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The microbiological profile of foods in the Republic of Cyprus: 1991–2000
Food Microbiology, 2002, 19, 463^471 Available online at http://www.idealibrary.com on
doi:10.1006/yfmic.508
ORIGINAL ARTICLE
The microbiological pro¢le of foods in the Republic of Cyprus: 1991^2000 Mary Eleftheriadou*, Andri Varnava-Tello, Maria Metta-Loizidou, Andri-Silvia Nikolaou and Dina Akkelidou
In Cyprus, as part of the exercised o⁄cial food microbiological control, 28 835 of a large variety of foodstu¡s were examined during the years 1991^2000. Parameters examined included Salmonella spp., Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus,Vibrio spp., E.coli and aerobic plate count.The results indicate a prevalence of1?8% for Salmonella spp. and L. monocytogenes, with desserts ranking ¢rst as the primary vehicle for Salmonella spp. and cured meats for L. monocytogenes. S. aureus was isolated in 0?7% of the samples examined in signi¢cant, potentially hazardous levels (4104 cfu g 1), whereas B. cereus in 1% of the samples in numbers (4104 cfu g 1). The most common food vehicle for both pathogens was found to be traditional Cyprus cheese.V. parahaemolyticus and V. cholerae non-01were isolated exclusively from imported frozen, raw prawns and shrimps at isolation rates of 6?9% and 1?3%, respectively. The incidence of E. coli at levels 4102 g 1 amounted to 6% of the samples examined, with traditional Cyprus cheese being the leading food vehicle. High aerobic plate counts were found in desserts, ready-to-eat meals, sandwiches and cured, non-fermented meats. The results presented in this paper indicate for the most part the good microbiological quality achieved by the majority of the food industry in Cyprus. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction The production of safe food is and will remain the responsibility of the producer. However, the authorities need to be regularly verifying and validating, through inspection and end-product testing, the e¡ectiveness of the controls implemented by the industry, both at the site of production and at the point of sale. In Cyprus, o⁄cial microbiological control of foodstu¡s is carried out by the State General Laboratory of the Ministry of Health and is *Corresponding author. Fax: E-mail: [email protected] 0740 -0020/02/050463+09 $35.00/0
+357-22316434;
aimed at verifying compliance with legislation and the protection of public health, consumer interests and fair trade practices. The importance of microbiologically safe food in Cyprus gains added importance as Cyprus is a country attracting many tourists with triple the number of its inhabitants visiting the island every year. This paper provides a basic evaluation of the results from the microbiological examination of 28 835 samples of a large variety of foodstu¡s over a 10 -year period (1991^2000) in the Republic of Cyprus and includes data on the prevalence of speci¢c pathogens and indicator bacteria. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 7 January 2002 Food Microbiology Laboratory, State General Laboratory, Ministry of Health, 44 Kimonos street, Nicosia1451, Cyprus
464 M. Eleftheriadou et al.
Materials and Methods Sampling During the years 1991^2000, 28 835 samples of foodstu¡s were examined microbiologically to ascertain their safety and quality status. Sampling of foods was undertaken by trained sampling o⁄cers/health inspectors of the Ministry of Health, the local municipalities and the Ministry of Industry, Commerce and Tourism, in accordance with the Cypriot food legislation. The sampling took place both from the production site (industry and mass catering establishments) and from retail outlets (shops, kiosks, school canteens, supermarkets, etc.). Sampling was based mainly on vulnerability to spoilage, consumption frequency with emphasis on high-risk population groups, and known microbiological safety/quality problems. The primary sources of the samples received were: routine surveillanceFincludes sampling from shops, kiosks, supermarkets, street vendors, schools, hotels, restaurants, take away shops, cruise ships and the food industryF (56?9%), National guard (28?4%), Imports/Exports (11?7%) and miscellaneous (3%)Fincludes, apart from privately submitted samples, food poisoning investigations and consumer complaints. All the samples were transported to the laboratory under low temperature (o81C) and either examined immediately or stored at 4o C until testing which took place within maximum 24 h of sampling. Table 1 lists the types of products sampled during this period.
cessful participation in external pro¢ciency testing schemes. For the detection methods (Salmonella spp., L. monocytogenes and Vibrio spp.), 25 g of each of the samples was weighed aseptically into sterile Stomacher bags and homogenized in a Stomacher for up to 2 min. The methodology applied for the detection of Salmonella spp. was based on NMKL Method No.71, 4th edn, 1991 (Nordic Committee on Food Analysis, 1991). The methodology for the detection of L. monocytogenes consisted of a non-selective preenrichment in Listeria enrichment broth with supplement containing Nalidixic acid and cycloheximide and incubation at 301C for 48 h, followed by plating onto Oxford agar which was then incubated for a further 48 h at 371C (Roberts et al. 1995). Aesculin-positive colonies, with black zones around them and with concave appearance, were con¢rmed by using the catalase reaction, Gram stain and the Listeria API biochemical identi¢cation Kit (Biomerieux 2000). For the detection of Vibrio spp. (V. cholerae and V. parahaemolyticus), the methods employed were ISO 8914:1990 and the method described in the FDA bacteriological Analytical Manual, 7th edn, 1992, Chapter 9. Suspicious colonies (green for V. parahaemolyticus, yellow for V. cholerae) were then tested biochemically
Table 1. Types of foods examined microbiologically: 1991^2000 Food type
Methodology The parameters examined included indicator bacteria (Escherichia coli and APCFaerobic plate count) and pathogenic organisms (Salmonella spp., Listeria monocytogenes, Vibrio spp., Staphylococcus aureus and Bacillus cereus). Not all samples were tested for all parameters. Selection of parameters depended on the type of food, the technology/processing received and microbial ecology. The methods used were o⁄cial methods of analysis and the laboratory has proved its competence repeatedly by suc-
Milk Milk products Juices and concentrates Desserts Cured meats Sandwiches Ready-to-eat meals Seafood Mixed salads and vegetables Eggs and egg products Pasta Other (miscellaneous)a Total a
Number Percentage of samples of total 6741 6507 5489 2402 1567 1459 1382 596 294 205 194 1999
23?4 22?6 19?0 8?3 5?4 5?1 4?8 2?1 1?0 0?7 0?7 6?9
28 835
100?0
Includes canned foods, coconut, baby foods, soft drinks, rose water, savory items, etc.
