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Surveillance of soft and semi-soft cheeses for the presence of Listeria spp.
International Journal of Food Microbiology, 5 (1987) 157-163 Elsevier
157
JFM 00171
Surveillance of soft and semi-soft cheeses for the presence of Listeria spp. J . M . F a r b e r , M . A . J o h n s t o n , U. P u r v i s a n d A. L o i t Bureau of Microbial Hazards, Ottawa, Ontario, Canada Received 22 June 1987; accepted 28 August 1987)
Recent outbreaks of listeriosis associated with dairy products prompted a survey to determine the incidence of Listeria in domestic and imported cheeses and to assess the manufacturing practices of the Canadian cheese industry. A total of 374 samples of soft and semi-soft cheeses from 61 Canadian and 98 foreign manufacturers were examined for Listeria and for phosphatase. Two samples contained Listeria monocytogenes, and one sample contained Listeria innocua. The three lots of cheese were all manufactured by one plant in France. Thirty-four samples from Canadian and foreign manufacturers gave positive phosphatase tests. Additional information confirmed that some of these cheeses were made from unpasteurized milk and were not held 60 days prior to sale. Five of 30 domestic manufacturers inspected at the time of sampling were not adhering to good manufacturing practices and were using unpasteurized milk to make cheese. Although Listeria was not found in Canadian cheese, the possibility of a Listeria outbreak occurring in Canada exists if conditions do not improve in a few plants. Continued surveillance by government and industry is recommended in order to ensure the microbiological safety of such cheeses. Key words: Listeria; Listeriosis; Cheese, soft and semi-soft; Canadian cheese industry
Introduction
Listeria monocytogenes, although known as a human pathogen for many years, has only recently become recognized as a foodborne pathogen. The organism, a Gram-positive, psychrotrophic rod, is widespread in nature and can be isolated from a variety of sources such as poor quality silage, vegetation, soil, sewage, stream water, mud, slaughter house waste, milk of normal and mastitic cows and the feces of healthy humans. In addition, Listeria spp. have been isolated from at least 37 species of mammals, 17 species of birds, flies, ticks, fish and crustaceans (Gray and Killinger, 1966; Seeliger and Jones, 1986). There have been four food-borne outbreaks of listeriosis in North America reported within the last 7 years, with two of these outbreaks involving dairy
Correspondence address: J.M. Farber, Health and Welfare Canada, Health Protection Branch, Bureau of Microbiol Hazards, Tunney's Pasture, Ottawa, Ontario, Canada K1AOL2. 0168-1605/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
158 products (Schlech et al., 1983; Centers for Disease Control, 1985; Fleming et al., 1985; Ho et al., 1986). In 1983, a listeriosis outbreak epidemiologically linked to pasteurized milk occurred in Massachusetts (Fleming et al., 1985). Forty-nine patients were involved with a fatality rate of 29%. Milk in the bulk tank of one of the farms supplying the incriminated processing plant contained L, monocytogenes serotype 4b, the same serotype as that found in 32 of 40 case isolates. In the latest outbreak occurring in the spring of 1985, Mexican-style soft cheese produced by a company in California was the incriminated vehicle (Centers for Disease Control, 1985). There were 181 cases in infant-mother pairs, with 65 deaths, and another 133 cases in other members of the population with an overall fatality rate of 33.4%. These recent outbreaks of listeriosis associated with dairy products along with the recent recalls of Listeria contaminated dairy products in the United States has prompted the Health Protection Branch of Health and Welfare Canada to conduct Listeria surveys on food. The present study was undertaken to determine whether domestically produced and imported soft and semi-soft cheeses in Canada contained Listeria spp., and also to assess the ability of the Canadian cheese industry to follow good manufacturing practices.
