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Microbial safety of cheese in Canada
International Journal of Food Microbiology 321 (2020) 108521
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbial safety of cheese in Canada
T
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Kyle Ganz , Etsuko Yamamoto, Kate Hardie, Christine Hum, Hussein Hussein, Annie Locas, Marina Steele Food Safety Science Directorate, Science Branch, Canadian Food Inspection Agency, 1400 Merivale Road, Ottawa, ON, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Raw-milk Salmonella L. monocytogenes E. coli S. aureus Surveillance
A profile of the microbial safety of cheese in Canada was established based on the analysis of 2955 pasteurized and raw-milk cheeses tested under Canada's National Microbiological Monitoring Program (NMMP) and 2009 raw-milk cheeses tested under the Targeted Survey Program. 97.8% of NMMP and 99.6% of Targeted Survey cheese samples were assessed as being of satisfactory microbiological safety. Under the NMMP, Salmonella spp. was detected in 2 samples, Listeria monocytogenes was detected in 15 samples and no Escherichia coli O157/ H7:NM (non-motile) was detected. Cheese samples assessed as having unsatisfactory levels of S. aureus and generic E. coli were found in 18 and 41 samples, respectively. Under the Targeted Survey, L. monocytogenes was detected in 2 samples, while no Salmonella spp. or E. coli O157/H7:NM were detected. Cheese samples assessed as having investigative and unsatisfactory levels of S. aureus were found in 4 and 2 samples respectively. No samples were found to have investigative or unsatisfactory levels of generic E. coli. For cheese samples collected under the NMMP, logistic regression models indicated that contamination was more frequent in raw-milk cheeses compared to pasteurized-milk cheeses (OR = 5.0, 95% CI (3.0, 8.3)), and in imported cheeses compared to domestic cheeses (OR = 8.2, 95% CI (4.1, 16.1)). A statistically significant association was found between cheese samples assessed as having unsatisfactory levels of generic E. coli and detection of L. monocytogenes, Salmonella spp. or levels of S. aureus that were assessed as unsatisfactory (p < .001). These test results will help support risk analysis and inform food safety decisions.
1. Introduction Cheese is an important nutritional component to the Canadian diet. Approximately 466 million kilograms of cheese were produced domestically and approximately 27 million kilograms of cheese were imported into Canada in 2016 (AAFC, 2017). The average Canadian was reported to consume 15.07 kg of cheese in 2016 (AAFC, 2017). Since 1984, there have been approximately 12 documented Canadian foodborne outbreaks associated with cheeses (PHAC, 2018). Five of these outbreaks were attributed to Salmonella spp., four were attributed to Escherichia coli O157:H7 and three were attributed to Listeria monocytogenes. All four outbreaks attributed to E. coli O157:H7, and half of the total outbreaks, were linked to raw-milk cheeses. Worldwide, these pathogens, along with Staphylococcus aureus have also been linked to multiple cheese outbreaks (Dominguez et al., 2009; Gould et al., 2014; Johler et al., 2015). The European Food Safety Authority found that 4.8% of foodborne outbreaks which occurred in the European Union in 2016 were associated with cheeses (European Food Safety Authority EFSA and European Centre for Disease Prevention and Control ECDC,
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2017). Overall, this indicates that cheese is a potential source of foodborne illness and that Salmonella spp., E. coli O157:H7, L. monocytogenes and S. aureus have been identified as causative agents. The Canadian Food Inspection Agency (CFIA) monitors the microbiological safety of imported and domestic cheeses in Canada under two different surveillance programs. The first of these programs is the National Microbiological Monitoring Program (NMMP), under which a wide variety of raw and pasteurized-milk cheeses were collected and tested between April 2012 and March 2017. The second program is the Targeted Surveys program. Targeted Surveys are studies of a finite period, performed to address specific data gaps and primarily focus on areas of highest health risk. Results from a microbiological Targeted Survey of raw-milk cheeses, sampled at retail establishments, between November 2014 and March 2018 were also included in this study. The objective of this study is to summarize results from these CFIA Food Microbial Surveillance Programs to provide a comprehensive profile of the microbial safety of cheese in Canada.
