Growth of Listeria monocytogenes as a Biofilm on Various Food-Processing Surfaces

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1 87 Jornal of Food Protection, Vol. 59, No.8, 1996, Pages Copyright, International Association of Milk. Food and Environmental Sanitarians Growth of Listeria monocytogenes as a Biofilm on Varios Food-Processing Srfaces ISABEL c. BLACKMAN and JOSEPH F. FRANK* Center for Food Safety and Qality Enhancement, Department of Food Science and Technology, University of Georgia, Athens, Georgia 36, USA (MS# 95-34: Received 14 September 1995/Accepted 4 Janary 1996) ABSTRACT The objective of this research was to determine the ability of Listeria monocytogenes to grow as a biofilm on varios foodprocessing srfaces inclding stainless steel, Teflon, nylon, and polyester floor sealant. Each of these srfaces was able to spport biofilm formation when incbation was at 1 C in Trypticase soy broth (TSB). Biofilm formation was greatest on polyester floor sealant (4% of srface area covered after 7 days of incbation) and least on nylon (3% coverage). The se of chemically defined minimal medim reslted in a lack of biofilm formation on polyester floor sealant, and redced biofilm levels on stainless steel. Biofilm formation was redced with incbation at 1 C, bt Teflon and stainless steel still allowed 3 to 4% coverage after incbation in TSB for 18 days. Biofilm growth of L. monocytogenes was sfficient to provide a sbstantial risk of this pathogen contaminating the food-processing plant environment if wet srfaces are not maintained in a sanitary condition. Key words: Listeria monocytogenes, biofilm, food processing, epiflorescence microscopy, SEM Listeria monocytogenes is a pathogen which cases life-threatening illness primarily in immnocompromised and pregnant individals. Evidence that listeriosis can be transmitted by food was reported after a Canadian otbreak was linked to consmption of contaminated coleslaw (18). Since then, other major otbreaks have been associated with milk, cheese, and eggs (17). Cox et al. () readily isolated L. monocytogenes from food-processing and hoseholdkitchen environments. Processed food prodcts can be contaminated by L. monocytogenes growing in the processing-plant environment (6, 17). Therefore, controlling the growth of this pathogen in food-processing plants is an important aspect of hygienic food processing. Herald and Zottola (8) observed that L. monocytogenes cold attach to stainless steel and prodce attachment fibrils. The pathogen also attaches to glass, polypropylene, and rbber (1), and prodces a sanitizer-resistant biofilm on * Athor for correspondence. Tel: ; Fax: ; cmsjoe@vga.cc.vga.ed glass, stainless steel, and Bna-N rbber (5, 11, 16). These biofilms are resistant to chlorine, iodine, acid anionic, and qaternary ammonim sanitizers. L. monocytogenes is capable of srvival and growth in mlti species biofilms (4, 9). The objective of this research was to determine the ability of L. monocytogenes to prodce biofilms on stainless steel, nylon, Teflon and polyester (floor sealant), materials typically fond in food-processing environments. Biofilm accmlation was determined in complex and chemically defined growth media at 1 and 1 C by sing epiflorescence and scanning electron microscopy. MATERIALS AND METHODS Cltre preparation L. monocytogenes Scott A was obtained from the cltre collection of the Department of Food Science and Technology, University of Georgia. The cltre was maintained on Trypticase soy agar (TSA) (Difco Laboratories, Detroit, MI) slants at 4 C. Before each experiment, the cltre was transferred three times in Trypticase soy broth (TSB) (Difco) with incbation at 1 C for 4h. Planktonic growth Planktonic growth crves at 1 and 1 C were determined in complex (TSB) and chemically defined minimal media. The chemically defined medim (DlO) was prepared as described by Trivett and Meyer (19) and inclded per liter: K HP 4, 8.5 g; NaH P 4 H,.5 g; NCl,.5 g; MgS 4 7 H,.41 g; FeC H,.48 g; NaOH,.4 g; nitriloacetic acid,.48 g; L-cysteine hydrochloride, 1 mg; L-lecine, 1 mg; DLisolecine, mg; L-arginine hydrochloride, mg; L-histidine hydrochloride, mg; riboflavin, 1, Ilg; thiamine hydrochloride, 1, Ilg; D-biotin, 1 Ilg; and a-lipoic acid, 1 Ilg (all vitamins and amino acids from Sigma Chemical Co., St. Lois, MO). A 1X soltion of the for vitamins was prepared separately, filter sterilized, and added to the atoclaved medim immediately before inoclation. Planktonic growth rates were determined by inoclating 5 ml of each medim with.5 ml of cltre and incbating at 1 and 1 C withot shaking. The optical density (4 nm) of the cltre was measred at varios intervals dring incbation. Generation

