dividend) in removing ECM from biofilm cells. Nonviable cells and ECM were shown in several instances to remain on sanitized surfaces. Cell debris may provide a hospitable environment for successive populations of adhering cells. In examining various strains individually, we found differences in their abilities to attach to under lower nutrient conditions. The strains that attached best to in were strains 02, 07, and Scott A. The biofilms formed by these strains contained higher numbers of cells than the other strains, and greater proportions of the biofilm populations survived sanitizer treatments. Several differences between S. typhimurium and L. monocytogenes were observed. S. typhimurium was less affected by the bacteriostatic component of. The ECM matrix of S. typhimurium grown under lower nutrient conditions was more tenacious though SEM fixation than the matrix formed by L. monocytogenes. LeChevallier et al. (70) observed that very low nutrient levels produced more resistant biofilms. However, we found that while L. monocytogenes biofilms grown in lower nutrient medium were no more resistant to sanitizers than those grown in higher nutrient conditions, cells and ECM of the biofilms grown in higher nutrient conditions seemed to persist better through the rigors of fixation and critical point drying for SEM. Nutrient level may have an influence on both the rate of production and chemical composition of the ECM. Such conditions might affect resistance of the ECM to various types of sanitizers. Further research on the interaction of these factors, and identification of surfaces that may mitigate the effects of sanitizers or detergents, would be helpful in improving the efficacy of cleaning procedures. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by contributions to the Food Research Institute. REFERENCES 1. Anwar, H., M. K. Dasgupta, and J. W. Costerton Testing the susceptibility of bacteria in biofilms to antibacterial agents. Antimicrob. Agents Chemother. 34: Best, M., M. E. Kennedy, and F. Coates Efficacy of a variety of disinfectants against Listeria spp. Appl. Environ. Microbiol. 56: Clesceri, L. S., A. E. Greenberg, and R. R Trussell (ed.) Standard method for the examination of water and wastewater, 17th ed. American Public Health Association, Washington, DC. 4. Czechowski, M. H Gasket and surface sanitation: environmental parameters affecting bacterial attachment. Aust. J. Dairy Technol. 45: Czechowski, M. H Bacterial attachment to gaskets in milk processing equipment. Aust. J. Dairy Technol. 45: Frank, J. F., and R. A. Koffi Surface-adherent growth of Listeria monocytogenes is associated with increased resistance to surfactant sanitizers and heat. J. Food Prot. 53: Herson, D., B. McGonigle, M. A. Payer, and K. H. Baker Attachment as a factor in the protection of Enlerobacter cloacae from chlorination. Appl. Environ. Microbiol. 53: Krysinski, E. P., L. J. Brown, and T. J. Marchisello Effect of cleaners and sanitizers on Listeria monocytogenes attached to product contact surfaces. J. Food Prot. 55: LaTouretteProsser, B.,D. Taylor, B. A. Dix, and R. Cleeland Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrob. Agents Chemother. 31: LeChevallier, M. W C. D. Cawthon, and R. G. Lee Factors promoting survival of bacteria in chlorinated water supplies. Appl. Environ. Microbiol. 54: LeChevallier, M. W., C. D. Cawthon, and R. G. Lee Inactivation of biofilm bacteria. Appl. Environ. Microbiol. 54: Lee, S. H., and J. F. Frank Inactivation of surface-adherent Listeria monocytogenes hypochlorite and heat. J. Food Prot. 54: Lee, W. H., and D. McClain Improved Listeria monocytogenes selective agar. Appl. Environ. Microbiol. 52: Lopes, I. A Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeria monocytogenes. I. Dairy Sci. 69: Mafu, A. A., D. Roy, I. Goulet, L. Savoie, and R. Roy Efficiency of sanitizing agents for destroying Listeria monocytogenes on contaminated surfaces. I. Dairy Sci. 73: Mafu, A. A., D. Roy, I. Goulet, and P. Magny Attachment of Listeria monocytogenes to, glass, polypropylene, and rubber surfaces after short contact times. I. Food Prot. 53: Mosley, E. B., P. R. Elliker, and H. Hays Destruction of food spoilage, indicator and pathogenic organisms by various germicides in solution and on a surface. I. Milk Food Technol. 39: Mustapha, A., and M. B. Liewen Destruction of Listeria monocytogenes by sodium hypochlorite an quaternary ammonium sanitizers. I. Food Prot. 52: Nickel, J. C, 1. Ruseska, J. B. Wright, and J. W. Costerton Tobramycin resistance of Pseudomonas aeruginosa cells growing as

9 RONNER AND WONG a biofilm on urinary catheter material. Antimicrob. Agents Chemother. 27: Spurlock. A. T and E. A. Zottola Growth and attachment of Listeria monocytogenes to cast iron. J. Food Prot. 54: Stanley, P. M Factors affecting the irreversible attachment of Pseudomonas aeruginosa to. Can. J. Microbiol. 29: Stickler, D.. i. Dolman, S. Rolfe, and 1. Chawla Activity of antiseptics against Escherichia coli growing as biofilms on silicone surfaces. Eur. J. Clin. Microbiol. Infect. Dis. 8: JOURNAL OF FOOD PROTECTION, VOL. 56. SEPTEMBER 1993