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Biofilm formation by Salmonella spp. on food contact surfaces and their sensitivity to sanitizers
International Journal of Food Microbiology 64 (2001) 367–372 www.elsevier.nl / locate / ijfoodmicro
Short communication
Biofilm formation by Salmonella spp. on food contact surfaces and their sensitivity to sanitizers B. Joseph, S.K. Otta, Indrani Karunasagar, I. Karunasagar* Department of Fishery Microbiology, College of Fisheries, Mangalore -575 002, India Received in revised form 16 March 2000; accepted 12 October 2000
Abstract Biofilm formation by two poultry isolates of Salmonella on three commonly used food contact surfaces viz plastic, cement and stainless steel were studied. Biofilm formation of both the isolates showed a similar trend with the highest density being on plastic followed by cement and steel. Salmonella weltevreden formed biofilm with a cell density of 3.4 3 10 7 , 1.57 3 10 6 and 3 3 10 5 cfu / cm 2 on plastic, cement and steel respectively while Salmonella FCM 40 biofilm on plastic, cement and steel were of the order of 1.2 3 10 7 , 4.96 3 10 6 and 2.23 3 10 5 cfu / cm 2 respectively. The sensitivity of the biofilm cells grown on these surfaces to different levels of two sanitizers namely hypochlorite and iodophor for varying exposure times was studied. Biofilm cells offered greater resistance when compared to their planktonic counterparts. Such biofilm cells in a food processing unit are not usually removed by the normal cleaning procedure and therefore could be a source of contamination of foods coming in contact with such surfaces. 2001 Elsevier Science B.V. All rights reserved. Keywords: Biofilm; Salmonella; Sanitizers; Hypochlorite; Iodophor
1. Introduction Microbial biofilms are attracting attention of scientists in different areas such as the medical field, aquatic environment, food processing industries etc. Microbial biofilms may be detrimental and undesirable in food processing premises. Biofilms by pathogenic bacteria such as Salmonella (Jones and Bradshaw, 1996; Dhir and Dodd, 1995; Humphery et al., 1995; Somers et al., 1994), Klebsiella (Jones and *Corresponding author. Tel.: 1 91-824-436-384. E-mail address: [email protected] (I. Karunasagar).
Bradshaw, 1996; Morin et al., 1996), Pseudomonas (Brown et al., 1995), Campylobacter and enterohaemorrhagic E.coli (Somers et al., 1994) and Listeria (Mafu et al., 1990; Ren and Frank, 1993) have been reported. Such biofilms could be a continuous source of contamination to foods coming in contact with them when formed on contact surfaces. Increased resistance of biofilm cells to antibacterial agents and sanitizers have also been observed (Costerton et al., 1987; Costerton and Lapin-Scott, 1989; Carpentier and Cerf, 1993; Ren and Frank, 1993). Salmonella are a group of food poisoning organisms which are of considerable significance to the
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00466-9
368
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
food processing industry. This study was undertaken to understand the ability of Salmonella to form biofilms on potential food contact surfaces. The resistance or otherwise of biofilm and planktonic cells to sanitizers such as iodophor and chlorine was investigated.
2. Materials and methods
2.1. Test organisms Two cultures of Salmonella isolated from poultry meat were used in this study. One culture was identified as Salmonella weltevreden and the other unspeciated one (due to minor variations in the biochemical characters) was designated as Salmonella FCM 40.
2.2. Biofilm development Three types of material namely plastic (high density polyethylene, HDPE), cement and stainless steel were used to develop the biofilm. The concrete slabs (4 cm 2 , locally made) were scrubbed with a brush and washed well with HPLC grade water. Stainless steel coupons (4 cm 2 , Bharath Steel Supplies, Mangalore, India) were cleaned with acetone to remove grease and were etched by submerging in 5N HCl for 15 min, cleaned in detergent solution and finally rinsed in HPLC grade water. The HDPE coupons (4 cm 2 , Brite plastics, Mumbai) were cleaned with detergent and rinsed with Type 1 reagent grade water. The prepared samples were placed individually in 250 ml glass beakers and 100 ml of 0.2% (w / v) tryptone soy broth (TSB, HiMedia, Mumbai, India) were added. Experiments were conducted wherein two samples of the same type viz plastic, cement or steel were placed in 1000 ml glass beakers and 200 ml 0.2%(w / v) TSB were added. Salmonella strains were grown in TSB for 24 h at 378C and 2 ml of this culture was added to the beakers with 0.2% (w / v) TSB and the samples. After incubation at ambient temperature (28628C) for 48 h, the samples were aseptically removed, washed in sterile phosphate buffer saline (PBS, pH 7.4) to remove unattached cells and placed in beakers with fresh sterile 0.2% (w / v)TSB (Ren and Frank, 1993).