2 88 BLACKMAN AND FRANK times were determined to be 1.5 and 3.5 h for TSBat 1 and 1 C respectively, and 1.6 and 15.5 h for DlO at 1 and 1 C respectively. Preparation of srfaces for biofilm growth Teflon and stainless-steel (type 34, no. 4 finish) sheets (ca. mm thickness) were ct into by 5 cm pieces (referred to as copons). Nylon 66 sheets were ct into 5-mm sqares. The floor sealant srface was prepared by coating by 5 cm glass microscope slides with polyester patch (Martin Chemical Co. Ltd., Happage, New York). Polyester patch is recommended for sealing floor cracks and forms a rogh skid-resistant srface. The slides were completely covered with the patch material so that no glass wold be exposed to the cltre dring biofilm formation. All copons were degreased before se by overnight immersion in acetone. Copons were cleaned before each experiment with indstrial cleaning chemicals. The procedre involved heating for 5 min at 75 C in g/liter of commercial chlorinated alkali (Monarch 13l3-SD; H. B. Fller Co., Minneapolis, MN) followed by similar heating in g/liter of commercial phosphoric acid cleaner (Monarch MP-). The copons were then rinsed thoroghly in deionized water. Biofilm accmlation experiments Biofilms were formed sing a semicontinos static system with transfer of copons to fresh media before the onset of stationary phase of growth as determined by the planktonic growth crve for each medim and temperatre combination. This procedre allowed biofilm cells to remain in a ntrient-rich environment throghot the experiment. Sterile copons were placed in test tbes containing 3 ml of sterile media (sfficient to completely immerse the copon). Tbes were inoclated with.1 ml of cltre, reslting in an inoclm of between 5 X 1 6 and 1 X 1 7 CFU/ml and incbated at the appropriate temperatre. The time between transfer of the copons to fresh media was 4 h for TSB and 36 h for D 1 incbated at 1 C, and 6 days for TSB and 1 days for D 1 incbated at 1 C. Dring the transfer to fresh media, nattached cells were removed from copons by rinsing with sterile phosphate bffer. At selected transfer periods, rinsed copons were retained for analysis. Determination of biofilm accmlation Biofilms on stainless steel, Teflon and floor-sealant copons were qantified sing epiflorescence microscopy. The biofilms were stained for 3 min in.5 g/liter of Hoechst 38 florescent ncleic acid stain (Sigma Chemical Co., St. Lois, MO), rinsed with water, and air dried. Digital images were obtained sing the l6x objective on a Zeiss IM- microscope and were analyzed sing Image I software (Universal Imaging Corporation, West Chester, PA). The brightness of florescent objects was measred in absolte grey vales ranging from to 55. Pixels in each object were assigned calibrated vales to sbtract backgrond florescence, and these vales were averaged. Florescent objects smaller than Listeria cells were deleted from the image. Data was obtained as percent area covered by florescent cells. At least 1 random fields were analyzed per copon. Biofilms on nylon copons cold not be analyzed sing epifiorescence microscopy becase of excessive backgrond florescence. Biofilm formation on these copons was determined by scanning electron microscopy. Biofilms on nylon were fixed sing Pardcz's fixation procedre (J 5), dried sing a Samdri critical-point dryer and coated with 6 nm of gold-palladim sing a Hmmer 1 sptter coater. Coated films were observed sing a Phillips 55 scanning electron mic!"oscope at 3,1X magnification. Data was collected as percent srface area covered by cells from ten photomicrographs randomly selected from each copon. Areas covered by cells were calclated by direct measrement on the photomicrographs. Data analysis All biofilm experiments tilized dplicate copons and were replicated twice. Data were analyzed sing two-way analysis of variance for all treatments and srfaces followed by a one-way analysis of variance for treatments which showed an interaction between srface type and time. Means were separated sing Dncan's mltiple range test. A significance level of P =.5 was employed. RESULTS Biofilm accmlation at JC Stainless steel, Teflon, and floor sealant spported a significant accmlation of Listeria