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The influence of temperature on the adhesion of mixed cultures of Staphylococcus aureus and Escherichia coli to polypropylene
Food Microbiology, 2000, 17, 361^365 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.1999.0291
ORIGINAL ARTICLE
The in£uence of temperature on the adhesion of mixed cultures of Staphylococcus aureus and Escherichia coli to polypropylene Daisy M. C. Pompermayer1 and Christine C. Gaylarde2,*
The adhesion of Staphylococcus aureus and Escherichia coli, in mixed cultures, to polypropylene surfaces was evaluated at 128C and 308C. The micro-organisms were isolated from a chicken carcass and cultured in an aqueous extract, prepared from the same carcass, for the production of bio¢lms on polypropylene coupons. Adhered cells were counted by epi£uorescence microscopy with acridine orange staining. Escherichia coli adhered in greater numbers to the coupons than S. aureus at both temperatures. Staphylococcus aureus adhered better at 128C than at 308C, while the reverse was true for E. coli. At 308C, there was no increase in the number of adherent cells of S. aureus over 8 h, while E. coli increased from a median of 5?0^19?0 per microscope ¢eld. At 128C, the major increase in adherent cell numbers for both species occurred between 2 and 4 h, so that leaving cleaning until 8 h, as is common, would not result in greatly increased bio¢lms, 2-hourly cleansing is clearly unrealistic. However, total adherent cell numbers were the same at 128 and 308C between 4 and 6 h incubation. Hence it seems that reduced temperature has little to o¡er for restricting bio¢lm formation on polypropylene work sur# 2000 Academic Press faces in a well-run food processing plant.
Introduction The adhesion of bacterial cells to food processing equipment can lead to a variety of problems, such as corrosion of metal surfaces (Beech and Gaylarde 1989) and cross-contamination of processed foods (Carpentier and Cerf 1993). The latter is especially important because of the hazard to human health. The processing of chickens for human consumption involves a number of steps, each of which involves the risk of microbial contamination, derived from the birds themselves or from the *Corresponding author. Fax: +55 51 316 6029. E-mail: [email protected] 0740 -0020/00/040361 +05 $35.00/0
equipment and handlers. Banwart (1989) emphasised that it is during processing that the microbial loading of chicken skin can increase dramatically and it has been shown that microorganisms adhering to tissues can be transferred to inert surfaces even under conditions which are not suitable for microbial growth (Schwach and Zottola 1982). If hygienic precautions are not adequate, a single bird carrying human pathogenic and food spoilage microorganisms could infect a huge number of carcasses via processing equipment surfaces, leading to reduced shelf life and economic and health problems. Factors which in£uence microbial adhesion to inert surfaces include growth phase of the # 2000 Academic Press
Received: 23 October1998 1
Science and Technology Foundation (CIENTEC), Porto Alegre, RS, Brazil. 2 MIRCEN, Dept. of Soils, UFRGS, Cx.P. 776, Porto Alegre, RS, 91000-970, Brazil
362 D. M. C. Pompermayer and C. C. Gaylarde
cells (Stone and Zottola 1985), the type and properties of the inert material (BoulangePetermann 1996), the presence of organic material (Zottola and Sasahara 1994), environmental pH and temperature (Herald and Zottola 1988a,b). There is, however, little information on the e¡ect of temperature on the adhesion of pathogenic micro-organisms to surfaces used in the food industry. Czechowski (1990) showed that bacteria adhere almost immediately to surfaces with which they come into contact, even at low temperatures (58C, 118C). However, the consolidation and subsequent production of biomass in the surface bio¢lm are timedependent (Notermans et al. 1991) and growth of most micro-organisms will be delayed at low temperatures. The processes of cleaning and sanitisation of surfaces are more di¤cult in the presence of adhered micro-organisms. This has become problematical with the increased use of automated procedures and complex equipment, which provides a variety of niches for bacterial adhesion. Stainless steel is the most frequently used material for food processing equipment, but it has several disadvantages, including high cost and susceptibility to corrosion. Polypropylene does not have these problems and is becoming more popular in the industry for the construction of tanks, pipework, accessories and cutting surfaces. Thus it is important to assess the likelihood of cross-contamination of foods via this route, by determining the rate of bacterial adhesion and bio¢lm formation on polypropylene surfaces. This article reports the results of experiments to measure the adhesion of two important food pathogens, Staphylococcus aureus and Escherichia coli, to polypropylene at 128C and 308C. These temperatures were chosen for their relevance to Brazilian food processing
Table 1. Physico-chemical
characteristics of chicken extract solution used as culture medium Parameter
Moisture content Total protein Mineral content Fats pH
Value 98?67% 1?12% 0?21% 0?023% 6?2
industries, where procedures may be carried out at room temperature (25^358C) or in cooled environments at 128C.