Corresponding author at: Floor 6, Room 244, 1400 Merivale Road, Tower 2, Ottawa, Ontario K1A 0Y9, Canada. E-mail address: [email protected] (K. Ganz).
https://doi.org/10.1016/j.ijfoodmicro.2020.108521 Received 20 June 2019; Received in revised form 27 September 2019; Accepted 12 January 2020 Available online 24 January 2020 0168-1605/ Crown Copyright © 2020 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
International Journal of Food Microbiology 321 (2020) 108521
K. Ganz, et al.
2. Materials and methods
Government of Canada's Food and Drug Act prohibits the sale of food containing any poisonous or harmful substance, therefore, detection of the pathogens Salmonella spp. or E. coli O157:H7/NM resulted in an unsatisfactory assessment. Samples in which the pathogen L. monocytogenes was detected were assessed in accordance with Health Canada's Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2011). According to this policy, no L. monocytogenes is permitted in Category 1 foods, which are defined as ready-to-eat foods in which growth of L. monocytogenes can occur throughout the stated shelf-life. Results of pH and water activity analyses were primarily used to make the Category determination. Assessment of the levels of generic E. coli and S. aureus in cheese samples was performed according to the assessment criteria for raw and pasteurized-milk cheeses outlined in the Health Products and Food Branch (HPFB) Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary (Health Canada, 2008). An investigative assessment was assigned to Targeted Survey samples where the levels of generic E.coli and S. aureus were found to be above the marginally acceptable level (m) but below the unacceptable level (M) (Table 1). A cheese sample was considered to be of satisfactory microbiological safety if all individual microbiological test results were considered satisfactory based on the assessment criteria outlined in Table 1, while a cheese sample was considered to be of unsatisfactory microbiological safety if one or more individual microbiological test results were unsatisfactory. A cheese sample was considered to be of investigative microbiological safety if an investigative assessment was given to one or more individual microbiological tests and all other analyses were satisfactory. All samples assessed as unsatisfactory and investigative were subject to appropriate follow-up actions by both industry and the CFIA.
2.1. Sample collection and analysis NMMP data from 595 raw-milk cheese samples (92 domestic and 503 imported) and 2360 pasteurized-milk cheese samples (1659 domestic and 701 imported) collected between April 2012 and March 2017 were assessed. NMMP cheese samples were collected at domestic cheese establishments (production facilities, packing facilities and warehouses) and import warehouses across all Canadian provinces by CFIA inspectors. Finished products in intact packaging was preferentially sampled, however, bulk products, such as cheese wheels, were occasionally sampled as well using aseptic techniques. Cheese samples were collected under sampling plans targeting a variety of cheese types. Each sample comprised five 200 g subunits that were randomly collected from each lot of cheese sampled. A random sample is one in which all elements in the lot have an equal and independent chance of being included in the sample. Targeted Survey data from 2009 raw-milk cheese samples (390 domestic and 1619 imported) collected between November 30, 2014 and March 31, 2018 were assessed. These cheese samples were collected from national retail chains and local/regional grocery stores located in 11 major cities across Canada. The number of samples collected from these cities was in proportion to the relative population of the respective areas. Each sample comprised either a single or multiple unit (s) of a single lot weighing a total of at least 250 g. Testing was performed in ISO 17025 accredited CFIA laboratories using methods validated in accordance with Health Canada's Compendium of Analytical Methods (Health Canada, 2019). These methods used for analysis are as follows: MFHPB-20, MFLP-29 and MFLP-40 for Salmonella spp.; MFHPB-10, MFLP-30 and MFLP-80 for E. coli O157:H7; MFHPB-30, MFLP-53, MFLP-74 