Microbiological pro¢le of Cyprus foods 465
using the API 20 E identi¢cation system, with inoculums containing 3% sodium chloride (Elliot et al. 1992, ISO 1990). All presumptive V. cholerae strains were sent to a reference laboratory abroad for con¢rmation and serological testing. For the enumeration methods (S. aureus, B. cereus, E. coli and APC) 11 g of each of the samples was weighed aseptically into Stomacher bags, diluted with 99 ml of maximum recovery diluent (MRD) and homogenized in a Stomacher for up to 2 min. Serial dilutions were then performed using MRD, as required. Aerobic plate counts were enumerated by pour plating appropriate dilutions in plate count agar and incubating the plates at 301C for 72 h (ISO 1978). The methods used for the enumeration and isolation of S. aureus were AOAC method 975?55 and ISO 6888:1999 (Association of O⁄cial Analytical Chemists (AOAC) 1976, ISO 1999). Enumeration of B. cereus was performed by surface plating of appropriate dilutions onto mannitol^egg yolk^polymyxin B agar (MYP) and incubated at 301C for 24 h.Typical B. cereus colonies (dry, rough surface with a pink to purple base, surrounded by a ring of dense precipitate) were Gram-stained and tested for glucose, mannitol, xylose and arabinose fermentation (Kramer et al. 1982). The enumeration of E. coli was carried out by the MPN technique using lauryl sulfate tryptose broth (48 h at 351C) followed by subculture of positive tubes (gas and cloudiness) in brilliant green broth incubated at 44?51C for a further 48 h (International Commission for the Microbiological Speci¢cation in Foods (ICMSF) 1982).
Results and Discussion Analysis of results Each year, all the results from the microbiological examination of foods are summarized, evaluated and published in the annual report of the State General Laboratory. Based on these results, the sampling schemes for the following year are then prepared. Indeed, the evaluation of results of the previous year has proved extre-
mely valuable in setting priorities for future inspections and sampling. This was the ¢rst time that cumulative evaluation of results for a 10 year period was retrospectively undertaken, and they have provided a much more precise picture of the problems encountered or are much more likely to appear, as well as of any trends evident in the food industry. These problems/trends could have gone unnoticed if only 1 year was evaluated.
Types of foods sampled Table 1 presents the types of foodstu¡s examined microbiologically during the 10 -year period (1991^2000). Sampling, as mentioned earlier, was based on factors such as vulnerability to microbial spoilage, increased consumption by the consumer, unsatisfactory results from previous samplings, export reasons and use by the military services. Milk and milk products constitute by far the largest percentage (46%) of the analysed items. The authorities in Cyprus lay strong emphasis on the monitoring of milk and milk products, especially pasteurized milk and traditional milk products since they are items consumed daily by the majority of consumers. The second most frequently sampled food item category was fruit juices (19%) (freshly squeezed, pasteurized and UHT juices for export). For many, this would seem as an unreasonably high percentage of sampling for a low pH product, but being a citrus fruit producing country, a juice exporting country and a tourist site, these items constitute a large piece in the purchasing basket of the consumer. In addition, there was evidence of serious quality problems, through consumer complaints, for the minimally processed items in this category (freshly squeezed juices). Desserts, cured meats, sandwiches and ready-to-eat meals follow in sampling frequency, with percentages ranging between 4?8% and 8?3% of the total.
Micro-organisms Salmonella spp. and L. monocytogenes. In Cyprus, a zero tolerance is considered acceptable in all types of foodstu¡s for Salmonella spp.
466 M. Eleftheriadou et al.
and L. monocytogenes. Salmonella spp. and L. monocytogenes were isolated from 1?8% of the samples examined. Even though, the percentage of isolation for the two pathogens is coincidentally identical, the types of foods from where these pathogens were isolated were very di¡erent. For Salmonella spp., the commonest implicated items were desserts, followed by imported desiccated coconut and raw poultry, meat and ¢sh. Figure 1 shows in detail the types of samples from where Salmonella spp. were isolated between the years 1991 and 2000. Even though, coconut, raw poultry and meat are well linked epidemiologically with Salmonella (D’Aoust 1997, Mortimore and Wallace 1994, ICMSF 1986), the food category of desserts as the primary vehicle of this food-poisoning organism in Cyprus was a surprising discovery, but not an inexplicable one. Desserts are prepared mainly with either whipped cream or with patisserie cream (heated milk cream) and a substantial amount of handling takes place during preparation. In addition, raw shell eggs are a main ingredient in every
cheese
confectionery preparation area, since the use of pasteurized liquid/powder egg is not yet widespread, and this can easily lead to crosscontamination of the work surfaces, utensils and ultimately the ready-to-eat desserts. In some recipes (i.e. mousses), raw eggs are added without further cooking and the confectioners have been advised to avoid such high-risk recipes. It is worthwhile noting that from the 13 248 samples of milk and milk products examined, only one sample of cheese contained Salmonella, establishing an almost zero prevalence of this pathogen in this broad and very important food category. L. monocytogenes, on the other hand, was isolated most frequently from cured meats (ham, smoked pork loin and sausages), mixed vegetable salads, traditional Cyprus cheese (Halloumi and FlaounaFsemihard, curd cheeses) and smoked salmon. All these items are well-documented vehicles for this organism (Beuchat 1995, Eklund et al. 1995, Fain 1996, Reed 1994,Velani and Roberts 1990). Figure 2 presents the allocation of positive samples for L. monocytogenes in the contaminated foods.
1
Bone meal
1
Raw eggs
2
Cured meat
2
Smoked Fish
2
Mixed salad
2
Ice cream
2
Ready-to-eat meals
4
Sandwiches
4
Raw pasta
4 8
Raw meat, fish
18
Coconut
38
Desserts 0
5
10
15
20
25
30
35
Numbers
Figure 1. Foods from where Salmonella was isolated (1991^2000).
40
Microbiological pro¢le of Cyprus foods 467
Ice cream 4%
Pate 3% Mixed salads 24%
Cured Meats Sausages 34%
Desserts 6%
Cyprus cheese 48%
Sandwich 17%
Cheese 21%
Ready-to-eat meals 2%
Smoked Salmon 18%
Figure 2. Allocation of Listeria monocytogenes in foods: 1991^2000. Raw pasta 23%
Vibrio parahaemolyticus and Vibro cholerae. These were only examined in seafood samples, mainly imported ¢sh and shell¢sh. Testing of imports was intensi¢ed in 1998 following EC decisions regarding the risk that ¢shery products originating from countries of East Africa and China may be contaminated with pathogenic vibrios (European Union (EU) 1997a, b). Both organisms were isolated exclusively from imported, raw, frozen prawns and shrimps.V. parahaemolyticus had the highest incidence of isolation out of the pathogens where zero tolerance is expected (6?9%). V. cholerae non- 01 was also isolated, but at a much lower frequency (1?3%). V. cholerae 01 was never isolated. All seafood imports containing these organisms were banned from entering into the country.