This procedure was repeated five times every alternate day to complete the biofilm formation.
2.3. Enumeration of biofilm cells To enumerate biofilm cells after ten days of incubation, the samples were washed with sterile PBS to remove unattached cells and the biofilm cells were removed by swabbing with sterile cotton swabs. The swabs were transferred to 100 ml physiological saline (0.85% NaCl, w / v prepared in the laboratory) shaken vigorously and enumerated by standard spread plate technique. Tryptone soy agar (TSA, HiMedia, Mumbai, India) was used for enumeration and plates were incubated at 378C for 48 h. To test the sensitivity of biofilm cells to sanitizers namely iodophor (Asian catalysts and chemicals, Bangalore, Active compound I 2 ) and hypochlorite (BDH, Active compound Cl 2 ), the samples were dipped in solutions containing 10, 20, 50 and 100 ppm of available Cl 2 solution and 1, 10, 20 and 50 ppm of available I 2 solution for 5, 10, 15, 20 and 25 min. The substrates were then transferred to neutralising solution (0.1 M Na 2 S 2 O 3 ) for 30 s, rinsed with PBS and cells were enumerated after swabbing as described above. The limit of detection by this method was 25 cells / cm 2 .
2.4. Sensitivity of planktonic cells Sensitivity of planktonic cells to sanitizers was tested as follows. 24 h old culture of the two species in TSB were centrifuged and the cell pellets were resuspended in same volume of sterile PBS. An aliquot was removed for enumeration by serial dilution and plated on TSA. The plates were incubated at 378C for 48 h. Chlorine at 10 ppm and Iodine at 1 ppm levels were added to the PBS suspension of Salmonella cells. Aliquots were taken at 5 and 10 min, serially diluted and plated to enumerate the cells.
3. Results
3.1. Sensitivity of Salmonella biofilm to chlorine Salmonella weltevreden formed biofilm on plastic with a cell density of 3.4 3 10 7 cfu / cm 2 . Exposure
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
to 10 ppm Cl 2 up to 25 min brought about less than 2 log reduction in counts. Similarly, counts ranging from 8.0 3 10 4 to 2 3 10 4 cells / cm 2 were observed after 25 min exposure to 20 ppm and 50 ppm Cl 2 respectively. Exposure to 100 ppm Cl 2 for 5, 10 and 15 min resulted in a 3, 4 and 5 log reduction in the cell density respectively. Biofilm cells of S.weltevreden could not be detected on plastic after exposure
369
to 100 ppm Cl 2 for 20 min (Fig. 1a). On cement S.weltevreden formed biofilm with a cell density of 1.57 3 10 6 cfu / cm 2 . The counts after exposure to 100 ppm Cl 2 for 5, 10 and 20 min were 2.16 3 10 3 , 8.72 3 10 2 and 4.64 3 10 2 respectively. There was a 3 log reduction in the biofilm cell count on cement when exposed for 25 min to 10, 20 and 50 ppm Cl 2 (Fig. 1b). The biofilm formed by S.weltevreden on
Fig. 1. Effect of chlorine on S.weltevreden biofilm on different surfaces (a) plastic (b) cement (c) stainless steel.