Materials and Methods Micro-organisms Staphylococcus aureus and E. coli were isolated from a chilled chicken carcass purchased in a supermarket. Isolation and identi¢cation procedures were those laid down in the Bacteriological Analytical Manual (FDA 1995). The bacteria were cultured in a sterile chicken extract, prepared as follows. Raw chicken breast (250 g) was homogenized in 2 l sterile distilled water in a stomacher. The resultant suspension was ¢ltered to remove large particles and then membrane sterilized (0?22 mm pore size). It was stored frozen, in small aliquots, until used in the experiments. The physico-chemical properties of this culture medium are shown in Table 1. Cultures of each micro-organism were prepared by inoculating 5 ml of nutrient broth containing 108 cells ml71 (E. coli) or 109 cells ml71 (S. aureus) into 20 ml of sterile chicken extract and incubating at 358C for 18 h, to the stationary phase of growth. The ¢nal cell concentration (1011 ml71) was determined by plate counts and optical density used to standardize the inocula for the experiments, using a previously prepared calibration curve. The two standard cultures were mixed in equal proportions to form a mixed suspension.
Adhesion to polypropylene Polypropylene coupons (3 mm61 cm62 cm) were immersed for 30 min at room temperature (c. 258C) in neutral detergent (Fliper, Fliper Com. E Ind. de Produtos de Limpeza Brazil, 30 ml in 1 l water), washed well in distilled water to remove all the detergent and dried at 608C for 2 h prior to autoclaving. They were placed in the mixed bacterial cultures and incubated at 128C or 308C for up to 8 h. After incubation they were removed, dipped three times into 10 ml sterile distilled water at room temperature to remove loosely adherent cells, and left to dry
Adhesion of bacteria to polypropylene 363
at room temperature. Three replicate coupons were used for each treatment time.
Analysis of adherent cells Coupons were stained with acridine orange (0?025%, 3 min) and examined using an epi£uorescence microscope (Zeiss MPC64). For each coupon, at least 300 orange-staining cells in at least 10 ¢elds of view were counted, using an oil immersion objective and a ¢nal magni¢cation of 61600. The area of the ¢eld of view was 8?6661073 mm2. Results were analysed statistically using the Kruskal^Wallis nonparametric ANOVA, as recommended by Woolfson (1993). A probability value (P) of 50?01 was accepted as indicating a signi¢cant di¡erence.
Results and Discussion In the mixed cultures employed in this investigation, both S. aureus and E. coli adhered to polypropylene surfaces in low numbers (Figs 1 and 2), in comparison with the concentrations seen with pure cultures (Pompermayer and Gaylarde 1998). In pure culture experiments, carried out under the same conditions as these currently reported, a median of 45?5 cells/¢eld and six cells/¢eld were found for E. coli and S. aureus, respectively, after 2 h at 308C, while in the mixed cultures the respective numbers were 5?0 and 2?0 cells/¢eld. These numbers are signi¢cantly di¡erent at the 1% level. This
Figure 1. Adhesion of E. coli to polypropylene at
12 and 308C. Area of ¢eld 8?6661073 mm2. & 308C+18C, & 128C+18C.