monocytogenes dring 7 days of incbation in TSB at 1 e (Fig. 1). Nylon only spported a minimal accmlation (3% of srface area covered); polyester floor sealant and stainless steel were the most rapid (33 and 4% coverage), with Teflon spporting an intermediate accmlation at % coverage. The se of a chemically defined minimal medim prodced different reslts (Fig. ). No biofilm accmlation or cell attachment was observed on the polyester floor sealant and only 1 to 1% coverage on stainless steel was achieved after 6 to 1 days of incbation. A similar level (8%) of accmlation was spported by the nylon srface. Biofilm accmlation on Teflon was significantly greater than on the other srfaces after 1 days of incbation in the defined medim. Biofilm accmlation at looc A significant accmlation of biofilm was observed on stainless steel, Teflon, and floor sealant srfaces dring incbation in TSB at looe (Fig. 3). Althogh attachment of.!!! " >, 4 a CJ Stainless steel a ISS"l Nylon IZI Floor sealant a 3. 5 ab - > ab " 15 a ab b 5 Time (Days) 4 7 FIGURE 1. Accmlation of Listeria monocytogenes biofilm on stainless steel, Tejlon, nylon, and polyester jloor sealant in Trypticase soy broth at 1 c. Bars marked with the same letters indicate no difference between srfaces at a specific time period. Bars marked with the same nmbers indicate no difference over time for a specific srface.

LISTERIA MONOCYTOGENES BIOFILMS 89 4 4 o Stainless steel..!!! &S':'l Nylon..!!! 3 3. 5 "tj 5 "tj a i; > 1 15 a b ill! 1 ill! 1 15 c 5 5. o Stainless &S':'l Nylon steel 1 a a 1, 1 3 a a b 1.

3 LISTERIA MONOCYTOGENES BIOFILMS o Stainless steel..!!! &S':'l Nylon..!!! "tj 5 "tj a i; > 1 15 a b ill! 1 ill! 1 15 c 5 5. o Stainless &S':'l Nylon steel 1 a a 1, 1 3 a a b FIGURE. Accmlation of Listeria monocytogenes biofilm on chemically defined medim at 1"e. Bars marked with the same letters indicate no difference between srfaces at a specific time period. Bars marked with the same nmbers indicate no difference over time for a specific srface. FIGURE 4. Accmlation of Listeria monocytogenes biofilm on chemically defined medim at IO e. Bars marked with the same letters indicate no difference between srfaces at a specific time period. Bars marked with the same nmbers indicate no difference over time for a specific srface. L. monocytogenes was observed on nylon copons, there was no significant accmlation on this srface between 6 and 18 days of incbation. Teflon and floor sealant allowed the greatest accmlation of Listeria biofilm at 3 to 4% of the total area covered after 18 days of incbation, whereas with stainless steel only 14% coverage was achieved at this time. The se of chemically defined growth medim again prodced different reslts. As for the l o C incbation, no attachment or accmlation of cells was observed on the floor-sealant srface incbated at wac. A significant biofilm accmlation over time was observed on nylon and stainless steel srfaces; however, accmlation was slow, with only a 7 to 1% coverage after 3 days of incbation (Fig. 4). Listeria attached to the Teflon srface bt no significant accmlation was observed between II and 3 days of incbation. Fig. 5 is a photomicrograph showing Listeria biofilm on nylon after 1 days of incbation in defined medim at 1 C. The photomicrograph provides clear evidence of microcolony formation, indicating that srface growth has occrred. Similar evidence of microcolony formation was obtained for other materials incbated nder conditions which allowed biofilm accmlation. DISCUSSION Reslts of this stdy demonstrate the ability of L. monocytogenes to accmlate as a biofilm on materials commonly fond in food-processing plants. These inclde both hydrophobic (Teflon ) and hydrophilic (stainless steel) srfaces. Helke et al. (7), Mostellar and Bishop (14), Herald and Zottola (8), and Maf et al. (1) previosly demonstrated the ability of this pathogen to adhere to varios 4 III Stainless steel Teflon &S':'l 'on..!!! loor sealant a a "tj > a 15 b " 1, " 1 b ll'1 5 c FIGURE 3. Accmlation of Listeria monocytogenes biofilm on Trypticase soy broth at IO e. Bars marked with the same letters indicate no difference between srfaces at a specific time period. Bars marked with the same nmbers indicate no difference over time for a specific srface. FIGURE 5. Scanning electron micrograph showing Listeria monocytogenes microcolonies formed on nylon after IO days of incbation in chemically defined medim at Ioe (bar = 1 JLm).