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B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
steel had a cell density of 3 3 10 5 cfu / cm 2 . Here the biofilm cells could survive a treatment of 100 ppm Cl 2 for only 15 min. Biofilm cells survived 25 min treatment to 10, 20 and 50 ppm Cl 2 with cell 4 4 numbers being 5.42 3 10 , 1.10 3 10 and 3.92 3 3 2 10 cfu / cm respectively (Fig. 1c). As in the case of S.weltevreden, the cell density of biofilm formed by Salmonella FCM 40 was highest on plastic followed by cement and steel. Cell density 7 2 of biofilm on plastic was 1.2 3 10 cfu / cm . Biofilm cells of Salmonella FCM 40 on plastic were found to be resistant to 10, 20, 50 and 100 ppm of Cl 2 with cell counts of 3.62 3 10 4 , 2.29 3 10 4 , 1.12 3 10 4 and 4.2 3 10 3 cfu / cm 2 respectively after 25 min exposure (results not shown). Salmonella FCM 40 biofilm on cement had a cell density of 4.96 3 10 5 cfu / cm 2 . Biofilm cells of Salmonella FCM 40 were partially inactivated after treatment with 10, 20, and 50 ppm Cl 2 for 25 min with cell counts being 1.36 3 10 5 , 2.52 3 10 4 and 1.4 3 10 3 cfu / cm 2 respectively. On the other hand, Salmonella FCM 40 biofilm could not be detected on cement after exposure to 100 ppm Cl 2 for 20 min (results not shown). Salmonella FCM 40 formed biofilm on steel with a cell density of 2.09 3 10 5 cfu / cm 2 . Treatment of biofilm cells with 10 ppm Cl 2 brought about 1 log reduction in counts after 25 min exposure. 20 ppm brought about 2 log reduction after 25 min exposure. However, the inactivation was complete with 100 ppm Cl 2 after 20 min exposure (results not shown).
3.2. Sensitivity of Salmonella biofilm to iodine On plastic, S.weltevreden biofilm was found to survive all concentrations of I 2 up to 25 min with a maximum of 4 log reduction in cell counts (Fig. 2a). The biofilm cells on cement were completely inactivated after 50 ppm I 2 treatment for 25 min. The cell counts after 25 min exposure to 1, 10 and 20 ppm I 2 4 4 3 2 were 8.4 3 10 , 3.84 3 10 and 2.25 3 10 cfu / cm respectively (Fig. 2b). On steel the biofilm cells were found to be more sensitive surviving only up to 15 min exposure to 50 ppm I 2 with a cell concentration of 2.63 3 10 1 cfu / cm 2 . Biofilm cells of S.weltevreden could not be detected on steel after 20 min exposure to 50 ppm I 2 . The cell density of biofilm after 25 min exposure to 1, 10 and 20 ppm I 2 was 3 3 3 2 3.86 3 10 , 4.26 3 10 and 2.29 3 10 cfu / cm respectively (Fig. 2c).
Exposure of Salmonella FCM 40 biofilm on plastic to 1 ppm I 2 resulted in partial inactivation of cells and the survival was comparable to that after treatment with 10 ppm Cl 2 . Increase of I 2 concentration to 10 ppm only marginally increased inactivation. About 10 3 cells survived even after treatment with 20 ppm I 2 for 25 min, whereas the biofilm cells could not be detected after 25 min exposure to 50 ppm I 2 (results not shown). 25 min exposure to 1, 10 and 20 ppm I 2 on cement resulted in partial inactivation with counts being 1.7 3 10 4 , 3.58 3 10 3 and 1.86 3 10 3 cfu / cm 2 respectively. However, the biofilm cells could not be detected after treatment with 50 ppm I 2 for 25 min (results not shown). About 2 log reduction in counts could be obtained after treatment with 1 ppm I 2 for 25 min on steel surface. Increase of I 2 concentration to 10 and 20 ppm resulted in only a marginal increase in the extent of inactivation. 50 ppm iodophor totally inactivated biofilm cells with a 25 min exposure (results not shown).