and MFLP-77 for L. monocytogenes; MFHPB-19, MFHPB-34 and Compact Dry EC medium count plates for generic E. coli; and MFHPB-21 for S. aureus. Enumeration of generic E. coli using Compact Dry EC medium count plates (HyServe GmbH & Co. KG) was performed by diluting 1 part cheese sample with 10 parts buffered peptone water, mixing thoroughly, plating 1 ml of diluted sample and incubating at 42 °C for 24 ± 2 h. Both NMMP and Targeted Survey samples were tested for the presence of Salmonella spp., L. monocytogenes (all samples) and E. coli O157:H7/ NM (non-motile) (raw-milk cheese samples only). If L. monocytogenes was detected, levels of L. monocytogenes were enumerated as colony forming units per gram (CFU/g) and the pH and water activity of the cheese was measured to determine the potential of the cheese to support growth of L. monocytogenes, as described in Health Canada's Policy on L. monocytogenes in Ready-to-Eat Foods (Health Canada, 2011). Levels of generic E. coli were enumerated as CFU/g or most probable number per gram (MPN/g), depending on the methodology used, and levels of S. aureus were enumerated as CFU/g. Generally, the manner in which these methods were applied for the analysis of cheese provided limit of detections of 1.8 MPN/g for generic E. coli, 25 CFU/g for S. aureus and 5 CFU/g for L. monocytogenes. An equal portion (25 g) of each of the five sub-units collected under the NMMP, were aseptically combined into a single composite sample (125 g) for each analysis performed. For the Targeted Survey, 25 g of the sample was tested. Samples collected during the initial 4 month period of the Targeted Survey were only analyzed for one microorganism (E. coli O157:H7/ NM).
2.3. Determination of cheese pasteurization status, origin and class Sample information on the pasteurization status, country of origin and product type was recorded at the time of sample collection. For NMMP samples this information was recorded as part of the sample description by CFIA inspectors, whereas for Targeted Surveys this information was derived from information on the product labels. Based on this recorded information, the cheese samples were classified as rawmilk or pasteurized-milk cheeses, and of domestic or imported origin and were assigned to one of seven cheese classes (Classes A to G) using the cheese classification scheme developed by Ottogalli (Ottogalli, 1998, 2000) (Table 2). The Ottogalli cheese classification scheme was selected because it is based on the microbiological aspects of cheese and cheese production and because it includes an extensive list of preclassified cheeses that were used to classify the cheese samples. The preclassified list classified some cheese types (e.g., Gouda, Edam, etc.) into two different classes due to large variations in their physical properties (e.g., moisture content and pH) and ripening times (e.g., 3–6 months for Gouda). As this type of detailed information on cheese samples was not captured, it was not possible to classify some of these cheese types, therefore they were placed in the unclassified group. A small number of additional cheese samples did not match the exact name of a cheese on the pre-classified list but were classified based on their similarity to a pre-classified cheese on this list (Table 2, footnotes 1–3). Overall, a total of 67.2% of cheeses within the NMMP dataset and 80.8% of the cheeses within the Targeted Survey dataset were assigned a cheese class. 2.4. Statistical analyses
2.2. Sample assessment Due to the differences in the design of the NMMP and Targeted Survey programs, results from these two programs were analyzed separately. Due to the small number of unsatisfactory samples found in the Targeted Survey data set, comparative statistical analyses of relationships between variables were not performed. Analyses were performed using Stata/SE version 15 (StataCorp, College Station, TX). For
NMMP and Targeted Survey test results were assessed as being of satisfactory, unsatisfactory, or investigative microbiological safety using assessment criteria outlined in Table 1, which are based on Canadian Acts, Regulations and policies as described below (Government of Canada, 2018, 2019; Health Canada, 2011). Section 4(1)a of the 2