Figure 2. Adhesion of S. aureus to polypropy-
lene at 12 and 308C. Area of ¢eld 8?6661073 mm2. & 308C+18C, & 128C+18C.
indicates that, as suggested by McEldowney and Fletcher (1987), there is a competition for attachment, which reduces the numbers of each species adhering to the surface. It is important to obtain further information on adhesion in controlled mixtures of micro-organisms in order to understand the factors in£uencing this process. In the studies reported here, the two bacteria could be readily di¡erentiated on the surface because of their di¡erent morphologies. Where this is not the case, techniques such as speci¢c immuno£uorescent or £uorescent gene probe labelling, like that employed by Ramsing et al. (1993) on trickling ¢lter bio¢lms, could be used. The overall adherence of E. coli was signi¢cantly greater than S. aureus at both temperatures (Figs 1 and 2, P50?01).This was expected, in view of the published superiority of Gram- negative cells to adhere to inert surfaces (Speers and Gilmour 1985; Pompermayer and Gaylarde, 1998). Gilbert et al. (1991) have shown greater adhesion of E. coli than S. epidermidis on glass surfaces and related this to surface electro-negativity of the cells. Another factor to be taken into account is that E. coli cells have a shorter generation time, which would enable them to develop and maintain their dominance (Oberhofer and Frazier, cited in Pelczar et al., 1981). Banks and Bryers (1991) suggest that the predominant organisms in a bio¢lm will be those with the highest growth rate, although these cells will never totally exclude more slowly growing organisms. At 128C the population of E. coli on the surfaces was generally lower than at the higher
364 D. M. C. Pompermayer and C. C. Gaylarde
temperature and more Gram-positive cells were present (Figs 1 and 2). The reduced adhesion of Gram-negative cells at lower temperatures has also been shown for Pseudomonas £uorescens (Czechowski 1990) and, indeed, would be expected for all organisms because of the e¡ects of temperature on chemical and physical adsorption processes, medium viscosity and microbial physiology (Fletcher 1977). Thus it is surprising to note that the adhesion of S. aureus to polypropylene was greater at 128C than at 308C (Fig. 2; P50?01).This positive e¡ect of such low temperatures on bacterial adhesion has not previously been reported, although Herald and Zottola (1988a,b) showed that Listeria monocytogenes and Yersinia enterocolitica adhered to stainless steel coupons in greater numbers at 218C than at 358C. These authors concluded that adhesion was directly related to £agellar movement and the production of exopolymer. As S. aureus is non-motile, the ¢rst of these suggestions can be ruled out. Certain bacteria are known to produce more exopolymer under conditions of stress, such as sub-optimal temperatures (Costerton et al. 1978), but this has not been reported for S. aureus. Future investigations to elucidate this phenomenon in S. aureus might centre on gene expression. It is known that sudden decreases in temperature can result in a reduction in the expression of some prokaryotic genes and induction of others (Singleton 1997) and that this may result in changes within the bacterial cell envelope (Mastronicolis et al. 1998). Such changes could in£uence adhesion in a positive or negative fashion. Although the molecular basis of this altered adhesion is unknown, it is an important observation from the point of view of bio¢lm formation in the food industry. Not only were the numbers of adhered cells of S. aureus higher at 128C than at 308C, but they increased signi¢cantly between 2 and 4 h at the former temperature, while remaining constant at 308C (Fig. 2). Czechowski (1990) studied the adhesion of P. £uorescens to various inert substrates and found that adhesion followed a biphasic pattern on stainless steel at all temperatures used (58C, 118C and 258C), but on the synthetic rubber Buna-N and on te£on adsorption was linear with time at 58C and 258C, showing the