3.3. Sensitivity of planktonic cells of Salmonella to chlorine and iodine Planktonic cells of S.weltevreden which were suspended in PBS to a level of 2.4 3 10 6 cells / ml were completely killed by exposure to 10 ppm Cl 2 for 10 min and a cell density of 1.9 3 10 6 cells / ml were completely killed after 5 min exposure to 10 ppm I 2 (results not shown). Planktonic cells of Salmonella FCM 40 suspended in PBS to a level of 2.26 3 10 6 were completely killed after exposure to 10 ppm Cl 2 for 10 min and 10 ppm I 2 for 5 min (results not shown). Salmonella could form biofilms on all the three substrates studied, viz. plastic, cement and steel. The cell density of Salmonella was highest on plastic followed by cement and steel reflecting the surface preference of Salmonella. Fletcher and Loeb (1979) noted that large numbers of bacteria attached to hydrophobic surfaces with little or no surface charge and moderate numbers attached to hydrophobic metals with positive charge or neutral charge and very few attached to hydrophilic negatively charged substrates. The importance of hydrophobicity in bacterial attachment has been highlighted by other investigators also (Characklis, 1990). Variation in biofilm density depending on surface was also
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
371
Fig. 2. Effect of iodine on S.weltevreden biofilm on different surfaces (a) plastic (b) cement (c) stainless steel.
reported by Karunasagar et al. (1996) with Vibrio harveyi. The results presented here show that biofilm cells of Salmonella are much more resistant to sanitizers compared to planktonic cells. The efficiency of
biofilm formation varied depending on the surface and the resistance to treatment with sanitizers also varied depending on the surfaces. Biofilm cells on stainless steel were most sensitive and those on plastic most resistant. These observations were con-
372
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
sistent with both strains of Salmonella studied. However, minor variations between strains were also observed. For instance, biofilm cells of Salmonella FCM 40 on plastic were not inactivated even after 25 min treatment with 100 ppm Cl 2 while in the case of S.weltevreden, treatment with the same level of Cl 2 inactivated a similar cell density in 20 min. Formation of biofilm by Salmonella on various surfaces has implications for the food processing industry. Salmonella strains entering the food processing environment either through the meat or through carriers handling meat may survive in the premises by forming biofilms on various surfaces. HDPE surfaces are generally available on crates which are used to carry food products. Sometimes the contact time between foods and HDPE surface may be 24–48 h depending on the processing conditions including design of equipment and cleaning and disinfection regimes. Once a biofilm is formed, this could be a source of contamination for foods passing through the same processing line. The results of the study have shown that Salmonella spp can form biofilms on food contact surfaces and that these biofilm cells are much more resistant to sanitizers compared to planktonic cells. Such biofilm cells which are not removed during normal cleaning procedure in a food processing unit could be a source of contamination for foods. The cleaning and disinfection protocols in food processing units should therefore consider Salmonella biofilms.
References Brown, M.L., Henry, C.A., Gauthier, J.J., 1995. Relation between Glycocalyx and Povidone-Iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms. Appl. Env. Microbiol. 61 (1), 187–193.
Carpentier, B., Cerf, O., 1993. Biofilms and their consequences with particular reference to hygiene in the food industry: A review. J. Appl. Bacteriol. 75, 499–511. Characklis, W.G., 1990. Process analysis. In: Characklis W.G Marshall, K.C. (Ed.), Biofilms. Wiley-Interscience, New York, pp. 17–54. Costerton, J.W., Lappin-Scott, H.M., 1989. Behavior 307 of bacteria in biofilms. ASM News 55, 650–654. Costerton, J.W., Cheng, K.J., Geesey, G.G., Ladd, T.L., Nickel, J.C., Dasgupta, M., Marrie, T.J., 1987. Bacterial biofilms in nature and disease. Ann. Rev. Microbiol. 41, 435–464. Dhir, V.K., Dodd, C.E.R., 1995. Susceptibility of suspended and surface attached Salmonella enteritidis to biocides at elevated temperatures. Appl. Environ. Microbiol. 61, 1731–1738. Fletcher, M., Loeb, G.I., 1979. Influence of substratum characteristics in the attachment of a marine psuedomonad to solid surfaces. Appl. Environ. Microbiol. 