International Journal of Food Microbiology 321 (2020) 108521
K. Ganz, et al.
Table 1 Microbiological assessment criteria used to assess the microbiological safety of cheese analyzed under the National Microbiological Monitoring Program and Targeted Survey Program. na
ca
ma
Ma
Satisfactory
Investigative
Unsatisfactory
Assessment criteria for Salmonella spp., E. coli O157:H7 and L. monocytogenes – Not detected Salmonella spp. 5/1b 0 0 E. coli O157:H7 5/1b 0 0 – Not detected L. monocytogenesc 5/1b 0 0 – Not detected
n/a n/a n/a
Detected Detected Detected
Assessment criteria for generic E. coli NMMP raw-milk cheeses 5
2
5 × 102
2 × 103
n/a
NMMP pasteurized-milk cheeses
5
2
102
2 × 103
Targeted Survey raw-milk cheeses
1
–
5 × 102
2 × 103
≤M in any subunit and if > m then in ≤c subunits ≤M in any subunit and if > m then in ≤c subunits ≤m
> M in any subunit or > m in > c subunits > M in any subunit or > m in > c subunits >M
Assessment criteria for S. aureus NMMP raw-milk cheeses
5
2
103
104
n/a
NMMP pasteurized-milk cheeses
5
2
102
104
Targeted Survey raw milk cheeses
1
–
103
104
≤M in any subunit and if > m then in ≤c subunits ≤M in any subunit and if > m then in ≤c subunits ≤m
n/a > m to ≤M
n/a > m to ≤M
> M in any subunit or > m in > c subunits > M in any subunit or > m in > c subunits >M
a
As described in Health Canada's Health Products and Food Branch Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary (Health Canada, 2008). b 5 subunits of 25 g were tested for NMMP samples and 1 unit of 25 g was tested for Targeted Survey samples. c All NMMP and Targeted Survey cheese samples in which L. monocytogenes was detected were category 1 foods for which there is no acceptable level of L. monocytogenes.
3. Results
the NMMP data set, chi-square tests were used to determine if there were associations between the assessment status of the samples (i.e. satisfactory or unsatisfactory) and sample variables (i.e., pasteurization status, origin and cheese class). The chi-squared test was also used to determine if there was an association between an unsatisfactory assessment due to levels of generic E. coli that exceed the assessment criteria and detection of pathogens. For the purpose of this analysis, pvalues < 0.05 were considered to be statistically significant. Regression modelling, using an unsatisfactory assessment as the dependent variable, was performed to further explore these relationships and generate odds ratios (OR). A multivariable logistic regression model was built using pasteurization status and origin as predictor variables, and variable significance was tested using Wald tests.
3.1. Sample assessment Under the NMMP, 2955 cheese samples were collected. Of these cheese samples, 97.8% (n = 2891) were assessed as being of satisfactory microbiological safety. In contrast, 2.2% (n = 64) of samples were assessed as being of unsatisfactory microbiological safety. Chi-square analysis indicated that there was no statistically significant relationship between the year of sampling and the frequency of unsatisfactory samples in an individual year (p = 0.45). The prevalence of Salmonella spp. and L. monocytogenes in cheeses sampled under the NMMP were found to be 0.1% (n = 2) and 0.5% (n = 15), respectively (Table 3). All 15 L. monocytogenes positive cheese samples were determined by pH and water activity to belong to
Table 2 Classification of cheese samples. Class
Common types
A: Fresh soft cheeses, practically amicrobial Rarely ripened, rennet-free, and amicrobial due to rapid coagulation by organic acids. B: Fresh soft cheeses, rich in lactic acid bacteria Unripened, rennet coagulated, and use biological acidification.