importance of the nature of the substrate on adsorption kinetics at di¡erent temperatures. In the current studies, only the adsorption of E. coli at 308C showed linearity. In all other cases where adherent cell numbers increased with time, a rapid increase up to 4 h was followed by a plateau up to the end of the observation time (8 h). The apparent reduction in adherent numbers of S. aureus at 6 h (128C) is clearly erroneous and, in fact, was caused by the presence in the data set of one coupon with very low adherence. There are obvious implications of these results for the chicken processing industry. The organisms used in the experiments were isolated directly from a chicken carcass and adhesion tests were carried out in a chicken extract medium. This system provides a reasonably simple, yet realistic model for investigating the e¡ects of environmental conditions, materials, cleansing and sanitizing treatments to be used in the food industry. The use of a high bacterial inoculum (well above that which would normally be expected in the real situation) decreases the time required for the formation of a substantial bio¢lm, enabling `accelerated' tests to be carried out. Experimental results obtained using pure cultures, even if the physico-chemical conditions approximate to those used in the factory, have been shown to be misleading if compared to a more realistic situation, with mixtures of micro-organisms. It is important to note that, even at 128C, signi¢cant bio¢lms form on polypropylene surfaces after 4 h. This is especially true for S. aureus. The lower adherent E. coli populations at 128C could be thought to indicate the superiority of these conditions for the avoidance of cross-contamination, but the total cell numbers adhering at this temperature are, in general, equal to, or greater than those at 308C. Only if cleaning and sanitization are carried out after 2 h (an unrealistic expectation in most factories), will the process be rendered easier at 128C because of the presence of fewer adhered organisms, although the reduced temperature could, of course, also a¡ect the ef¢cacy of the sanitising agent used. The importance of this work lies in its extension of our knowledge of potential
Adhesion of bacteria to polypropylene 365
contamination conditions in the chicken processing industry. Cross-contamination is one of the major worries of the food industry. Micro-organisms from raw materials, the environment, the workforce, or insects and rodents gaining entry to the plant can become endemic in the equipment, surviving the cleaning and sanitization processes (Notermans et al. 1991). Our results reenforce the fact that, even when working at lower temperatures, it is important to use good quality raw materials, well trained sta¡ and frequent (and e¤cient) cleaning routines.
References Banks, M. K. and Bryers, J. D. (1991) Bacterial species dominance within a binary culture bio¢lm. Appl. Environ. Microbiol. 57, 1974^1979. Banwart, G. J. (1989) Sources of micro-organisms. In Basic Food Microbiology Ed. G. J. Banwart, New York,Van Nostrand Reinhold, pp. 165^194. Beech, I. B. and Gaylarde, C. C. (1989) Adhesion of Desulfovibrio desulfuricans and Pseudomonas £uorescens to mild steel surfaces. J. Appl. Bacteriol. 67, 201^207. Boulange-Petermann, L. (1996) Processes of bioadhesion on stainless steel surfaces and cleanability: a review with special reference to the food industry. Biofouling. 10, 275^300. Carpentier, B. and Cerf, O. (1993) Bio¢lms and their consequences, with particular reference to hygiene in the food industry. J. Appl. Bacteriol. 75, 499^511. Costerton, J.W., Geesey, G. G. and Cheng, K. J. (1978) How bacteria stick. Sci. Amer. 238, 86^95. Czechowski, M. H. (1990) Gasket and stainless steel surface sanitation: environmental parameters affecting bacterial attachment. Austr. J. Dairy Technol. pp. 38^39. Fletcher, M. (1977) The e¡ects of culture concentration and age, time and temperature on bacterial attachment to polystyrene. Can. J. Microbiol. 23, 1^6. Food and Drug Administration. (1995) Bacteriological analytical manual. 8th edn. Washington, Association of O¤cial Analytical Chemists, p. 12. Gilbert, P., Evans, D. J., Evans, E., Duguid, I. G. and Brown, M. R. W. (1991) Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis. J. Appl. Bacteriol. 71, 72^77. Herald, P. J. and Zottola, E. A. (1988a) Attachment of Listeria monocytogenes to stainless steel surfaces at various temperatures and pH values. J. Food Sci. 53, 1549^1552.