37, 67–72. Humphery, T.J., Slater, E., McAlpine, K., Rowbury, R.J., Gilbert, R.J., 1995. Salmonella enteritidis Phage type 4 isolated more tolerant of heat, acid or hydrogen peroxide also survive longer on surfaces. Appl. Env. Microbiol. 61 (8), 3161–3164. Jones, K., Bradshaw, S.B., 1996. Biofilm formation by the Enterobacteriaceae:A comparison between Salmonella enteritidis, E. coli and a Nitrogen fixing strain of Klebsiella pneumoniae. J. Appl. Bacteriol. 80, 458–464. Karunasagar, I., Otta, S.K., Karunasagar, I., 1996. Bioflm formation by Vibrio harveyi on surfaces. Aquaculture 140, 241–245. Mafu, A.A., Roy, D., Gonlet, J., Magny, P., 1990. Attachment of Listeria monocytogenes to stainless steel, glass, polypropylene, rubber surfaces after short contact times. J. Food. Prot. 53, 742–746. Morin, P., Camper, A., Jones, W., Gatel, D., Goldman, J., 1996. Colonization and disinfection of biofilms hosting coliform colonized carbon fines. Appl. Env. Microbiol. 62 (12), 4428– 4432. Ren, T.J., Frank, J.F., 1993. Susceptibility of starved palnktonic and biofilm Listeria monocytogenes to quaternary ammonium sanitizer as determined by direct viable and agar plate counts. J. Food. Prot. 56, 573–576. Somers, E.B., Schoeni, S.L., Wong, A.C.L., 1994. Effect of Trisodium phosphate on biofilm and planktonic cells of Campylobacter jejuni, E. coli O157:H7, Listeria monocytogenes and Salmonella typhimurium. Int. J. Food. Microbiol. 22, 269–276.
Short communication
Biofilm formation by Salmonella spp. on food contact surfaces and their sensitivity to sanitizers B. Joseph, S.K. Otta, Indrani Karunasagar, I. Karunasagar* Department of Fishery Microbiology, College of Fisheries, Mangalore -575 002, India Received in revised form 16 March 2000; accepted 12 October 2000
Abstract Biofilm formation by two poultry isolates of Salmonella on three commonly used food contact surfaces viz plastic, cement and stainless steel were studied. Biofilm formation of both the isolates showed a similar trend with the highest density being on plastic followed by cement and steel. Salmonella weltevreden formed biofilm with a cell density of 3.4 3 10 7 , 1.57 3 10 6 and 3 3 10 5 cfu / cm 2 on plastic, cement and steel respectively while Salmonella FCM 40 biofilm on plastic, cement and steel were of the order of 1.2 3 10 7 , 4.96 3 10 6 and 2.23 3 10 5 cfu / cm 2 respectively. The sensitivity of the biofilm cells grown on these surfaces to different levels of two sanitizers namely hypochlorite and iodophor for varying exposure times was studied. Biofilm cells offered greater resistance when compared to their planktonic counterparts. Such biofilm cells in a food processing unit are not usually removed by the normal cleaning procedure and therefore could be a source of contamination of foods coming in contact with such surfaces. 2001 Elsevier Science B.V. All rights reserved. Keywords: Biofilm; Salmonella; Sanitizers; Hypochlorite; Iodophor
1. Introduction Microbial biofilms are attracting attention of scientists in different areas such as the medical field, aquatic environment, food processing industries etc. Microbial biofilms may be detrimental and undesirable in food processing premises. Biofilms by pathogenic bacteria such as Salmonella (Jones and Bradshaw, 1996; Dhir and Dodd, 1995; Humphery et al., 1995; Somers et al., 1994), Klebsiella (Jones and *Corresponding author. Tel.: 1 91-824-436-384. E-mail address: [email protected] (I. Karunasagar).
Bradshaw, 1996; Morin et al., 1996), Pseudomonas (Brown et al., 1995), Campylobacter and enterohaemorrhagic E.coli (Somers et al., 1994) and Listeria (Mafu et al., 1990; Ren and Frank, 1993) have been reported. Such biofilms could be a continuous source of contamination to foods coming in contact with them when formed on contact surfaces. Increased resistance of biofilm cells to antibacterial agents and sanitizers have also been observed (Costerton et al., 1987; Costerton and Lapin-Scott, 1989; Carpentier and Cerf, 1993; Ren and Frank, 1993). Salmonella are a group of food poisoning organisms which are of considerable significance to the
0168-1605 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00466-9
368
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
food processing industry. This study was undertaken to understand the ability of Salmonella to form biofilms on potential food contact surfaces. The resistance or otherwise of biofilm and planktonic cells to sanitizers such as iodophor and chlorine was investigated.