Materials and Methods
Survey A total of 182 domestic (61 manufacturers) and 192 imported (98 manufacturers) samples of cheese were analyzed for Listeria spp. between October, 1985 and March, 1987. In addition, the phosphatase test was done on 79 of the 182 domestic and 108 of the 192 imported samples. For the domestically produced cheeses, the majority ( > 80%) of the plants included in this survey were from Quebec and Ontario, since most of the soft cheese industry in Canada is centered in these two provinces. Two lots of domestic cheese, each lot from a different day's production, were sampled from each manufacturer. A sample consisted of 5 sample units randomly selected from each lot. Imported cheeses were obtained at the import or distribution level. No more than two lots of a product type from the same foreign manufacturer were analyzed. Samples were obtained from 12 of the 22 exporting countries that send major shipments of cheese to Canada on a regular basis. Cheeses from France, Denmark and Germany were sampled more heavily than cheeses from other countries since these are the 3 largest exporters of soft cheeses. A nalysis For the analysis, five 25 g subsamples were composited and analyzed by the method of Lovett et al. (1987). A composite (25 g X 5) was added to 1225 ml of FDA enrichment broth which consisted of the following: tryptone soya broth (Oxoid Canada Inc., Nepean, Ontario, Canada) 30 g; yeast extract (Difco) 6 g;
159 acriflavine hydrochloride (Sigma Chemical Co., St. Louis, MO) 15 mg; nalidixic acid (Sigma) 40 mg; cycloheximide (Sigma) 50 mg; and distilled water 1 1. The composite was blended for 2 min and then incubated at 30 ° C. After 24 and 48 h of incubation, enrichment cultures were both (a) streaked directly onto Modified McBride's Agar (MMA) made up of phenylethanol agar (Difco Laboratories, Detroit, MI) 35.5 g, glycine anhydride 10 g; lithium chloride 0.5 g; cycloheximide 200 mg and distilled water 1 1 (McBride and Girard, 1960; Lovett et al., 1987) and (b) alkali treated (1 ml into 9 ml of 0.5% KOH) and then surface streaked onto MMA. Isolation procedures and confirmatory tests
MMA plates were incubated for 48 h at 35 °C. Typical colonies (bluish gray, ground glass appearance) were selected for initial confirmation by performing the hanging drop motility test (22°C), B-hemolysis test on trypticase soy agar with 7% defibrinated sheep blood, catalase test (positive), and Gram stain. Further confirmatory tests for L. monocytogenes (reactions in parenthesis) included motility on semisolid medium (Bacto SIM Medium) with 0.6% yeast extract (+), Methyl-red and Voges-Proskauer (+), nitrate reduction ( - ) , triple sugar iron agar ( + / + , no H2S), and carbohydrate fermentation tests including dextrose (+), esculin (+), maltose (+), mannitol ( - ) , rhamnose (+), xylose ( - ) and a-methyl-D-mannoside (+). Serology was performed using Difco polyvalent, type 1 and type 4 antisera according to the manufacturer's instructions. Listeria species other than L. monotytogenes were identified according to the procedures described by Seeliger (1984). Mouse pathogenicity test
Bacterial cultures were grown for 24 h at 35°C in 10 ml tryptone soya broth (Oxoid Canada Ltd) containing 0.6% yeast extract. The cells were centrifuged (3000 rpm, 30 rain) and the pellets re-suspended in 1 ml 0.1% peptone water. Swiss CD-1 female mice weighing 16-18 g (Charles River Canada, Inc.) were then inoculated intraperitoneally (I.P.) with 0.1 ml of the bacterial suspension. Known pathogenic and nonpathogenic cultures were included as controls. Pathogenic strains killed all 5 mice inoculated, while nonpathogenic strains had no effect. Survival of Listeria in cheese
Naturally contaminated cheese samples, which when received were approx. 4 months old, were stored at 4 ° C for 8 months. The cheeses were sampled four times during this latter storage period using an MPN procedure. 10 g of cheese were aseptically removed, and added to 90 ml of sterile 0.1% peptone water in a stomacher bag. Samples were stomached for 1 min (Colworth Stomacher 400; Canadian Lab. Supplies Ltd.), with further dilutions being made in peptone water. Samples from each of three lots of brand C and one lot of brand D were analyzed (see Table II). The MPN procedure consisted of placing 1 ml of the various diluted
160 cheese suspensions into each of three tubes (per dilution) containing 4.5 ml double-strength trypticase soy broth and 0.6% yeast extract. The tubes were incubated at 3 0 ° C for 18 h and then 4.5 ml of double strength F D A enrichment broth was added to the tubes. These tubes were then incubated at 3 0 ° C for 48 h and the broth streaked onto MMA which was incubated at 35 ° C for 48 h.