Herald, P. J. and Zottola, E. A. (1988b) Scanning electron microscopic examination of Yersinia enterocolitica attached to stainless steel at selected temperatures and pH values. J. Food Prot. 51, 445^448. McEldowney, S. and Fletcher, M. (1987) Adhesion of bacteria from mixed cell suspension to solid surfaces. Arch. Microbiol. 148, 57^62. Mastronicolis, S. K., German, J. B., Megoulas, N., Petrou, E., Foka, P. and Smith, G. M. (1998) In£uence of cold shock on the fatty-acid composition of di¡erent lipid classes of the food-borne pathogen Listeria monocytogenes. Food Microbiol. 15, 299^306. Notermans, S., Dormans, J. and Mead, G. C. (1991) Contribution of surface attachment to the establishment of micro-organisms in food processing plant: a review. Biofouling. 5, 21^36. Pelczar, M. J., Reid, R. and Chan, E. C. S. (1981) Fundamentos da Ecologia Microbiana. In Microbiologia Vol. 2 (translated by M. A. May Pereira and M. R. S. Borges), pp. 807^829. Sa¬o Paulo, McGraw-Hill, Brazil. Pompermayer, D. C. and Gaylarde, C. (1998) Assessment and control of adhesion of bacterial cells to polypropylene surfaces. In LABS 3 ö Biodegradation and Biodeterioration in Latin America (Eds. C. C. Gaylarde, T. C. P. Barbosa and N. H. Gabilan) paper No. 71. UK, The British Phycological Society. Ramsing, N. B., KÏhl, M. and Jorgensen, B. B. (1993) Distribution of sulfate reducing bacteria, oxygen and hydrogen sul¢de in photosynthetic bio¢lms determined by oligonucleotide probes and microelectrodes. Appl. Environ. Microbiol. 59, 3840^384. Schwach, T. S. and Zottola, E. A. (1982) Use of scanning electron microscopy to demonstrate microbial attachment to beef and beef contact surfaces. J. Food Sci. 47, 1401^1405. Singleton, P. (1997) Molecular biology I: genes and gene expression. In Bacteria in Biology, Biotechnology and Medicine, (Ed. P. Singleton) pp. 97^139. New York, J.Wiley. Speers, J. G. S. and Gilmour, A. (1985) The in£uence of milk and milk components on the attachment of bacteria to farm dairy equipment surfaces. J. Appl. Bacteriol. 59, 325^332. Stone, L. S. and Zottola, E. A. (1985) Relationship between the growth phase of Pseudomonas fragi and its attachment to stainless steel. J. Food Sci. 50, 957^960. Woolfson, A. D. (1993) The statistical evaluation of adherence assays. In Microbial Bio¢lms: Formation and Control. (Eds. S. P. Denyer, S. P. Gorman, M. Sussman) pp. 315^325. London, Blackwell Scienti¢c. Zottola, E. A. and Sasahara, K. C. (1994) Microbial bio¢lms in the food processing industry ö Should they be a concern? Internat. J. Food Microbiol. 23, 125^148.
doi:10.1006/fmic.1999.0291
ORIGINAL ARTICLE
The in£uence of temperature on the adhesion of mixed cultures of Staphylococcus aureus and Escherichia coli to polypropylene Daisy M. C. Pompermayer1 and Christine C. Gaylarde2,*
The adhesion of Staphylococcus aureus and Escherichia coli, in mixed cultures, to polypropylene surfaces was evaluated at 128C and 308C. The micro-organisms were isolated from a chicken carcass and cultured in an aqueous extract, prepared from the same carcass, for the production of bio¢lms on polypropylene coupons. Adhered cells were counted by epi£uorescence microscopy with acridine orange staining. Escherichia coli adhered in greater numbers to the coupons than S. aureus at both temperatures. Staphylococcus aureus adhered better at 128C than at 308C, while the reverse was true for E. coli. At 308C, there was no increase in the number of adherent cells of S. aureus over 8 h, while E. coli increased from a median of 5?0^19?0 per microscope ¢eld. At 128C, the major increase in adherent cell numbers for both species occurred between 2 and 4 h, so that leaving cleaning until 8 h, as is common, would not result in greatly increased bio¢lms, 2-hourly cleansing is clearly unrealistic. However, total adherent cell numbers were the same at 128 and 308C between 4 and 6 h incubation. Hence it seems that reduced temperature has little to o¡er for restricting bio¢lm formation on polypropylene work sur# 2000 Academic Press faces in a well-run food processing plant.