2. Materials and methods
2.1. Test organisms Two cultures of Salmonella isolated from poultry meat were used in this study. One culture was identified as Salmonella weltevreden and the other unspeciated one (due to minor variations in the biochemical characters) was designated as Salmonella FCM 40.
2.2. Biofilm development Three types of material namely plastic (high density polyethylene, HDPE), cement and stainless steel were used to develop the biofilm. The concrete slabs (4 cm 2 , locally made) were scrubbed with a brush and washed well with HPLC grade water. Stainless steel coupons (4 cm 2 , Bharath Steel Supplies, Mangalore, India) were cleaned with acetone to remove grease and were etched by submerging in 5N HCl for 15 min, cleaned in detergent solution and finally rinsed in HPLC grade water. The HDPE coupons (4 cm 2 , Brite plastics, Mumbai) were cleaned with detergent and rinsed with Type 1 reagent grade water. The prepared samples were placed individually in 250 ml glass beakers and 100 ml of 0.2% (w / v) tryptone soy broth (TSB, HiMedia, Mumbai, India) were added. Experiments were conducted wherein two samples of the same type viz plastic, cement or steel were placed in 1000 ml glass beakers and 200 ml 0.2%(w / v) TSB were added. Salmonella strains were grown in TSB for 24 h at 378C and 2 ml of this culture was added to the beakers with 0.2% (w / v) TSB and the samples. After incubation at ambient temperature (28628C) for 48 h, the samples were aseptically removed, washed in sterile phosphate buffer saline (PBS, pH 7.4) to remove unattached cells and placed in beakers with fresh sterile 0.2% (w / v)TSB (Ren and Frank, 1993).
This procedure was repeated five times every alternate day to complete the biofilm formation.
2.3. Enumeration of biofilm cells To enumerate biofilm cells after ten days of incubation, the samples were washed with sterile PBS to remove unattached cells and the biofilm cells were removed by swabbing with sterile cotton swabs. The swabs were transferred to 100 ml physiological saline (0.85% NaCl, w / v prepared in the laboratory) shaken vigorously and enumerated by standard spread plate technique. Tryptone soy agar (TSA, HiMedia, Mumbai, India) was used for enumeration and plates were incubated at 378C for 48 h. To test the sensitivity of biofilm cells to sanitizers namely iodophor (Asian catalysts and chemicals, Bangalore, Active compound I 2 ) and hypochlorite (BDH, Active compound Cl 2 ), the samples were dipped in solutions containing 10, 20, 50 and 100 ppm of available Cl 2 solution and 1, 10, 20 and 50 ppm of available I 2 solution for 5, 10, 15, 20 and 25 min. The substrates were then transferred to neutralising solution (0.1 M Na 2 S 2 O 3 ) for 30 s, rinsed with PBS and cells were enumerated after swabbing as described above. The limit of detection by this method was 25 cells / cm 2 .
2.4. Sensitivity of planktonic cells Sensitivity of planktonic cells to sanitizers was tested as follows. 24 h old culture of the two species in TSB were centrifuged and the cell pellets were resuspended in same volume of sterile PBS. An aliquot was removed for enumeration by serial dilution and plated on TSA. The plates were incubated at 378C for 48 h. Chlorine at 10 ppm and Iodine at 1 ppm levels were added to the PBS suspension of Salmonella cells. Aliquots were taken at 5 and 10 min, serially diluted and plated to enumerate the cells.