Ricotta Panir/Paneer Queso Frescoa Mozzarella Goat Bocconcinib Burratab Feta Halloumi Brie Camembert Blue Cheese Roquefort Raclette Monterey Cheddar Swissc
C: Short ripened soft cheeses Soft or semi-soft consistency that are usually rindless or have a thin rind. D: Soft surface ripened cheeses Surface microflora ripen the cheese from the outside surface towards the center. E: Blue cheeses Soft or semi-soft, blue veined due to the growth of mycelia and spores of Penicillia. F: Semi-hard cheeses Semi-hard cheeses with medium ripening time G: Hard cheeses Hard consistency cheeses with long ripening time Unclassified Total samples a b c
NMMP cheese samples Pasteurized
Raw
79
0
0
440
0
5
180
2
0
194
102
2
107
68
136
89
47
18
518
159
1463
753 2955
217
385 2009
13 Queso Fresco samples were classified as class A based on similarity to Queso Blanco. 20 Burrata and 6 Bocconcini cheese samples were classified as class B based on similarity to Mozzarella. 31 Swiss cheese samples were classified as class G based on similarity to Emmental cheese. 3
Targeted Survey raw-milk cheese samples
International Journal of Food Microbiology 321 (2020) 108521
K. Ganz, et al.
Table 3 Proportion of NMMP and Targeted Survey cheese samples assessed as being of unsatisfactory microbiological safety by analysis. National Microbiological Monitoring Program
Targeted Survey on raw-milk cheese
Analyte
Unsatisfactory
n
Occurrence
Unsatisfactory
n
Occurrence
E. coli O157:H7/NM Salmonella spp. L. monocytogenes Unsatisfactory levelsa of S. aureus Unsatisfactory levelsa of generic E. coli
0 2 15 18 41
600 2953 2954 2948 2953
0% 0.1% 0.5% 0.6% 1.4%
0 0 2 2 0
2009 1723 1723 1723 1723
0% 0% 0.1% 0.1% 0%
a
As described in Table 1 and in Health Canada's Health Products and Food Branch Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary.
safety on the basis of one or more microbial analyses. Statistical analysis indicated that raw-milk cheese samples are more likely to be of unsatisfactory microbiological safety compared to pasteurized-milk cheese samples. The 29 pasteurized-milk NMMP cheese samples assessed as being of unsatisfactory microbiological safety were based on the results of 32 unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 21), while the remaining were due to the presence of L. monocytogenes (n = 9) and levels of S. aureus (n = 2). The 35 raw-milk NMMP cheese samples of unsatisfactory microbiological safety were based on the results of 44 unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 20) and levels of S. aureus (n = 16) which exceeded the levels specified in the assessment criteria, followed by the presence of L. monocytogenes (n = 6) and Salmonella spp. (n = 2). When stratified by microorganism, the relationship between pasteurization status and unsatisfactory assessment remained statistically significant.
category 1 as per Health Canada's Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2011), i.e. supporting the growth of L. monocytogenes. Enumeration of L. monocytogenes in these samples revealed levels ranging from < 5 to 630 CFU/g with 9 of the 15 samples containing levels below the limit of detection. E. coli O157:H7/NM was not detected in any of the samples. Unsatisfactory levels of generic E. coli and S. aureus were observed in 41 and 18 of the samples, respectively. Levels of generic E. coli ranging from < 1.8 MPN/g to 6.2 × 104 CFU/g and levels of S. aureus ranging from < 23 to 4.3 × 105 CFU/g were observed in all cheese samples tested under the NMMP. Of the 41 cheese samples assessed as having unsatisfactory levels for generic E. coli, a pathogen was also detected in 11 of these samples. A chi-squared test demonstrated an association between samples assessed as having unsatisfactory levels of generic E. coli and those containing a pathogen (p < 0.001). This association was also observed when raw-milk and pasteurized-milk cheeses were analyzed separately (p < 0.001). Under the Targeted Survey, 2009 raw-milk cheese samples were collected. Of the cheese samples analyzed, 99.6% (n = 2001) were assessed as being of satisfactory microbiological safety. Four (0.2%) samples were assessed as being of unsatisfactory microbiological safety (Table 3) and four samples (0.2%) with levels of S. aureus between 103 and 104 CFU/g were assessed as investigative. Of the four samples assessed as unsatisfactory, two samples (0.1%) were positive for L. monocytogenes with levels < 5 CFU/g and two samples (0.1%) had unsatisfactory levels of S. aureus > 104 CFU/g. Levels of S. aureus ranging from < 25 to 8.7 × 105 CFU/g and levels of generic E. coli ranging from < 1.8 to < 50 CFU/g were observed in all cheese samples tested under the Targeted Survey.