Staphylococcus aureus and B. cereus. The percentage of isolation of S. aureus and B. cereus in signi¢cant, potentially hazardous levels (o104 cfu g 1 ) were 0?7% and 1?0%, respectively. Traditional Cyprus cheese (Halloumi, Flaouna and Anari Cheese) was the most common food vehicle for both the pathogens. In the case of S. aureus, frozen raw pasta (ravioli with cheese) and sandwiches followed traditional cheeses in the frequency of isolation (Fig. 3). In all three of these foods (traditional cheese, ravioli and sandwiches) there is, in many cases, substantial amount of human handling during preparation and being that S. aureus can contaminate foods following handling (Jacob 1989), this is the assumption made
Figure 3. Allocation of S. aureus in foods in levels 4104 cfu g 1 : 1991^2000.
for its presence in these food categories. The percentage of isolation, (0?7%), however, is not alarming. Table 2 presents in detail the prevalence of S. aureus in foodstu¡s for the 10 -year period, and shows that 98?2% of all samples examined contained low numbers of the organism (o103 cfu g 1 ), whereas 1?1% of the samples contained the organisms in borderline levels of acceptability (103 ^104 cfu g 1 ). For B. cereus, desserts were the second most commonly contaminated food item after Cyprus traditional cheese (Fig. 4). As in the case of S. aureus, the majority of samples examined (97?3%) contained B. cereus in low, insigni¢cant levels (o103 cfu g 1 ). Table 2 shows analytically the number of samples examined and the levels of B. cereus found.
E. coli. The isolation of E. coli in foods, as an indicator of fecal contamination, was considered of signi¢cance when levels exceeded 102 cfu g 1. In the 9679 samples examined, the incidence of E. coli was higher (6%) than that of isolation of S. aureus and B. cereus. Traditional Cyprus cheese was the leading item for E. coli, followed by sandwiches and mixed salads (Fig. 5). This indicates a need to improve simple hygienic practices in the preparation of these items and in the washing procedures of vegetables to be used in salads or sandwiches.
468 M. Eleftheriadou et al.
Table 2. Incidence of isolation of Staphylococcus aureus and Bacillus cereus in foods: 1991^2000 Total number examined Containing o103 cfu g 1 Containing 103 ^104 cfu g Containing 4104 cfu g 1
S. aureus
%
B. cereus
%
12 415 12 193 132 90
98?2 1?1 0?7
10 310 10 029 180 101
97?3 1?7 1?0
1
40 35 No. of samples
30 25 20 15 10 5 0 Cyprus cheese
Desserts
Sandwiches Ready-to- Egg powder eat meals
Salad
Figure 4. Allocation of B. cereus in foods in levels 4104 cfu g 1.
200 180 160
120 100 80 60 40 20
ea d R
Figure 5. Allocation of E. coli in foods in levels 4100 g 1.
c. is M
ea ls yto -e
at m
Pa st a R aw
e C he es
er ts D es s
la ds M
ix
ed
Sa
he s nd w ic Sa
yp
ru s
C he es
e
0
C
No. of samples
140
Microbiological pro¢le of Cyprus foods 469
Table 3. Unsatisfactory APC in foodstu¡s: 1991^2000 Type of food
With increased APC (%) 1991^1995
1996^2000
Milk
17?7
5?1
Milk products
20?5
11?5
Desserts
37?2
31?4
Sandwiches
48?5
5?3
Cured meats Non-fermented
15?4
29?2
Juices/concentrates
12?5
4?0
Ready-to-eat meals
32?6
23?5
Criteriaa used in evaluating results for unacceptability
Pasteurized milk and cream: 45 104 cfu ml UHT milk: 4102 cfu ml 1c , Raw milk: 45 105 cfu ml 1d Ice cream: 4105 cfu g 1, Anari cheese: 4105 cfu g
1b
1
With and without dairy cream 4105 cfu g 1 With syrup, oven-baked and bakery products: 4103 cfu g 1 With and without salad: 4105 cfu g Toasted: 4102 cfu g 1
1
Cooked ham: 4106 cfu g 1 Frankfurters and other non-fermented meats: 42 105 cfu g 1e Freshly squeezed: 4105 cfu ml 1 Pasteurized: 4104 cfu ml 1 , UHT juices: 410 cfu ml Concentrates: 4103 cfu ml 1 4104 cfu g
1
1
a
Intra-laboratory, unpublished criteria, if not otherwise speci¢ed. Cyprus Standard for pasteurized milk CYS 93:1989 (Cyprus Government 1989c). c Speci¢cations for UHT milk 3(p)(i) and 3(p) (ii), Ministry of Defense (Cyprus Government 1989a,b). d Cyprus Standard for raw milk CYS 92:1989 (Cyprus Government 1989d). e Speci¢cation for frankfurters and smoked ham 10(a) and 10(b), Ministry of Defense (Cyprus Government 1990a,b). b
Total bacterial count. As shown in Table 3, the percentages of unsatisfactory APC as an indicator of good hygiene practice and temperature and process control were the highest in sandwiches between 1991 and 1995, amounting to 48?5% and fell dramatically to 5?3% in 1996^ 2000.This fall can be explained partially, apart from the intensi¢cation of education of foodhandlers in street vending outlets and school canteens, by the fact that ham was omitted as an ingredient in sandwiches sold in schools canteens due to its vulnerability to spoilage and to the very high APCs found in many samples during routine surveillance of this item. Indeed, the increase in the percentage of unsatisfactory APC in the category of cured meats in the second period (Table 3) is due to the sampling and testing of a large number of samples of cooked sliced ham. The APC of desserts, in both periods examined was high, 37?2% and 31?4%, respectively, indicating signi¢cant problems in hygiene practices, temperature control and the use of
raw ingredients, such as poorly washed fruit and nuts, that may have contributed signi¢cantly to the APC. The fall of unsatisfactory APCs in milk samples from 17?7% to 5?1% in 1995^2000 can be attributed to signi¢cant improvements in the milk production industry as a result of coordinated e¡orts by the Cyprus Milk Industry Organization and the private sector and also to the refrigerated transport of all raw milk from the farms to the processing facilities.