3. Results
3.1. Sensitivity of Salmonella biofilm to chlorine Salmonella weltevreden formed biofilm on plastic with a cell density of 3.4 3 10 7 cfu / cm 2 . Exposure
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
to 10 ppm Cl 2 up to 25 min brought about less than 2 log reduction in counts. Similarly, counts ranging from 8.0 3 10 4 to 2 3 10 4 cells / cm 2 were observed after 25 min exposure to 20 ppm and 50 ppm Cl 2 respectively. Exposure to 100 ppm Cl 2 for 5, 10 and 15 min resulted in a 3, 4 and 5 log reduction in the cell density respectively. Biofilm cells of S.weltevreden could not be detected on plastic after exposure
369
to 100 ppm Cl 2 for 20 min (Fig. 1a). On cement S.weltevreden formed biofilm with a cell density of 1.57 3 10 6 cfu / cm 2 . The counts after exposure to 100 ppm Cl 2 for 5, 10 and 20 min were 2.16 3 10 3 , 8.72 3 10 2 and 4.64 3 10 2 respectively. There was a 3 log reduction in the biofilm cell count on cement when exposed for 25 min to 10, 20 and 50 ppm Cl 2 (Fig. 1b). The biofilm formed by S.weltevreden on
Fig. 1. Effect of chlorine on S.weltevreden biofilm on different surfaces (a) plastic (b) cement (c) stainless steel.
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B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
steel had a cell density of 3 3 10 5 cfu / cm 2 . Here the biofilm cells could survive a treatment of 100 ppm Cl 2 for only 15 min. Biofilm cells survived 25 min treatment to 10, 20 and 50 ppm Cl 2 with cell 4 4 numbers being 5.42 3 10 , 1.10 3 10 and 3.92 3 3 2 10 cfu / cm respectively (Fig. 1c). As in the case of S.weltevreden, the cell density of biofilm formed by Salmonella FCM 40 was highest on plastic followed by cement and steel. Cell density 7 2 of biofilm on plastic was 1.2 3 10 cfu / cm . Biofilm cells of Salmonella FCM 40 on plastic were found to be resistant to 10, 20, 50 and 100 ppm of Cl 2 with cell counts of 3.62 3 10 4 , 2.29 3 10 4 , 1.12 3 10 4 and 4.2 3 10 3 cfu / cm 2 respectively after 25 min exposure (results not shown). Salmonella FCM 40 biofilm on cement had a cell density of 4.96 3 10 5 cfu / cm 2 . Biofilm cells of Salmonella FCM 40 were partially inactivated after treatment with 10, 20, and 50 ppm Cl 2 for 25 min with cell counts being 1.36 3 10 5 , 2.52 3 10 4 and 1.4 3 10 3 cfu / cm 2 respectively. On the other hand, Salmonella FCM 40 biofilm could not be detected on cement after exposure to 100 ppm Cl 2 for 20 min (results not shown). Salmonella FCM 40 formed biofilm on steel with a cell density of 2.09 3 10 5 cfu / cm 2 . Treatment of biofilm cells with 10 ppm Cl 2 brought about 1 log reduction in counts after 25 min exposure. 20 ppm brought about 2 log reduction after 25 min exposure. However, the inactivation was complete with 100 ppm Cl 2 after 20 min exposure (results not shown).
3.2. Sensitivity of Salmonella biofilm to iodine On plastic, S.weltevreden biofilm was found to survive all concentrations of I 2 up to 25 min with a maximum of 4 log reduction in cell counts (Fig. 2a). The biofilm cells on cement were completely inactivated after 50 ppm I 2 treatment for 25 min. The cell counts after 25 min exposure to 1, 10 and 20 ppm I 2 4 4 3 2 were 8.4 3 10 , 3.84 3 10 and 2.25 3 10 cfu / cm respectively (Fig. 2b). On steel the biofilm cells were found to be more sensitive surviving only up to 15 min exposure to 50 ppm I 2 with a cell concentration of 2.63 3 10 1 cfu / cm 2 . Biofilm cells of S.weltevreden could not be detected on steel after 20 min exposure to 50 ppm I 2 . The cell density of biofilm after 25 min exposure to 1, 10 and 20 ppm I 2 was 3 3 3 2 3.86 3 10 , 4.26 3 10 and 2.29 3 10 cfu / cm respectively (Fig. 2c).