Phosphatase test The phosphatase test was performed using Health Protection Branch official method MFO-3 (1981). In some regions samples were initially screened using the Scherer rapid test standard method kit (Produit de Recherche Appliqu6 Limit6e, Montreal, Canada) and then weak or strongly positive samples confirmed using method MFO-3.
Inspectional procedures Inspections of domestic cheese plants were carried out using Health Protection Branch procedures (Code of Practice, General Principles of Food Hygiene For Use By the Food Industry in Canada, 1983). The selection of soft cheese manufacturers for good manufacturing practice (GMP) assessment was based primarily upon location, i.e. within a 1 h drive of a district office. Thirty of the 74 manufacturers in Ontario and Quebec were given in-depth inspections designed to assess GMPs related to microbiological hazards such as Listeria.
Results and Discussion
None of the domestic samples contained Listeria spp. (Table I). Based upon the phosphatase test 60 out of 79 samples which were analyzed both for Listeria and for phosphatase were made from pasteurized milk. The remaining 19 samples were followed up at the manufacturing level to determine if the cheeses were held 60 days prior to sale. The follow-up identified 7 samples as satisfactory and 12 samples as unsatisfactory, the latter being in violation of Health Protection Branch regulations (Canadian Food and Drug Regulations, Minister of Supply and Services). Inspectors assessed the various steps in the processing, handling and storage of cheese to determine whether the 30 plants examined in detail were abiding by GMP. Storage of milk and other raw ingredients was considered substandard for 8 plants. Listeria, a psychrotrophic organism, if present in the milk and other ingredients could have multiplied during storage at these plants because of inadequate temperature control. It has recently been shown (Donnelly and Briggs, 1986) that some strains of L. monocytogenes can grow as quickly at 10 ° C as at 2 2 ° C in whole milk, in some instances cells increasing 6 logs in number in whole milk stored at 10 o C for 48 h. Proper pasteurization treatments were observed in 20 out of the 30 plants. Of the remaining 10 plants, 7 required further assessment related to controls over pasteuri-
161 TABLE I Analysis of domestic and imported soft and semi-soft cheeses for the presence of Listeria spp. and phosphatase Country
No. samples
No. Listeria positive samples
No. phosphatase positive samples/ total number of samples ~
Austria Canada Denmark Finland France Germany Greece Holland Italy Norway Portugal Sweden Switzerland USA
2 182 43 1 104 16 2 2 3 7 2 2 3 5
0 0 0 0 3 0 0 0 0 0 0 0 0 0
0/2 19/79 5/16 1/1 7/67 0/7 0/2 0/2 0/3 0/3 0/1 0/2 2/3 0/4
a Samples which were analyzed both for Listeria and for phosphatase.