83 BLACKMAN AND FRANK srfaces inclding stainless steel, polypropylene, rbber and Teflon, bt did not report the relative ability of these materials to spport biofilm development.

4 83 BLACKMAN AND FRANK srfaces inclding stainless steel, polypropylene, rbber and Teflon, bt did not report the relative ability of these materials to spport biofilm development. Development of a biofilm is a reslt of both adherence and growth following adherence. Microcolonies were observed on all srfaces when incbated with TSB, indicating that srface growth as well as attachment had occrred. Maf et al. (13) determined that L. monocytogenes Scott A exhibits low cell hydrophobicity except nder conditions of low ph and high ionic strength. However, the srface free energy of materials that were tested (glass, stainless steel, polypropylene, and rbber) did not correlate with adherence. Their stdy indicates that factors other than hydrophobic interactions (sch as electrostatic and exopolymer interactions) accont for the attachment of L. monocytogenes to varios materials. The type of growth medim exerted a major inflence on biofilm accmlation. The best example of this effect is the total lack of biofilm accmlation on the polyester floor sealant in the presence of the defined medim, even thogh planktonic growth was observed in this medim in the presence of the floor sealant. In contrast, complex media spported extensive biofilm formation on this srface. Complex growth media contain organic polymers sch as hydrolyzed protein which adsorb onto a srface. These polymers are absent in chemically defined media. Therefore, srfaces exposed to these different media wold have very different preadsorbed conditioning films to which the Listeria cold attach. The importance of preadsorbed films of organic polymers on microbial attachment has been discssed by Baier (1). The slower biofilm accmlation observed with L. monocytogenes in the defined medim (Fig. and 4) as compared to the complex medim (Fig. 1 and 3) is not entirely explained by differing planktonic growth rates, since the time of each experiment was increased proportionally as growth rate decreased. For example, the time to stationary phase in defined medim at 1 C was 1 days, and biofilm accmlation was measred over a 3-day period. In the complex medim the time to stationary phase was 6 days at 1 C, and biofilm accmlation was measred over an 18-day period. However, there was greater biofilm development on Teflon and stainless steel after 18 days in the complex medim than after 3 days in the defined medim. Growth media can inflence cell srface chemistry by stimlating exocelllar polymer prodction or by casing changes in other srface strctres (3). The relatively low levels of biofilm accmlation on nylon may, in part, be a reslt of sing measrements from SEM rather than epiflorescence images. We fond that cell coverage on stainless steel was estimated at 5% sing epiflorescence measrement, whereas by sing SEM measrements it was estimated at %. Epiflorescence data leads to overestimates of the area covered by cells, since some extracelllar polymer is stained (). In conclsion, L. monocytogenes prodces biofilms on hydrophobic and hydrophilic srfaces in the presence of complex growth ntrients. Biofilm formation by this organism is redced bt not eliminated by growth in chemically defined media and at low temperatre. The practical implication of this research for the food indstry is the need to decrease levels of complex ntrients (food resides) on wet srfaces in the plant environment as one means of controlling biofilm formation. Or data indicate that L. monocytogenes can, given sfficient time, accmlate on