Exposure of Salmonella FCM 40 biofilm on plastic to 1 ppm I 2 resulted in partial inactivation of cells and the survival was comparable to that after treatment with 10 ppm Cl 2 . Increase of I 2 concentration to 10 ppm only marginally increased inactivation. About 10 3 cells survived even after treatment with 20 ppm I 2 for 25 min, whereas the biofilm cells could not be detected after 25 min exposure to 50 ppm I 2 (results not shown). 25 min exposure to 1, 10 and 20 ppm I 2 on cement resulted in partial inactivation with counts being 1.7 3 10 4 , 3.58 3 10 3 and 1.86 3 10 3 cfu / cm 2 respectively. However, the biofilm cells could not be detected after treatment with 50 ppm I 2 for 25 min (results not shown). About 2 log reduction in counts could be obtained after treatment with 1 ppm I 2 for 25 min on steel surface. Increase of I 2 concentration to 10 and 20 ppm resulted in only a marginal increase in the extent of inactivation. 50 ppm iodophor totally inactivated biofilm cells with a 25 min exposure (results not shown).
3.3. Sensitivity of planktonic cells of Salmonella to chlorine and iodine Planktonic cells of S.weltevreden which were suspended in PBS to a level of 2.4 3 10 6 cells / ml were completely killed by exposure to 10 ppm Cl 2 for 10 min and a cell density of 1.9 3 10 6 cells / ml were completely killed after 5 min exposure to 10 ppm I 2 (results not shown). Planktonic cells of Salmonella FCM 40 suspended in PBS to a level of 2.26 3 10 6 were completely killed after exposure to 10 ppm Cl 2 for 10 min and 10 ppm I 2 for 5 min (results not shown). Salmonella could form biofilms on all the three substrates studied, viz. plastic, cement and steel. The cell density of Salmonella was highest on plastic followed by cement and steel reflecting the surface preference of Salmonella. Fletcher and Loeb (1979) noted that large numbers of bacteria attached to hydrophobic surfaces with little or no surface charge and moderate numbers attached to hydrophobic metals with positive charge or neutral charge and very few attached to hydrophilic negatively charged substrates. The importance of hydrophobicity in bacterial attachment has been highlighted by other investigators also (Characklis, 1990). Variation in biofilm density depending on surface was also
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
371
Fig. 2. Effect of iodine on S.weltevreden biofilm on different surfaces (a) plastic (b) cement (c) stainless steel.
reported by Karunasagar et al. (1996) with Vibrio harveyi. The results presented here show that biofilm cells of Salmonella are much more resistant to sanitizers compared to planktonic cells. The efficiency of
biofilm formation varied depending on the surface and the resistance to treatment with sanitizers also varied depending on the surfaces. Biofilm cells on stainless steel were most sensitive and those on plastic most resistant. These observations were con-
372
B. Joseph et al. / International Journal of Food Microbiology 64 (2001) 367 – 372
sistent with both strains of Salmonella studied. However, minor variations between strains were also observed. For instance, biofilm cells of Salmonella FCM 40 on plastic were not inactivated even after 25 min treatment with 100 ppm Cl 2 while in the case of S.weltevreden, treatment with the same level of Cl 2 inactivated a similar cell density in 20 min. Formation of biofilm by Salmonella on various surfaces has implications for the food processing industry. Salmonella strains entering the food processing environment either through the meat or through carriers handling meat may survive in the premises by forming biofilms on various surfaces. HDPE surfaces are generally available on crates which are used to carry food products. Sometimes the contact time between foods and HDPE surface may be 24–48 h depending on the processing conditions including design of equipment and cleaning and disinfection regimes. Once a biofilm is formed, this could be a source of contamination for foods passing through the same processing line. The results of the study have shown that Salmonella spp can form biofilms on food contact surfaces and that these biofilm cells are much more resistant to sanitizers compared to planktonic cells. Such biofilm cells which are not removed during normal cleaning procedure in a food processing unit could be a source of contamination for foods. The cleaning and disinfection protocols in food processing units should therefore consider Salmonella biofilms.
References Brown, M.L., Henry, C.A., Gauthier, J.J., 1995. Relation between Glycocalyx and Povidone-Iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms. Appl. Env. Microbiol. 61 (1), 187–193.
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