zation, while three plants were not in compliance with Health Protection Branch regulations. Generally, the majority of the 30 plants examined were making soft and semi-soft cheese according to acceptable manufacturing practices. However, five manufacturers, all in Ontario, were in urgent need of major improvement in order to reduce the risk from Listeria contamination. As for the imported cheeses, only three samples were found to be positive for Listeria spp., with all three being manufactured by one producer in France (Tables I and II). Two different brands (brands C and D) contained L. rnonocytogenes, with brand D containing in addition Listeria innocua in 1 lot, All five packages of brand D (250 g size; same lot number) and three different lots of brand C were found to be positive for L. monocytogenes. These cheeses were stored at 4 ° C and the survival of the organism in these cheeses studied. Initial numbers of L. monocytogenes (first analysis done ca. 4 months after date of manufacture) in brand C were around 104-10 5 C F U / g . After one year's storage of the cheese at 4 ° C , Listeria was still recovered from lot numbers 1, 2 and 3, with either no decrease, a 1 log or a 2 log decrease in numbers, respectively (results not shown). It is evident that the 60-day holding period required for cheeses made from unpasteurized milk, a requirement under Canadian food and drug regulations, would not eliminate this organism. Despite label claims to the contrary, all samples found to contain Listeria were either weakly or strongly positive in the phosphatase test (Table II). Other countries which were found to be producing phosphatase-positive cheeses included France, Denmark, Switzerland and Finland (Table I).
162 TABLE II Analysis of cheeses manufactured by Plant I in France for the presence of Brand No.
A(1)
a
(2)
Listeria
spp. and phosphatase
Number of Samples Label claims (pasteurization)
Phosphatase test
Listeria
_}_
_
_
+
-
-
B
+
+
-
C
+
+
+c
D(I)
+
+
+
+/_
(2)
+ ~ b
_}_ d
Lot numbers. b Weakly positive. c L. monocytogenes. d L. innocua.
A l l c o n t a m i n a t e d cheeses were recalled f r o m the m a r k e t as soon as possible, a l t h o u g h some cheese which h a d b e e n d i s t r i b u t e d p r i o r to the recall, c o u l d n o t b e recovered. T h e r e were no illnesses r e p o r t e d f r o m the c o n s u m p t i o n of this cheese, a l t h o u g h isolated i n c i d e n t s w o u l d p r o b a b l y have b e e n missed. It is evident that a n u m b e r o f foreign a n d d o m e s t i c soft cheese m a n u f a c t u r e r s are p r o d u c i n g cheeses in which the m i l k m a y n o t b e sufficiently h e a t e d to d e s t r o y p a t h o g e n s such as L i s t e r i a . W h e n p r e s e n t in cheeses in large wedges (such as b r a n d C in our study) which are n o r m a l l y cut u p i n t o sections for sale to the c o n s u m e r , the p o s s i b i l i t y of c r o s s - c o n t a m i n a t i o n to o t h e r f o o d s a n d w o r k e r s w i t h i n retail stores exists. A l t h o u g h we d i d not detect L i s t e r i a in cheese m a n u f a c t u r e d in C a n a d a , there is a p o s s i b i l i t y of an o u t b r e a k of listeriosis or o t h e r m i l k - b o r n e diseases o c c u r r i n g if c o n d i t i o n s d o n o t i m p r o v e in a few plants. C o n t i n u e d surveillance b y r e g u l a t o r y agencies a n d b y i n d u s t r y is r e c o m m e n d e d in o r d e r to ensure the safety of soft cheeses.
Acknowledgements T h e authors a c k n o w l e d g e the c o l l a b o r a t i o n of H e a l t h P r o t e c t i o n Branch, inspectors and analysts within the F i e l d O p e r a t i o n s D i r e c t o r a t e .
References
Anon., 1981. Determination of phosphatase activity in dairy products. MFO-3. Health and Welfare Canada, Ottawa. Centers for Disease Control, 1985. Listeriosis outbreak associated with Mexican-Style cheese-California. Morb. Mortal. WKly. Rep. 34, 357-359.