Introduction The adhesion of bacterial cells to food processing equipment can lead to a variety of problems, such as corrosion of metal surfaces (Beech and Gaylarde 1989) and cross-contamination of processed foods (Carpentier and Cerf 1993). The latter is especially important because of the hazard to human health. The processing of chickens for human consumption involves a number of steps, each of which involves the risk of microbial contamination, derived from the birds themselves or from the *Corresponding author. Fax: +55 51 316 6029. E-mail: [email protected] 0740 -0020/00/040361 +05 $35.00/0
equipment and handlers. Banwart (1989) emphasised that it is during processing that the microbial loading of chicken skin can increase dramatically and it has been shown that microorganisms adhering to tissues can be transferred to inert surfaces even under conditions which are not suitable for microbial growth (Schwach and Zottola 1982). If hygienic precautions are not adequate, a single bird carrying human pathogenic and food spoilage microorganisms could infect a huge number of carcasses via processing equipment surfaces, leading to reduced shelf life and economic and health problems. Factors which in£uence microbial adhesion to inert surfaces include growth phase of the # 2000 Academic Press
Received: 23 October1998 1
Science and Technology Foundation (CIENTEC), Porto Alegre, RS, Brazil. 2 MIRCEN, Dept. of Soils, UFRGS, Cx.P. 776, Porto Alegre, RS, 91000-970, Brazil
362 D. M. C. Pompermayer and C. C. Gaylarde
cells (Stone and Zottola 1985), the type and properties of the inert material (BoulangePetermann 1996), the presence of organic material (Zottola and Sasahara 1994), environmental pH and temperature (Herald and Zottola 1988a,b). There is, however, little information on the e¡ect of temperature on the adhesion of pathogenic micro-organisms to surfaces used in the food industry. Czechowski (1990) showed that bacteria adhere almost immediately to surfaces with which they come into contact, even at low temperatures (58C, 118C). However, the consolidation and subsequent production of biomass in the surface bio¢lm are timedependent (Notermans et al. 1991) and growth of most micro-organisms will be delayed at low temperatures. The processes of cleaning and sanitisation of surfaces are more di¤cult in the presence of adhered micro-organisms. This has become problematical with the increased use of automated procedures and complex equipment, which provides a variety of niches for bacterial adhesion. Stainless steel is the most frequently used material for food processing equipment, but it has several disadvantages, including high cost and susceptibility to corrosion. Polypropylene does not have these problems and is becoming more popular in the industry for the construction of tanks, pipework, accessories and cutting surfaces. Thus it is important to assess the likelihood of cross-contamination of foods via this route, by determining the rate of bacterial adhesion and bio¢lm formation on polypropylene surfaces. This article reports the results of experiments to measure the adhesion of two important food pathogens, Staphylococcus aureus and Escherichia coli, to polypropylene at 128C and 308C. These temperatures were chosen for their relevance to Brazilian food processing
Table 1. Physico-chemical
characteristics of chicken extract solution used as culture medium Parameter
Moisture content Total protein Mineral content Fats pH
Value 98?67% 1?12% 0?21% 0?023% 6?2
industries, where procedures may be carried out at room temperature (25^358C) or in cooled environments at 128C.
Materials and Methods Micro-organisms Staphylococcus aureus and E. coli were isolated from a chilled chicken carcass purchased in a supermarket. Isolation and identi¢cation procedures were those laid down in the Bacteriological Analytical Manual (FDA 1995). The bacteria were cultured in a sterile chicken extract, prepared as follows. Raw chicken breast (250 g) was homogenized in 2 l sterile distilled water in a stomacher. The resultant suspension was ¢ltered to remove large particles and then membrane sterilized (0?22 mm pore size). It was stored frozen, in small aliquots, until used in the experiments. The physico-chemical properties of this culture medium are shown in Table 1. Cultures of each micro-organism were prepared by inoculating 5 ml of nutrient broth containing 108 cells ml71 (E. coli) or 109 cells ml71 (S. aureus) into 20 ml of sterile chicken extract and incubating at 358C for 18 h, to the stationary phase of growth. The ¢nal cell concentration (1011 ml71) was determined by plate counts and optical density used to standardize the inocula for the experiments, using a previously prepared calibration curve. The two standard cultures were mixed in equal proportions to form a mixed suspension.