a variety of srfaces to levels which might lead to the spread of the pathogen throghot a food-processing plant. Sch transmission cold reslt from aerosols prodced dring cleaning of these srfaces (1). ACKNOWLEDGMENTS This research was spported by a grant from the International Life Sciences Institte (ILSI) North American Committee on Food Microbiology, and by State and Hatch fnds allocated to the Georgia Agricltral Experiment Station. REFERENCES I. Baier, R. E Sbstrata inflences on adhesion of microorganisms and their resltant new srface properties, p In G. Bitton and K. C. Marshall (.), Adsorption of microorganisms to srfaces. John Wiley & Sons, New York.. Cox, L. J., T. Kleiss, J. L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Bemer, and A. Seibenga Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: Fletcher, M., and S. McEldowney Microbial attachment to nonbiological srfaces, p In M. J. Klg and C. A. Reddy (ed.), Crrent perspectives in microbial ecology. American Society for Microbiology, Washington, D.C. 4. Frank, J. F., R. A. N. Gillett, and G. O. Ware Association of Listeria spp. contamination in the dairy processing plant environment with the presence of staphylococci. J. Food Prot. 53: Frank, J. F., and R. A. Koffi Srface-adherent growth of Listeria monocytogenes is associated with increased resistance to srfactant sanitizers and heat. J. Food Prot. 53: Gabis, D., and R. E. Fast Controlling microbial growth in food processing environments. Food Technol. 4(1):81-8, Helke, D. M., E. B. Somers, anda. C. L. Wong Attachment of Listeria monocytogenes and Salmonella typhimrim to stainless steel and Bna-N in the presence of milk and individal milk components. J. Food Prot. 56: Herald, P. J., and E. A. Zottola Attachment of Listeria monocytogenes to stainless steel srfaces at varios temperatres and ph vales. J. Food Sci. 53: , Jeong, D. K. and J. F. Frank Growth of Listeria monocytogenes at 1 C in biofilms with microorganisms isolated from meat and dairy processing environments. J. Food Prot. 57: Kang, Y.-J., and J. F. Frank Characteristics of biological aerosols in dairy processing plants. J. Dairy Sci. 73: II. Lee, S.-H., and J. F. Frank Inactivation of srface-adherent Listeria monocytogenes by hypochlorite and heat. J. Food Prot. 54: Maf, A. A., D. Roy, J. Golet, and P. Magny Attachment of Listeria monocytogenes to stainless steel, glass, polypropylene, and rbber srfaces after short contact times. J. Food Prot. 53: Maf, A. A., D. Roy, J. Golet, and L. Savoie Characterization of physicochemical forces involved in adhesion of Listeria monocytogenes to srfaces. Appl. Environ. Microbiol. 57: Mosteller, T., and J. R. Bishop Sanitizer efficacy against attached bacteria in milk biofilm. J. Food Prot Pardcz, B Ciliary movement and coordination in ciliates. Int. Rev. Cytol. 1: Ronner, A., and A. C. L. Wong Biofilm development and sanitizer inactivation of Listeria monocytogenes and Salmonella typhimrim on stainless steel and Bna-N rbber. J. Food Prot. 56:

5 LISTERIA MONOCITOGENES BIOFILMS Ryser, E. T., and E. H. Marth Listeria, listeriosis and food safety. Marcel Dekker, Inc., New York. 18. Sch1ech, W. E, P. M. Lavigne, R. A. Bortolssi, A. C. Allen, E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome Epidemic listeriosis: evidence for transmission by food. N. Eng!. J. Med. 38: Trivett, T. L. and E. A. Meyer Citrate cycle and related metabolism of Listeria monocytogenes. J. Bacteriol. 17: Wirtanen, G., and T. MattiIa-Sandholm Epiflorescence image analysis and cltivation of foodborne biofilm bacteria grown on stainless steel srfaces. J. Food Prot. 56:

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