3.3. Associations between assessment status and origin Of the 2955 samples analyzed under the NMMP, 59.3% (n = 1751) cheeses were of domestic origin and 40.7% (n = 1204) were imported. A total of 10 domestic cheese samples, representing 0.6% of the domestic total (95% CI, 0.3–1.1%), and 54 imported cheese samples, representing 4.5% of the imported total (95% CI, 3.4–5.8%), were assessed as being of unsatisfactory microbiological safety (Table 5). Statistical analysis indicated that imported cheese samples are more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples. The 10 domestic NMMP cheese samples of unsatisfactory microbiological safety were assessed as unsatisfactory based on the results of 10 unsatisfactory analyses. The majority of these analyses were unsatisfactory due to levels of generic E. coli (n = 6), while the remaining were unsatisfactory due to the presence of L. monocytogenes (n = 2) or levels S. aureus (n = 2). The 54 imported NMMP cheese samples of unsatisfactory microbiological safety were based on the results of 66
3.2. Associations between assessment status and pasteurization status of the milk Of the 2955 samples analyzed under the NMMP, 2360 cheeses were made with pasteurized milk and 595 cheeses were made with raw-milk. As shown in Table 4, 29 pasteurized-milk cheese samples, representing 1.2% (95% CI, 0.8–1.8%) of the total pasteurized cheese samples tested under the NMMP, and 35 raw-milk cheese samples, representing 5.9% (95% CI, 4.1–8.1%) of the total raw-milk cheese samples tested under the NMMP, were assessed as being of unsatisfactory microbiological
Table 5 Unsatisfactory cheese samples tested under the National Microbiological Monitoring Program by the origin of the cheese. Origin
Table 4 Cheese samples assessed as being of unsatisfactory microbiological safety tested under the National Microbiological Monitoring Program by the pasteurization status of the milk. Pasteurization status
Unsatisfactory
n
Proportion
95% CI
Pasteurized-milk cheese Raw-milk cheese
29 35
2360 595
1.2% 5.9%a
0.8–1.8% 4.1–8.1%
Domestic pasteurized-milk cheese Domestic raw-milk cheese All domestic cheese Imported pasteurized-milk cheese Imported raw-milk cheese All imported cheese
a A logistic regression model indicated that raw-milk cheese samples were more likely to be of unsatisfactory microbiological safety compared to pasteurized-milk cheese samples (OR = 5.0, 95% CI (3.0, 8.3)).
Unsatisfactory
n
Proportion
95% CI
8
1659
0.5%
0.2–0.9%
2 10 21
92 1751 701
2.2% 0.6% 3.0%
0.6–7.6% 0.3–1.1% 1.9–4.5%
33 54
503 1204
6.6% 4.5%a
4.6–9.1% 3.4–5.8%
a A logistic regression model indicated that imported cheese samples were more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples (OR = 8.18, 95% CI (4.1, 16.1)).