Adhesion to polypropylene Polypropylene coupons (3 mm61 cm62 cm) were immersed for 30 min at room temperature (c. 258C) in neutral detergent (Fliper, Fliper Com. E Ind. de Produtos de Limpeza Brazil, 30 ml in 1 l water), washed well in distilled water to remove all the detergent and dried at 608C for 2 h prior to autoclaving. They were placed in the mixed bacterial cultures and incubated at 128C or 308C for up to 8 h. After incubation they were removed, dipped three times into 10 ml sterile distilled water at room temperature to remove loosely adherent cells, and left to dry
Adhesion of bacteria to polypropylene 363
at room temperature. Three replicate coupons were used for each treatment time.
Analysis of adherent cells Coupons were stained with acridine orange (0?025%, 3 min) and examined using an epi£uorescence microscope (Zeiss MPC64). For each coupon, at least 300 orange-staining cells in at least 10 ¢elds of view were counted, using an oil immersion objective and a ¢nal magni¢cation of 61600. The area of the ¢eld of view was 8?6661073 mm2. Results were analysed statistically using the Kruskal^Wallis nonparametric ANOVA, as recommended by Woolfson (1993). A probability value (P) of 50?01 was accepted as indicating a signi¢cant di¡erence.
Results and Discussion In the mixed cultures employed in this investigation, both S. aureus and E. coli adhered to polypropylene surfaces in low numbers (Figs 1 and 2), in comparison with the concentrations seen with pure cultures (Pompermayer and Gaylarde 1998). In pure culture experiments, carried out under the same conditions as these currently reported, a median of 45?5 cells/¢eld and six cells/¢eld were found for E. coli and S. aureus, respectively, after 2 h at 308C, while in the mixed cultures the respective numbers were 5?0 and 2?0 cells/¢eld. These numbers are signi¢cantly di¡erent at the 1% level. This
Figure 1. Adhesion of E. coli to polypropylene at
12 and 308C. Area of ¢eld 8?6661073 mm2. & 308C+18C, & 128C+18C.
Figure 2. Adhesion of S. aureus to polypropy-
lene at 12 and 308C. Area of ¢eld 8?6661073 mm2. & 308C+18C, & 128C+18C.
indicates that, as suggested by McEldowney and Fletcher (1987), there is a competition for attachment, which reduces the numbers of each species adhering to the surface. It is important to obtain further information on adhesion in controlled mixtures of micro-organisms in order to understand the factors in£uencing this process. In the studies reported here, the two bacteria could be readily di¡erentiated on the surface because of their di¡erent morphologies. Where this is not the case, techniques such as speci¢c immuno£uorescent or £uorescent gene probe labelling, like that employed by Ramsing et al. (1993) on trickling ¢lter bio¢lms, could be used. The overall adherence of E. coli was signi¢cantly greater than S. aureus at both temperatures (Figs 1 and 2, P50?01).This was expected, in view of the published superiority of Gram- negative cells to adhere to inert surfaces (Speers and Gilmour 1985; Pompermayer and Gaylarde, 1998). Gilbert et al. (1991) have shown greater adhesion of E. coli than S. epidermidis on glass surfaces and related this to surface electro-negativity of the cells. Another factor to be taken into account is that E. coli cells have a shorter generation time, which would enable them to develop and maintain their dominance (Oberhofer and Frazier, cited in Pelczar et al., 1981). Banks and Bryers (1991) suggest that the predominant organisms in a bio¢lm will be those with the highest growth rate, although these cells will never totally exclude more slowly growing organisms. At 128C the population of E. coli on the surfaces was generally lower than at the higher
364 D. M. C. Pompermayer and C. C. Gaylarde
temperature and more Gram-positive cells were present (Figs 1 and 2). The reduced adhesion of Gram-negative cells at lower temperatures has also been shown for Pseudomonas £uorescens (Czechowski 1990) and, indeed, would be expected for all organisms because of the e¡ects of temperature on chemical and physical adsorption processes, medium viscosity and microbial physiology (Fletcher 1977). Thus it is surprising to note that the adhesion of S. aureus to polypropylene was greater at 128C than at 308C (Fig. 2; P50?01).This positive e¡ect of such low temperatures on bacterial adhesion has not previously been reported, although Herald and Zottola (1988a,b) showed that Listeria monocytogenes and