Conclusion The microbiological examination of foodstu¡s plays an important role in assuring the safety and quality of foods. Even though the implementation of Hazard Analysis and Critical Control Point (HACCP) system places emphasis in the proactive approach to critical steps in the production of foods, end-product testing will still be exercised and be signi¢cant in
470 M. Eleftheriadou et al.
terms of validation and veri¢cation of the implemented proactive measures and controls at production. The o⁄cial results presented here re£ect for the most part the good microbiological quality achieved by the majority of the food industry in Cyprus. This was substantiated indirectly by Cartwright and Chaled (1997) who reported that the incidence of travellers’diarrhea among tourists in Cyprus is very low compared to many other countries. The realization that desserts are the leading vehicles of contamination with Salmonella spp., the second most frequent item from where B. cereus was isolated in signi¢cant numbers and the item in which over 30% of the samples tested contained increased, unjusti¢able APC, has led to the intensi¢cation of controls in confectionery shops and other places selling desserts, as well as to the promotion of stringent education of the foodhandlers and owners of such premises. Traditional Cyprus cheese (Anari, Flaouna, Halloumi) was the leading item for contamination with S. aureus, B. cereus and E. coli in signi¢cant numbers. This is indicative of the need to monitor and improve existing practices in the production of these traditional foods, which in many cases may be produced in small, family sized establishments. The results for the APC, especially for desserts, sandwiches, cured non-fermented meats and ready-to-eat meals, indicate that emphasis should be given during inspection and education to enforce GMPs and temperature control vigorously in order to bring bacteria levels down to more acceptable, yet achievable levels. As regards the sources of the samples, it is evident that the samples submitted for food poisoning investigations and consumer complaints amounted to less than 3% of the total number analysed. This suggests that consumers should be educated to report problems associated with the buying and consumption of foodstu¡s and that the existing foodborne disease surveillance system must receive attention and upgrading. The body of information gathered here can be very useful as a base where microbiological risk assessment, both quantitative and qualitative, can be based. Also, it can provide valuable
information for the design of monitoring and surveillance programs for the o⁄cial food microbiological control in Cyprus.
Acknowledgements The authors are grateful to Prof. Richard J. Gilbert for his advice and assistance in the preparation of this manuscript.
References Association of O⁄cial Analytical Chemists (1975) Staphylococcus aureus in Foods. Surface Plating Method for Isolation and Enumeration. O⁄cial Method 975?55, JAOAC 58, 1154^1158, Washington DC. Beuchat, L. R. (1995) Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59, 204^216. Biomerieux, Inc. (2000) API Listeria System for Identi¢cation of Listeria. Package Insert. Cartwright, R. Y. and Chahed, M. (1997) Foodborne diseases in travellers. World Health Organization Statist. Quart. 50 (a/b), 102^110. Cyprus Government (1989a) Ministry of Defense Speci¢cation for UHT Milk No. 3 (p)(PP i). Cyprus Government. Cyprus Government (1989b) Ministry of Defense Speci¢cation for UHT Milk with Cocoa No. 3 (p)(PP iPP i). Cyprus Government. Cyprus Government (1989c) Ministry of Commerce, Industry and Tourism Standard for Pasteurized milk CYS 93:1989. Cyprus Government. Cyprus Government (1989d) Ministry of Commerce, Industry and Tourism Standard for raw milk CYS 92:1989. Cyprus Government. Cyprus Government (1990a) Ministry of Defense Speci¢cation for Frankfurters No. 10 (a). Cyprus Government. Cyprus Government (1990b) Ministry of Defense Speci¢cation for Smoked Ham No. 10 (b). Cyprus Government. D’Aoust, J. Y. (1997) Salmonella species. In Food MicrobiologyFFundamentals and Frontiers (Eds M. Doyle, K. Beuchat and T. Montville) pp. 129^158. ASM Press, Oslo, Norway. Eklund, M.W., Pousky, F. K., Paranjpye, R. N., Lashbrook, L. C., Peterson, M. E. and G. A. Pelroy (1995) Incidence and source of Listeria monocytogenes in cold smoked ¢shery products and processing plants. J. Food Prot. 58, 502^508. Elliot, E. L., Kaysner, C. A. and Tamplim, M. L. (1992) V. cholerae, V. parahaemolyticus, V. vulni¢cus and other Vibrio spp. FDA Bacteriological Analytical Manual, 7th edn. AOAC International.
Microbiological pro¢le of Cyprus foods 471
European Union (1997a) Commission Decision (97/ 368/EC) concerning certain protective measures with regard to certain ¢shery products originating in China. O⁄c. J. Eur. Commun. L156/57, 57^58. European Union (1997b) Commission Decision (97/ 878/EC) concerning certain protective measures with regard to certain ¢shery products originating in Uganda, Kenya, Tanzania and Mozambique. O⁄c. J. Eur. Commun. L356/64, 64. Fain, A. R. (1996) A review of the microbiological safety of fresh salads. Dairy Food and Environ. Sanit. 16 (3), 146^149. Jacob, M. (1989) Safe Food Handling. A Training Guide for Managers of Food Service-Establishments, 16pp.World Health Organization. International Commission for the Microbiological Speci¢cation in Foods (ICMSF) (1982) Coliform bacteria. Micro-organisms in Foods:1.Their Significance and Methods of Enumeration, 2nd edn pp. 125^139. Toronto, University of Toronto Press. International Commission for the Microbiological Speci¢cation in Foods (ICMSF) (1986) Sampling plans for vegetables, fruits, and nuts. Micro-organisms in foods, 2. Sampling for Microbiological Analysis: Principles and Speci¢c Applications, 2nd edn pp. 197^202. Toronto, University of Toronto Press. ISO (1978) MicrobiologyFGeneral Guidance for Enumeration of Micro-organismsFColony Count Technique at 301C, ISO 4833 -1978. International Organization for Standardization. ISO (1990) MicrobiologyFGeneral Guidance for the detection of Vibrio parahaemolyticus, ISO 8914 -
1990. International Organization for Standardization. ISO (1999) Microbiology of food and animal feeding stu¡sFhorizontal method for the enumeration of coagulase-positive Staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird^Parker medium, ISO 6888 -1-1999. International Organization for Standardization. Kramer, J. M., Turnbull, P. C. B, Munshi, G. and Gilbert, R. J. (1982) Identi¢cation and characterization of Bacillus cereus and other Bacillus species associated with foods and food poisoning. In Isolation and Identi¢cation Methods for Food-Poisoning Organisms (Eds J. E. L. Corry, D. Roberts and F. A. Skinner) pp. 261^286. New York, Academic Press Inc. Mortimore, S. and Wallace, C. (1994) Appendix C pathogen pro¢les. HACCPFA Practical Approach, pp. 271^281. London, Chapman & Hall. Nordic Committee on Food Analysis (1991) Salmonella bacteria. Detection in Foods. Method 71, 4th edn. Reed, H. G. (1994) Foodborne illness (Part 10) Listeria monocytogenes. Dairy Food Environ. Sanit. 14, 482^483. Roberts, D., Hooper, W. and Greenwood, M. (1995) Practical Food Microbiology. London, PHLS. Velani, S. and Roberts D. (1990) Listeria monocytogenes and other Listeria spp. in prepacked mixed salads and individual salad ingredients. PHLS Microbiol. Dig. 8(1), 21^23.