4
International Journal of Food Microbiology 321 (2020) 108521
K. Ganz, et al.
unsatisfactory NMMP samples by class was not performed due to the large proportion of NMMP cheese samples which could not be classified into Ottogalli cheese classes.
unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 35) and levels S. aureus (n = 16) which exceeded the levels specified in the assessment criteria, followed by the presence of L. monocytogenes (n = 13) and Salmonella spp. (n = 2). When stratified by microorganism, the relationship between import status and unsatisfactory assessment remained statistically significant for generic E. coli, L.monocytogenes, and S. aureus. Analyses indicated that samples of unsatisfactory microbiological safety collected under the NMMP were more frequently associated with raw-milk cheeses and imported cheeses. Since a higher proportion of raw-milk cheeses were imported (84.5%) compared to being produced domestically (15.5%), additional chi-square tests were performed to determine if an association between unsatisfactory assessment and rawmilk cheeses was still observed when the origin of the cheeses was controlled. It was observed that within domestic cheeses, raw-milk cheeses were still more frequently associated with samples of unsatisfactory microbiological safety, and this was shown to be statistically significant (p = 0.04). Within imported cheeses, raw-milk cheeses were also more frequently associated with samples of unsatisfactory microbiological safety, and this was shown to be statistically significant (p = 0.003). A logistic regression model was built to explore the associations when both the origin and pasteurization status as predictors are considered. Both variables remain significant in the model when controlling for the other, with an OR of 5.55 for origin status (p < 0.001, 95% CI 2.67–11.52) and an OR of 2.43 for pasteurization status (p = 0.001, 95% CI 1.42–4.18). Of the 2009 raw-milk cheese samples analyzed under the Targeted Survey, 80.6% (n = 1619) of samples were of imported origin and 19.4% (n = 390) were of domestic origin. The 4 samples assessed as being of unsatisfactory microbiological safety were of imported origin.
4. Discussion Under the NMMP, 94.1% of raw-milk cheeses and 98.8% of pasteurized-milk cheeses were assessed as being of satisfactory microbiological safety and under the Targeted Survey, 99.6% of the raw-milk cheese samples were assessed as being of satisfactory microbiological safety. These results suggest that the vast majority of cheeses on the Canadian marketplace are of good microbiological safety. Cheese contamination has previously been found to occur at various points in the cheese making process such as from contaminated input ingredients, introduced during unsanitary manufacturing processes or during postprocessing (Fox and McSweeney, 2004). E. coli O157:H7/NM was not found in any of the raw-milk cheeses sampled under the two programs examined in this study. This is consistent with a recent study of 1606 domestic and imported raw-milk cheeses collected at manufacturers, distribution centers and retail stores in the United States (USFDA, 2016), for which no E. coli O157:H7 was detected. However, several international (Gould et al., 2014) and Canadian (Currie et al., 2018; Gaulin et al., 2012; Honish et al., 2005) foodborne outbreaks of E. coli O157:H7 have been associated with raw-milk cheeses. Considering the ready-to-eat nature of cheese, the relatively low infectious dose (Teunis et al., 2008), and the severity of illness (Pennington, 2010), the presence of any level of E. coli O157:H7 in cheese remains a concern to public health. Salmonella spp. was not detected in any of the Targeted Survey samples and was detected in 0.1% of the NMMP samples. These two cheese samples were raw-milk cheeses. This is consistent with the recent study of raw-milk cheeses in the United States (USFDA, 2016) where Salmonella spp. was detected in 0.19% of samples. Considering the ready-to-eat nature of cheese, the relatively low infectious dose of certain serovars of Salmonella spp. (D'Aoust, 1994) and the severity of illness (Thomas et al., 2013), the presence of any level of Salmonella spp. in cheese remains a concern to public health. L. monocytogenes was detected in 0.5% of NMMP samples and 0.1% of Targeted Survey samples. This is consistent with recent international studies on the prevalence of L. monocytogenes in cheeses at retail, which revealed prevalence rates of 0% in soft cheese in Greece (Angelidis et al., 2012), 0.4% in soft and semi-soft cheeses in Sweden (Lambertz et al., 2012), 1.6% in pasteurized-milk cheeses in Portugal (Mena et al., 2004), and 2.7% in hard cheeses in the United Kingdom (Little et al., 2009). The recent study on raw-milk cheeses in the United States (USFDA, 2016) found a prevalence of L. monocytogenes of 0.62%. Results of pH and water activity analyses indicated that the cheeses