Yersinia enterocolitica adhered to stainless steel coupons in greater numbers at 218C than at 358C. These authors concluded that adhesion was directly related to £agellar movement and the production of exopolymer. As S. aureus is non-motile, the ¢rst of these suggestions can be ruled out. Certain bacteria are known to produce more exopolymer under conditions of stress, such as sub-optimal temperatures (Costerton et al. 1978), but this has not been reported for S. aureus. Future investigations to elucidate this phenomenon in S. aureus might centre on gene expression. It is known that sudden decreases in temperature can result in a reduction in the expression of some prokaryotic genes and induction of others (Singleton 1997) and that this may result in changes within the bacterial cell envelope (Mastronicolis et al. 1998). Such changes could in£uence adhesion in a positive or negative fashion. Although the molecular basis of this altered adhesion is unknown, it is an important observation from the point of view of bio¢lm formation in the food industry. Not only were the numbers of adhered cells of S. aureus higher at 128C than at 308C, but they increased signi¢cantly between 2 and 4 h at the former temperature, while remaining constant at 308C (Fig. 2). Czechowski (1990) studied the adhesion of P. £uorescens to various inert substrates and found that adhesion followed a biphasic pattern on stainless steel at all temperatures used (58C, 118C and 258C), but on the synthetic rubber Buna-N and on te£on adsorption was linear with time at 58C and 258C, showing the
importance of the nature of the substrate on adsorption kinetics at di¡erent temperatures. In the current studies, only the adsorption of E. coli at 308C showed linearity. In all other cases where adherent cell numbers increased with time, a rapid increase up to 4 h was followed by a plateau up to the end of the observation time (8 h). The apparent reduction in adherent numbers of S. aureus at 6 h (128C) is clearly erroneous and, in fact, was caused by the presence in the data set of one coupon with very low adherence. There are obvious implications of these results for the chicken processing industry. The organisms used in the experiments were isolated directly from a chicken carcass and adhesion tests were carried out in a chicken extract medium. This system provides a reasonably simple, yet realistic model for investigating the e¡ects of environmental conditions, materials, cleansing and sanitizing treatments to be used in the food industry. The use of a high bacterial inoculum (well above that which would normally be expected in the real situation) decreases the time required for the formation of a substantial bio¢lm, enabling `accelerated' tests to be carried out. Experimental results obtained using pure cultures, even if the physico-chemical conditions approximate to those used in the factory, have been shown to be misleading if compared to a more realistic situation, with mixtures of micro-organisms. It is important to note that, even at 128C, signi¢cant bio¢lms form on polypropylene surfaces after 4 h. This is especially true for S. aureus. The lower adherent E. coli populations at 128C could be thought to indicate the superiority of these conditions for the avoidance of cross-contamination, but the total cell numbers adhering at this temperature are, in general, equal to, or greater than those at 308C. Only if cleaning and sanitization are carried out after 2 h (an unrealistic expectation in most factories), will the process be rendered easier at 128C because of the presence of fewer adhered organisms, although the reduced temperature could, of course, also a¡ect the ef¢cacy of the sanitising agent used. The importance of this work lies in its extension of our knowledge of potential
Adhesion of bacteria to polypropylene 365
contamination conditions in the chicken processing industry. Cross-contamination is one of the major worries of the food industry. Micro-organisms from raw materials, the environment, the workforce, or insects and rodents gaining entry to the plant can become endemic in the equipment, surviving the cleaning and sanitization processes (Notermans et al. 1991). Our results reenforce the fact that, even when working at lower temperatures, it is important to use good quality raw materials, well trained sta¡ and frequent (and e¤cient) cleaning routines.
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