163 Donnelly, C.W. and E.H. Briggs, 1986. Psychrotrophic growth and thermal inactivation of Listeria monocytogenes as a function of milk composition. J. Food Protect. 49, 994-998. Fleming, D.W., S.L. Cochi, K.L. Macdonald, J. Brondum, P.S. Hayes, B.D. Plikaytis, M.B. Holmes, A. Audurier, C.V. Broome and A.L. Reingold, 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Eng. J. Med. 312, 404-407. Gray, M.L. and A.H. Killinger, 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30, 309-382. Ho, J.L., K.N. Shands, G. Friedland, P. Eckind and D.W. Fraser, 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146, 520-524. Lovett, J., D.W. Francis and J.M. Hunt, 1987. Listeria monocytogenes in raw milk: Detection, incidence, and pathogenicity. J. Food Protect. 50, 188-192. McBride, M.E. and K.F. Girard, 1960. A selective method for the isolation of Listeria monocytogenes from mixed bacterial populations. J. Lab. Clin. Med. 55, 153-157. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls and C.V. Broome, 1983. Epidemic listeriosis - evidence for transmission by food. New Eng. J. Med. 308, 203-206. Seeliger, H.P.R., 1984. Modern taxonomy of the Listeria group relationship to its pathogenicity. Clin. Investig. Med. 7, 217-221. Seeliger, H.P.R. and D. Jones, 1986. Genus Listeria Pirie. In: Bergey's Manual of Systematic Bacteriology, edited by P.H.A. Sheath, N.S. Mair and E. Sharpe, 7th edn. Vol. 2, Williams and Wilkins, Baltimore, MD, pp. 1235-1245.
157
JFM 00171
Surveillance of soft and semi-soft cheeses for the presence of Listeria spp. J . M . F a r b e r , M . A . J o h n s t o n , U. P u r v i s a n d A. L o i t Bureau of Microbial Hazards, Ottawa, Ontario, Canada Received 22 June 1987; accepted 28 August 1987)
Recent outbreaks of listeriosis associated with dairy products prompted a survey to determine the incidence of Listeria in domestic and imported cheeses and to assess the manufacturing practices of the Canadian cheese industry. A total of 374 samples of soft and semi-soft cheeses from 61 Canadian and 98 foreign manufacturers were examined for Listeria and for phosphatase. Two samples contained Listeria monocytogenes, and one sample contained Listeria innocua. The three lots of cheese were all manufactured by one plant in France. Thirty-four samples from Canadian and foreign manufacturers gave positive phosphatase tests. Additional information confirmed that some of these cheeses were made from unpasteurized milk and were not held 60 days prior to sale. Five of 30 domestic manufacturers inspected at the time of sampling were not adhering to good manufacturing practices and were using unpasteurized milk to make cheese. Although Listeria was not found in Canadian cheese, the possibility of a Listeria outbreak occurring in Canada exists if conditions do not improve in a few plants. Continued surveillance by government and industry is recommended in order to ensure the microbiological safety of such cheeses. Key words: Listeria; Listeriosis; Cheese, soft and semi-soft; Canadian cheese industry
Introduction
Listeria monocytogenes, although known as a human pathogen for many years, has only recently become recognized as a foodborne pathogen. The organism, a Gram-positive, psychrotrophic rod, is widespread in nature and can be isolated from a variety of sources such as poor quality silage, vegetation, soil, sewage, stream water, mud, slaughter house waste, milk of normal and mastitic cows and the feces of healthy humans. In addition, Listeria spp. have been isolated from at least 37 species of mammals, 17 species of birds, flies, ticks, fish and crustaceans (Gray and Killinger, 1966; Seeliger and Jones, 1986). There have been four food-borne outbreaks of listeriosis in North America reported within the last 7 years, with two of these outbreaks involving dairy
Correspondence address: J.M. Farber, Health and Welfare Canada, Health Protection Branch, Bureau of Microbiol Hazards, Tunney's Pasture, Ottawa, Ontario, Canada K1AOL2. 0168-1605/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
158 products (Schlech et al., 1983; Centers for Disease Control, 1985; Fleming et al., 1985; Ho et al., 1986). In 1983, a listeriosis outbreak epidemiologically linked to pasteurized milk occurred in Massachusetts (Fleming et al., 1985). Forty-nine patients were involved with a fatality rate of 29%. Milk in the bulk tank of one of the farms supplying the incriminated processing plant contained L, monocytogenes serotype 4b, the same serotype as that found in 32 of 40 case isolates. In the latest outbreak occurring in the spring of 1985, Mexican-style soft cheese produced by a company in California was the incriminated vehicle (Centers for Disease Control, 1985). There were 181 cases in infant-mother pairs, with 65 deaths, and another 133 cases in other members of the population with an overall fatality rate of 33.4%. These recent outbreaks of listeriosis associated with dairy products along with the recent recalls of Listeria contaminated dairy products in the United States has prompted the Health Protection Branch of Health and Welfare Canada to conduct Listeria surveys on food. The present study was undertaken to determine whether domestically produced and imported soft and semi-soft cheeses in Canada contained Listeria spp., and also to assess the ability of the Canadian cheese industry to follow good manufacturing practices.