doi:10.1006/yfmic.508
ORIGINAL ARTICLE
The microbiological pro¢le of foods in the Republic of Cyprus: 1991^2000 Mary Eleftheriadou*, Andri Varnava-Tello, Maria Metta-Loizidou, Andri-Silvia Nikolaou and Dina Akkelidou
In Cyprus, as part of the exercised o⁄cial food microbiological control, 28 835 of a large variety of foodstu¡s were examined during the years 1991^2000. Parameters examined included Salmonella spp., Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus,Vibrio spp., E.coli and aerobic plate count.The results indicate a prevalence of1?8% for Salmonella spp. and L. monocytogenes, with desserts ranking ¢rst as the primary vehicle for Salmonella spp. and cured meats for L. monocytogenes. S. aureus was isolated in 0?7% of the samples examined in signi¢cant, potentially hazardous levels (4104 cfu g 1), whereas B. cereus in 1% of the samples in numbers (4104 cfu g 1). The most common food vehicle for both pathogens was found to be traditional Cyprus cheese.V. parahaemolyticus and V. cholerae non-01were isolated exclusively from imported frozen, raw prawns and shrimps at isolation rates of 6?9% and 1?3%, respectively. The incidence of E. coli at levels 4102 g 1 amounted to 6% of the samples examined, with traditional Cyprus cheese being the leading food vehicle. High aerobic plate counts were found in desserts, ready-to-eat meals, sandwiches and cured, non-fermented meats. The results presented in this paper indicate for the most part the good microbiological quality achieved by the majority of the food industry in Cyprus. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction The production of safe food is and will remain the responsibility of the producer. However, the authorities need to be regularly verifying and validating, through inspection and end-product testing, the e¡ectiveness of the controls implemented by the industry, both at the site of production and at the point of sale. In Cyprus, o⁄cial microbiological control of foodstu¡s is carried out by the State General Laboratory of the Ministry of Health and is *Corresponding author. Fax: E-mail: [email protected] 0740 -0020/02/050463+09 $35.00/0
+357-22316434;
aimed at verifying compliance with legislation and the protection of public health, consumer interests and fair trade practices. The importance of microbiologically safe food in Cyprus gains added importance as Cyprus is a country attracting many tourists with triple the number of its inhabitants visiting the island every year. This paper provides a basic evaluation of the results from the microbiological examination of 28 835 samples of a large variety of foodstu¡s over a 10 -year period (1991^2000) in the Republic of Cyprus and includes data on the prevalence of speci¢c pathogens and indicator bacteria. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 7 January 2002 Food Microbiology Laboratory, State General Laboratory, Ministry of Health, 44 Kimonos street, Nicosia1451, Cyprus
464 M. Eleftheriadou et al.
Materials and Methods Sampling During the years 1991^2000, 28 835 samples of foodstu¡s were examined microbiologically to ascertain their safety and quality status. Sampling of foods was undertaken by trained sampling o⁄cers/health inspectors of the Ministry of Health, the local municipalities and the Ministry of Industry, Commerce and Tourism, in accordance with the Cypriot food legislation. The sampling took place both from the production site (industry and mass catering establishments) and from retail outlets (shops, kiosks, school canteens, supermarkets, etc.). Sampling was based mainly on vulnerability to spoilage, consumption frequency with emphasis on high-risk population groups, and known microbiological safety/quality problems. The primary sources of the samples received were: routine surveillanceFincludes sampling from shops, kiosks, supermarkets, street vendors, schools, hotels, restaurants, take away shops, cruise ships and the food industryF (56?9%), National guard (28?4%), Imports/Exports (11?7%) and miscellaneous (3%)Fincludes, apart from privately submitted samples, food poisoning investigations and consumer complaints. All the samples were transported to the laboratory under low temperature (o81C) and either examined immediately or stored at 4o C until testing which took place within maximum 24 h of sampling. Table 1 lists the types of products sampled during this period.
cessful participation in external pro¢ciency testing schemes. For the detection methods (Salmonella spp., L. monocytogenes and Vibrio spp.), 25 g of each of the samples was weighed aseptically into sterile Stomacher bags and homogenized in a Stomacher for up to 2 min. The methodology applied for the detection of Salmonella spp. was based on NMKL Method No.71, 4th edn, 1991 (Nordic Committee on Food Analysis, 1991). The methodology for the detection of L. monocytogenes consisted of a non-selective preenrichment in Listeria enrichment broth with supplement containing Nalidixic acid and cycloheximide and incubation at 301C for 48 h, followed by plating onto Oxford agar which was then incubated for a further 48 h at 371C (Roberts et al. 1995). Aesculin-positive colonies, with black zones around them and with concave appearance, were con¢rmed by using the catalase reaction, Gram stain and the Listeria API biochemical identi¢cation Kit (Biomerieux 2000). For the detection of Vibrio spp. (V. cholerae and V. parahaemolyticus), the methods employed were ISO 8914:1990 and the method described in the FDA bacteriological Analytical Manual, 7th edn, 1992, Chapter 9. Suspicious colonies (green for V. parahaemolyticus, yellow for V. cholerae) were then tested biochemically
Table 1. Types of foods examined microbiologically: 1991^2000 Food type
Methodology The parameters examined included indicator bacteria (Escherichia coli and APCFaerobic plate count) and pathogenic organisms (Salmonella spp., Listeria monocytogenes, Vibrio spp., Staphylococcus aureus and Bacillus cereus). Not all samples were tested for all parameters. Selection of parameters depended on the type of food, the technology/processing received and microbial ecology. The methods used were o⁄cial methods of analysis and the laboratory has proved its competence repeatedly by suc-
Milk Milk products Juices and concentrates Desserts Cured meats Sandwiches Ready-to-eat meals Seafood Mixed salads and vegetables Eggs and egg products Pasta Other (miscellaneous)a Total a
Number Percentage of samples of total 6741 6507 5489 2402 1567 1459 1382 596 294 205 194 1999
23?4 22?6 19?0 8?3 5?4 5?1 4?8 2?1 1?0 0?7 0?7 6?9
28 835
100?0
Includes canned foods, coconut, baby foods, soft drinks, rose water, savory items, etc.