Materials and Methods
Survey A total of 182 domestic (61 manufacturers) and 192 imported (98 manufacturers) samples of cheese were analyzed for Listeria spp. between October, 1985 and March, 1987. In addition, the phosphatase test was done on 79 of the 182 domestic and 108 of the 192 imported samples. For the domestically produced cheeses, the majority ( > 80%) of the plants included in this survey were from Quebec and Ontario, since most of the soft cheese industry in Canada is centered in these two provinces. Two lots of domestic cheese, each lot from a different day's production, were sampled from each manufacturer. A sample consisted of 5 sample units randomly selected from each lot. Imported cheeses were obtained at the import or distribution level. No more than two lots of a product type from the same foreign manufacturer were analyzed. Samples were obtained from 12 of the 22 exporting countries that send major shipments of cheese to Canada on a regular basis. Cheeses from France, Denmark and Germany were sampled more heavily than cheeses from other countries since these are the 3 largest exporters of soft cheeses. A nalysis For the analysis, five 25 g subsamples were composited and analyzed by the method of Lovett et al. (1987). A composite (25 g X 5) was added to 1225 ml of FDA enrichment broth which consisted of the following: tryptone soya broth (Oxoid Canada Inc., Nepean, Ontario, Canada) 30 g; yeast extract (Difco) 6 g;
159 acriflavine hydrochloride (Sigma Chemical Co., St. Louis, MO) 15 mg; nalidixic acid (Sigma) 40 mg; cycloheximide (Sigma) 50 mg; and distilled water 1 1. The composite was blended for 2 min and then incubated at 30 ° C. After 24 and 48 h of incubation, enrichment cultures were both (a) streaked directly onto Modified McBride's Agar (MMA) made up of phenylethanol agar (Difco Laboratories, Detroit, MI) 35.5 g, glycine anhydride 10 g; lithium chloride 0.5 g; cycloheximide 200 mg and distilled water 1 1 (McBride and Girard, 1960; Lovett et al., 1987) and (b) alkali treated (1 ml into 9 ml of 0.5% KOH) and then surface streaked onto MMA. Isolation procedures and confirmatory tests
MMA plates were incubated for 48 h at 35 °C. Typical colonies (bluish gray, ground glass appearance) were selected for initial confirmation by performing the hanging drop motility test (22°C), B-hemolysis test on trypticase soy agar with 7% defibrinated sheep blood, catalase test (positive), and Gram stain. Further confirmatory tests for L. monocytogenes (reactions in parenthesis) included motility on semisolid medium (Bacto SIM Medium) with 0.6% yeast extract (+), Methyl-red and Voges-Proskauer (+), nitrate reduction ( - ) , triple sugar iron agar ( + / + , no H2S), and carbohydrate fermentation tests including dextrose (+), esculin (+), maltose (+), mannitol ( - ) , rhamnose (+), xylose ( - ) and a-methyl-D-mannoside (+). Serology was performed using Difco polyvalent, type 1 and type 4 antisera according to the manufacturer's instructions. Listeria species other than L. monotytogenes were identified according to the procedures described by Seeliger (1984). Mouse pathogenicity test
Bacterial cultures were grown for 24 h at 35°C in 10 ml tryptone soya broth (Oxoid Canada Ltd) containing 0.6% yeast extract. The cells were centrifuged (3000 rpm, 30 rain) and the pellets re-suspended in 1 ml 0.1% peptone water. Swiss CD-1 female mice weighing 16-18 g (Charles River Canada, Inc.) were then inoculated intraperitoneally (I.P.) with 0.1 ml of the bacterial suspension. Known pathogenic and nonpathogenic cultures were included as controls. Pathogenic strains killed all 5 mice inoculated, while nonpathogenic strains had no effect. Survival of Listeria in cheese
Naturally contaminated cheese samples, which when received were approx. 4 months old, were stored at 4 ° C for 8 months. The cheeses were sampled four times during this latter storage period using an MPN procedure. 10 g of cheese were aseptically removed, and added to 90 ml of sterile 0.1% peptone water in a stomacher bag. Samples were stomached for 1 min (Colworth Stomacher 400; Canadian Lab. Supplies Ltd.), with further dilutions being made in peptone water. Samples from each of three lots of brand C and one lot of brand D were analyzed (see Table II). The MPN procedure consisted of placing 1 ml of the various diluted
160 cheese suspensions into each of three tubes (per dilution) containing 4.5 ml double-strength trypticase soy broth and 0.6% yeast extract. The tubes were incubated at 3 0 ° C for 18 h and then 4.5 ml of double strength F D A enrichment broth was added to the tubes. These tubes were then incubated at 3 0 ° C for 48 h and the broth streaked onto MMA which was incubated at 35 ° C for 48 h.
Phosphatase test The phosphatase test was performed using Health Protection Branch official method MFO-3 (1981). In some regions samples were initially screened using the Scherer rapid test standard method kit (Produit de Recherche Appliqu6 Limit6e, Montreal, Canada) and then weak or strongly positive samples confirmed using method MFO-3.
Inspectional procedures Inspections of domestic cheese plants were carried out using Health Protection Branch procedures (Code of Practice, General Principles of Food Hygiene For Use By the Food Industry in Canada, 1983). The selection of soft cheese manufacturers for good manufacturing practice (GMP) assessment was based primarily upon location, i.e. within a 1 h drive of a district office. Thirty of the 74 manufacturers in Ontario and Quebec were given in-depth inspections designed to assess GMPs related to microbiological hazards such as Listeria.
Results and Discussion
None of the domestic samples contained Listeria spp. (Table I). Based upon the phosphatase test 60 out of 79 samples which were analyzed both for Listeria and for phosphatase were made from pasteurized milk. The remaining 19 samples were followed up at the manufacturing level to determine if the cheeses were held 60 days prior to sale. The follow-up identified 7 samples as satisfactory and 12 samples as unsatisfactory, the latter being in violation of Health Protection Branch regulations (Canadian Food and Drug Regulations, Minister of Supply and Services). Inspectors assessed the various steps in the processing, handling and storage of cheese to determine whether the 30 plants examined in detail were abiding by GMP. Storage of milk and other raw ingredients was considered substandard for 8 plants. Listeria, a psychrotrophic organism, if present in the milk and other ingredients could have multiplied during storage at these plants because of inadequate temperature control. It has recently been shown (Donnelly and Briggs, 1986) that some strains of L. monocytogenes can grow as quickly at 10 ° C as at 2 2 ° C in whole milk, in some instances cells increasing 6 logs in number in whole milk stored at 10 o C for 48 h. Proper pasteurization treatments were observed in 20 out of the 30 plants. Of the remaining 10 plants, 7 required further assessment related to controls over pasteuri-
161 TABLE I Analysis of domestic and imported soft and semi-soft cheeses for the presence of Listeria spp. and phosphatase Country
No. samples
No. Listeria positive samples
No. phosphatase positive samples/ total number of samples ~
Austria Canada Denmark Finland France Germany Greece Holland Italy Norway Portugal Sweden Switzerland USA
2 182 43 1 104 16 2 2 3 7 2 2 3 5
0 0 0 0 3 0 0 0 0 0 0 0 0 0
0/2 19/79 5/16 1/1 7/67 0/7 0/2 0/2 0/3 0/3 0/1 0/2 2/3 0/4
a Samples which were analyzed both for Listeria and for phosphatase.