Microbiological pro¢le of Cyprus foods 465
using the API 20 E identi¢cation system, with inoculums containing 3% sodium chloride (Elliot et al. 1992, ISO 1990). All presumptive V. cholerae strains were sent to a reference laboratory abroad for con¢rmation and serological testing. For the enumeration methods (S. aureus, B. cereus, E. coli and APC) 11 g of each of the samples was weighed aseptically into Stomacher bags, diluted with 99 ml of maximum recovery diluent (MRD) and homogenized in a Stomacher for up to 2 min. Serial dilutions were then performed using MRD, as required. Aerobic plate counts were enumerated by pour plating appropriate dilutions in plate count agar and incubating the plates at 301C for 72 h (ISO 1978). The methods used for the enumeration and isolation of S. aureus were AOAC method 975?55 and ISO 6888:1999 (Association of O⁄cial Analytical Chemists (AOAC) 1976, ISO 1999). Enumeration of B. cereus was performed by surface plating of appropriate dilutions onto mannitol^egg yolk^polymyxin B agar (MYP) and incubated at 301C for 24 h.Typical B. cereus colonies (dry, rough surface with a pink to purple base, surrounded by a ring of dense precipitate) were Gram-stained and tested for glucose, mannitol, xylose and arabinose fermentation (Kramer et al. 1982). The enumeration of E. coli was carried out by the MPN technique using lauryl sulfate tryptose broth (48 h at 351C) followed by subculture of positive tubes (gas and cloudiness) in brilliant green broth incubated at 44?51C for a further 48 h (International Commission for the Microbiological Speci¢cation in Foods (ICMSF) 1982).
Results and Discussion Analysis of results Each year, all the results from the microbiological examination of foods are summarized, evaluated and published in the annual report of the State General Laboratory. Based on these results, the sampling schemes for the following year are then prepared. Indeed, the evaluation of results of the previous year has proved extre-
mely valuable in setting priorities for future inspections and sampling. This was the ¢rst time that cumulative evaluation of results for a 10 year period was retrospectively undertaken, and they have provided a much more precise picture of the problems encountered or are much more likely to appear, as well as of any trends evident in the food industry. These problems/trends could have gone unnoticed if only 1 year was evaluated.
Types of foods sampled Table 1 presents the types of foodstu¡s examined microbiologically during the 10 -year period (1991^2000). Sampling, as mentioned earlier, was based on factors such as vulnerability to microbial spoilage, increased consumption by the consumer, unsatisfactory results from previous samplings, export reasons and use by the military services. Milk and milk products constitute by far the largest percentage (46%) of the analysed items. The authorities in Cyprus lay strong emphasis on the monitoring of milk and milk products, especially pasteurized milk and traditional milk products since they are items consumed daily by the majority of consumers. The second most frequently sampled food item category was fruit juices (19%) (freshly squeezed, pasteurized and UHT juices for export). For many, this would seem as an unreasonably high percentage of sampling for a low pH product, but being a citrus fruit producing country, a juice exporting country and a tourist site, these items constitute a large piece in the purchasing basket of the consumer. In addition, there was evidence of serious quality problems, through consumer complaints, for the minimally processed items in this category (freshly squeezed juices). Desserts, cured meats, sandwiches and ready-to-eat meals follow in sampling frequency, with percentages ranging between 4?8% and 8?3% of the total.
Micro-organisms Salmonella spp. and L. monocytogenes. In Cyprus, a zero tolerance is considered acceptable in all types of foodstu¡s for Salmonella spp.
466 M. Eleftheriadou et al.
and L. monocytogenes. Salmonella spp. and L. monocytogenes were isolated from 1?8% of the samples examined. Even though, the percentage of isolation for the two pathogens is coincidentally identical, the types of foods from where these pathogens were isolated were very di¡erent. For Salmonella spp., the commonest implicated items were desserts, followed by imported desiccated coconut and raw poultry, meat and ¢sh. Figure 1 shows in detail the types of samples from where Salmonella spp. were isolated between the years 1991 and 2000. Even though, coconut, raw poultry and meat are well linked epidemiologically with Salmonella (D’Aoust 1997, Mortimore and Wallace 1994, ICMSF 1986), the food category of desserts as the primary vehicle of this food-poisoning organism in Cyprus was a surprising discovery, but not an inexplicable one. Desserts are prepared mainly with either whipped cream or with patisserie cream (heated milk cream) and a substantial amount of handling takes place during preparation. In addition, raw shell eggs are a main ingredient in every
cheese
confectionery preparation area, since the use of pasteurized liquid/powder egg is not yet widespread, and this can easily lead to crosscontamination of the work surfaces, utensils and ultimately the ready-to-eat desserts. In some recipes (i.e. mousses), raw eggs are added without further cooking and the confectioners have been advised to avoid such high-risk recipes. It is worthwhile noting that from the 13 248 samples of milk and milk products examined, only one sample of cheese contained Salmonella, establishing an almost zero prevalence of this pathogen in this broad and very important food category. L. monocytogenes, on the other hand, was isolated most frequently from cured meats (ham, smoked pork loin and sausages), mixed vegetable salads, traditional Cyprus cheese (Halloumi and FlaounaFsemihard, curd cheeses) and smoked salmon. All these items are well-documented vehicles for this organism (Beuchat 1995, Eklund et al. 1995, Fain 1996, Reed 1994,Velani and Roberts 1990). Figure 2 presents the allocation of positive samples for L. monocytogenes in the contaminated foods.
1
Bone meal
1
Raw eggs
2
Cured meat
2
Smoked Fish
2
Mixed salad
2
Ice cream
2
Ready-to-eat meals
4
Sandwiches
4
Raw pasta
4 8
Raw meat, fish
18
Coconut
38
Desserts 0
5
10
15
20
25
30
35
Numbers
Figure 1. Foods from where Salmonella was isolated (1991^2000).
40
Microbiological pro¢le of Cyprus foods 467
Ice cream 4%
Pate 3% Mixed salads 24%
Cured Meats Sausages 34%
Desserts 6%
Cyprus cheese 48%
Sandwich 17%
Cheese 21%
Ready-to-eat meals 2%
Smoked Salmon 18%
Figure 2. Allocation of Listeria monocytogenes in foods: 1991^2000. Raw pasta 23%
Vibrio parahaemolyticus and Vibro cholerae. These were only examined in seafood samples, mainly imported ¢sh and shell¢sh. Testing of imports was intensi¢ed in 1998 following EC decisions regarding the risk that ¢shery products originating from countries of East Africa and China may be contaminated with pathogenic vibrios (European Union (EU) 1997a, b). Both organisms were isolated exclusively from imported, raw, frozen prawns and shrimps.V. parahaemolyticus had the highest incidence of isolation out of the pathogens where zero tolerance is expected (6?9%). V. cholerae non- 01 was also isolated, but at a much lower frequency (1?3%). V. cholerae 01 was never isolated. All seafood imports containing these organisms were banned from entering into the country.
Staphylococcus aureus and B. cereus. The percentage of isolation of S. aureus and B. cereus in signi¢cant, potentially hazardous levels (o104 cfu g 1 ) were 0?7% and 1?0%, respectively. Traditional Cyprus cheese (Halloumi, Flaouna and Anari Cheese) was the most common food vehicle for both the pathogens. In the case of S. aureus, frozen raw pasta (ravioli with cheese) and sandwiches followed traditional cheeses in the frequency of isolation (Fig. 3). In all three of these foods (traditional cheese, ravioli and sandwiches) there is, in many cases, substantial amount of human handling during preparation and being that S. aureus can contaminate foods following handling (Jacob 1989), this is the assumption made
Figure 3. Allocation of S. aureus in foods in levels 4104 cfu g 1 : 1991^2000.
for its presence in these food categories. The percentage of isolation, (0?7%), however, is not alarming. Table 2 presents in detail the prevalence of S. aureus in foodstu¡s for the 10 -year period, and shows that 98?2% of all samples examined contained low numbers of the organism (o103 cfu g 1 ), whereas 1?1% of the samples contained the organisms in borderline levels of acceptability (103 ^104 cfu g 1 ). For B. cereus, desserts were the second most commonly contaminated food item after Cyprus traditional cheese (Fig. 4). As in the case of S. aureus, the majority of samples examined (97?3%) contained B. cereus in low, insigni¢cant levels (o103 cfu g 1 ). Table 2 shows analytically the number of samples examined and the levels of B. cereus found.
E. coli. The isolation of E. coli in foods, as an indicator of fecal contamination, was considered of signi¢cance when levels exceeded 102 cfu g 1. In the 9679 samples examined, the incidence of E. coli was higher (6%) than that of isolation of S. aureus and B. cereus. Traditional Cyprus cheese was the leading item for E. coli, followed by sandwiches and mixed salads (Fig. 5). This indicates a need to improve simple hygienic practices in the preparation of these items and in the washing procedures of vegetables to be used in salads or sandwiches.
468 M. Eleftheriadou et al.
Table 2. Incidence of isolation of Staphylococcus aureus and Bacillus cereus in foods: 1991^2000 Total number examined Containing o103 cfu g 1 Containing 103 ^104 cfu g Containing 4104 cfu g 1
S. aureus
%
B. cereus
%
12 415 12 193 132 90
98?2 1?1 0?7
10 310 10 029 180 101
97?3 1?7 1?0
1
40 35 No. of samples
30 25 20 15 10 5 0 Cyprus cheese
Desserts
Sandwiches Ready-to- Egg powder eat meals
Salad
Figure 4. Allocation of B. cereus in foods in levels 4104 cfu g 1.
200 180 160
120 100 80 60 40 20
ea d R
Figure 5. Allocation of E. coli in foods in levels 4100 g 1.
c. is M
ea ls yto -e
at m
Pa st a R aw
e C he es
er ts D es s
la ds M
ix
ed
Sa
he s nd w ic Sa
yp
ru s
C he es
e
0
C
No. of samples
140
Microbiological pro¢le of Cyprus foods 469
Table 3. Unsatisfactory APC in foodstu¡s: 1991^2000 Type of food
With increased APC (%) 1991^1995
1996^2000
Milk
17?7
5?1
Milk products
20?5
11?5
Desserts
37?2
31?4
Sandwiches
48?5
5?3
Cured meats Non-fermented
15?4
29?2
Juices/concentrates
12?5
4?0
Ready-to-eat meals
32?6
23?5
Criteriaa used in evaluating results for unacceptability
Pasteurized milk and cream: 45 104 cfu ml UHT milk: 4102 cfu ml 1c , Raw milk: 45 105 cfu ml 1d Ice cream: 4105 cfu g 1, Anari cheese: 4105 cfu g
1b
1
With and without dairy cream 4105 cfu g 1 With syrup, oven-baked and bakery products: 4103 cfu g 1 With and without salad: 4105 cfu g Toasted: 4102 cfu g 1
1
Cooked ham: 4106 cfu g 1 Frankfurters and other non-fermented meats: 42 105 cfu g 1e Freshly squeezed: 4105 cfu ml 1 Pasteurized: 4104 cfu ml 1 , UHT juices: 410 cfu ml Concentrates: 4103 cfu ml 1 4104 cfu g
1
1
a
Intra-laboratory, unpublished criteria, if not otherwise speci¢ed. Cyprus Standard for pasteurized milk CYS 93:1989 (Cyprus Government 1989c). c Speci¢cations for UHT milk 3(p)(i) and 3(p) (ii), Ministry of Defense (Cyprus Government 1989a,b). d Cyprus Standard for raw milk CYS 92:1989 (Cyprus Government 1989d). e Speci¢cation for frankfurters and smoked ham 10(a) and 10(b), Ministry of Defense (Cyprus Government 1990a,b). b
Total bacterial count. As shown in Table 3, the percentages of unsatisfactory APC as an indicator of good hygiene practice and temperature and process control were the highest in sandwiches between 1991 and 1995, amounting to 48?5% and fell dramatically to 5?3% in 1996^ 2000.This fall can be explained partially, apart from the intensi¢cation of education of foodhandlers in street vending outlets and school canteens, by the fact that ham was omitted as an ingredient in sandwiches sold in schools canteens due to its vulnerability to spoilage and to the very high APCs found in many samples during routine surveillance of this item. Indeed, the increase in the percentage of unsatisfactory APC in the category of cured meats in the second period (Table 3) is due to the sampling and testing of a large number of samples of cooked sliced ham. The APC of desserts, in both periods examined was high, 37?2% and 31?4%, respectively, indicating signi¢cant problems in hygiene practices, temperature control and the use of
raw ingredients, such as poorly washed fruit and nuts, that may have contributed signi¢cantly to the APC. The fall of unsatisfactory APCs in milk samples from 17?7% to 5?1% in 1995^2000 can be attributed to signi¢cant improvements in the milk production industry as a result of coordinated e¡orts by the Cyprus Milk Industry Organization and the private sector and also to the refrigerated transport of all raw milk from the farms to the processing facilities.