zation, while three plants were not in compliance with Health Protection Branch regulations. Generally, the majority of the 30 plants examined were making soft and semi-soft cheese according to acceptable manufacturing practices. However, five manufacturers, all in Ontario, were in urgent need of major improvement in order to reduce the risk from Listeria contamination. As for the imported cheeses, only three samples were found to be positive for Listeria spp., with all three being manufactured by one producer in France (Tables I and II). Two different brands (brands C and D) contained L. rnonocytogenes, with brand D containing in addition Listeria innocua in 1 lot, All five packages of brand D (250 g size; same lot number) and three different lots of brand C were found to be positive for L. monocytogenes. These cheeses were stored at 4 ° C and the survival of the organism in these cheeses studied. Initial numbers of L. monocytogenes (first analysis done ca. 4 months after date of manufacture) in brand C were around 104-10 5 C F U / g . After one year's storage of the cheese at 4 ° C , Listeria was still recovered from lot numbers 1, 2 and 3, with either no decrease, a 1 log or a 2 log decrease in numbers, respectively (results not shown). It is evident that the 60-day holding period required for cheeses made from unpasteurized milk, a requirement under Canadian food and drug regulations, would not eliminate this organism. Despite label claims to the contrary, all samples found to contain Listeria were either weakly or strongly positive in the phosphatase test (Table II). Other countries which were found to be producing phosphatase-positive cheeses included France, Denmark, Switzerland and Finland (Table I).
162 TABLE II Analysis of cheeses manufactured by Plant I in France for the presence of Brand No.
A(1)
a
(2)
Listeria
spp. and phosphatase
Number of Samples Label claims (pasteurization)
Phosphatase test
Listeria
_}_
_
_
+
-
-
B
+
+
-
C
+
+
+c
D(I)
+
+
+
+/_
(2)
+ ~ b
_}_ d
Lot numbers. b Weakly positive. c L. monocytogenes. d L. innocua.
A l l c o n t a m i n a t e d cheeses were recalled f r o m the m a r k e t as soon as possible, a l t h o u g h some cheese which h a d b e e n d i s t r i b u t e d p r i o r to the recall, c o u l d n o t b e recovered. T h e r e were no illnesses r e p o r t e d f r o m the c o n s u m p t i o n of this cheese, a l t h o u g h isolated i n c i d e n t s w o u l d p r o b a b l y have b e e n missed. It is evident that a n u m b e r o f foreign a n d d o m e s t i c soft cheese m a n u f a c t u r e r s are p r o d u c i n g cheeses in which the m i l k m a y n o t b e sufficiently h e a t e d to d e s t r o y p a t h o g e n s such as L i s t e r i a . W h e n p r e s e n t in cheeses in large wedges (such as b r a n d C in our study) which are n o r m a l l y cut u p i n t o sections for sale to the c o n s u m e r , the p o s s i b i l i t y of c r o s s - c o n t a m i n a t i o n to o t h e r f o o d s a n d w o r k e r s w i t h i n retail stores exists. A l t h o u g h we d i d not detect L i s t e r i a in cheese m a n u f a c t u r e d in C a n a d a , there is a p o s s i b i l i t y of an o u t b r e a k of listeriosis or o t h e r m i l k - b o r n e diseases o c c u r r i n g if c o n d i t i o n s d o n o t i m p r o v e in a few plants. C o n t i n u e d surveillance b y r e g u l a t o r y agencies a n d b y i n d u s t r y is r e c o m m e n d e d in o r d e r to ensure the safety of soft cheeses.
Acknowledgements T h e authors a c k n o w l e d g e the c o l l a b o r a t i o n of H e a l t h P r o t e c t i o n Branch, inspectors and analysts within the F i e l d O p e r a t i o n s D i r e c t o r a t e .
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