Conclusion The microbiological examination of foodstu¡s plays an important role in assuring the safety and quality of foods. Even though the implementation of Hazard Analysis and Critical Control Point (HACCP) system places emphasis in the proactive approach to critical steps in the production of foods, end-product testing will still be exercised and be signi¢cant in
470 M. Eleftheriadou et al.
terms of validation and veri¢cation of the implemented proactive measures and controls at production. The o⁄cial results presented here re£ect for the most part the good microbiological quality achieved by the majority of the food industry in Cyprus. This was substantiated indirectly by Cartwright and Chaled (1997) who reported that the incidence of travellers’diarrhea among tourists in Cyprus is very low compared to many other countries. The realization that desserts are the leading vehicles of contamination with Salmonella spp., the second most frequent item from where B. cereus was isolated in signi¢cant numbers and the item in which over 30% of the samples tested contained increased, unjusti¢able APC, has led to the intensi¢cation of controls in confectionery shops and other places selling desserts, as well as to the promotion of stringent education of the foodhandlers and owners of such premises. Traditional Cyprus cheese (Anari, Flaouna, Halloumi) was the leading item for contamination with S. aureus, B. cereus and E. coli in signi¢cant numbers. This is indicative of the need to monitor and improve existing practices in the production of these traditional foods, which in many cases may be produced in small, family sized establishments. The results for the APC, especially for desserts, sandwiches, cured non-fermented meats and ready-to-eat meals, indicate that emphasis should be given during inspection and education to enforce GMPs and temperature control vigorously in order to bring bacteria levels down to more acceptable, yet achievable levels. As regards the sources of the samples, it is evident that the samples submitted for food poisoning investigations and consumer complaints amounted to less than 3% of the total number analysed. This suggests that consumers should be educated to report problems associated with the buying and consumption of foodstu¡s and that the existing foodborne disease surveillance system must receive attention and upgrading. The body of information gathered here can be very useful as a base where microbiological risk assessment, both quantitative and qualitative, can be based. Also, it can provide valuable
information for the design of monitoring and surveillance programs for the o⁄cial food microbiological control in Cyprus.
Acknowledgements The authors are grateful to Prof. Richard J. Gilbert for his advice and assistance in the preparation of this manuscript.
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European Union (1997a) Commission Decision (97/ 368/EC) concerning certain protective measures with regard to certain ¢shery products originating in China. O⁄c. J. Eur. Commun. L156/57, 57^58. European Union (1997b) Commission Decision (97/ 878/EC) concerning certain protective measures with regard to certain ¢shery products originating in Uganda, Kenya, Tanzania and Mozambique. O⁄c. J. Eur. Commun. L356/64, 64. Fain, A. R. (1996) A review of the microbiological safety of fresh salads. Dairy Food and Environ. Sanit. 16 (3), 146^149. Jacob, M. (1989) Safe Food Handling. A Training Guide for Managers of Food Service-Establishments, 16pp.World Health Organization. International Commission for the Microbiological Speci¢cation in Foods (ICMSF) (1982) Coliform bacteria. Micro-organisms in Foods:1.Their Significance and Methods of Enumeration, 2nd edn pp. 125^139. Toronto, University of Toronto Press. International Commission for the Microbiological Speci¢cation in Foods (ICMSF) (1986) Sampling plans for vegetables, fruits, and nuts. Micro-organisms in foods, 2. Sampling for Microbiological Analysis: Principles and Speci¢c Applications, 2nd edn pp. 197^202. Toronto, University of Toronto Press. ISO (1978) MicrobiologyFGeneral Guidance for Enumeration of Micro-organismsFColony Count Technique at 301C, ISO 4833 -1978. International Organization for Standardization. ISO (1990) MicrobiologyFGeneral Guidance for the detection of Vibrio parahaemolyticus, ISO 8914 -
1990. International Organization for Standardization. ISO (1999) Microbiology of food and animal feeding stu¡sFhorizontal method for the enumeration of coagulase-positive Staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird^Parker medium, ISO 6888 -1-1999. International Organization for Standardization. Kramer, J. M., Turnbull, P. C. B, Munshi, G. and Gilbert, R. J. (1982) Identi¢cation and characterization of Bacillus cereus and other Bacillus species associated with foods and food poisoning. In Isolation and Identi¢cation Methods for Food-Poisoning Organisms (Eds J. E. L. Corry, D. Roberts and F. A. Skinner) pp. 261^286. New York, Academic Press Inc. Mortimore, S. and Wallace, C. (1994) Appendix C pathogen pro¢les. HACCPFA Practical Approach, pp. 271^281. London, Chapman & Hall. Nordic Committee on Food Analysis (1991) Salmonella bacteria. Detection in Foods. Method 71, 4th edn. Reed, H. G. (1994) Foodborne illness (Part 10) Listeria monocytogenes. Dairy Food Environ. Sanit. 14, 482^483. Roberts, D., Hooper, W. and Greenwood, M. (1995) Practical Food Microbiology. London, PHLS. Velani, S. and Roberts D. (1990) Listeria monocytogenes and other Listeria spp. in prepacked mixed salads and individual salad ingredients. PHLS Microbiol. Dig. 8(1), 21^23.