3.4. Distribution of samples assessed as unsatisfactory by type of cheese class The proportion of cheese samples assessed as being of unsatisfactory microbiological safety in each class for the NMMP samples is shown in Fig. 1. Class D: soft surfaced ripened cheeses, and Class F: semi-hard cheeses, contained the highest proportion of cheese samples of unsatisfactory microbiological safety at 5.1% (95% CI, 2.9–8.2%) and 6.6% (95% CI, 3.1–12.2%), respectively. Class G: hard cheeses, had the lowest proportion of cheese samples of unsatisfactory microbiological safety with 0.6% (95% CI, 0.1–1.5%). Of the 4 samples assessed as being of unsatisfactory microbiological safety under the Targeted Survey, two were from Class G and two were unclassified. The vast majority of the Targeted survey samples were Class G (72.8%) or unclassified (19.2%) (Table 2). Comparative analysis on the proportion of 7.0
n=136
Unsatisfactory Samples (%)
6.0 n=296 5.0 4.0 3.0
n=79
n=440
n=182 n=970
n=175
2.0 1.0
n=677
0.0 A: Fresh soft cheeses, amicrobial
B: Fresh soft cheeses, lactic acid bacteria
C: Short ripened soft cheeses
D: Soft surface ripened cheeses
E: Blue cheeses
F: Semi-hard cheeses
G: Hard cheeses
Unclassified
Fig. 1. Cheese samples assessed as being of unsatisfactory microbiological safety under the National Microbiological Monitoring Program by Class (n = total number of samples tested per class). 5
International Journal of Food Microbiology 321 (2020) 108521
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have been aged for 60 days, which likely accounts for this observed bias against unripened raw-milk cheeses. The frequency of samples of unsatisfactory microbiological safety in each class was compared. For the NMMP samples, Class D, the soft surfaced ripened cheeses, and Class F, the semi-hard cheeses, were observed to contain higher proportions of samples of unsatisfactory microbiological safety (Fig. 1). Class G, the hard cheeses, contained the fewest samples of unsatisfactory microbiological safety. In contrast to the NMMP samples, the majority of Targeted Survey samples belonged to Class G (72.8%) and the unclassified group (19.2%) (Table 2). This may explain why fewer samples of unsatisfactory or investigative microbiological safety were detected in the Targeted Survey when compared to the NMMP. The Targeted Survey samples assessed as of either unsatisfactory or investigative microbiological safety were distributed between Class G and the unclassified group. These results suggest that some classes of cheeses may be more susceptible to microbial contamination and/or more supportive of bacterial growth. The Ottogalli cheese classes were based on the microbiological aspects of cheese and cheese production, thus these differences likely reflect differences in how these cheeses were produced and the different inherent microbial barriers to growth. Lastly, we observed that the same cheese types and brands were often represented by multiple samples in the dataset. As samples were collected as part of our randomly sampled NMMP and Targeted sampling plan by different samplers over several years, we would expect that more widely available cheese types and brands may be more represented at higher levels than less widely available products. Of the 64 NMMP samples that were assessed as unsatisfactory, 24 samples were of the same cheese type and/or brand as at least one other unsatisfactory cheese sample. None of these samples were of the same lot of cheese. Of the 8 Targeted Survey cheese samples that were assessed as either unsatisfactory or investigative, 5 were of the same cheese type and of these 5, 4 were of the same brand and of these 4, 3 were of the same lot. Without detailed knowledge of the market share that these cheese types represent in Canada, it was not possible to assess if these cheese types and brands were over-represented in the dataset. These cheese samples were assessed as unsatisfactory or investigative due to levels of generic E. coli and/or S. aureus and L. monocytogenes. In summary, the microbiological safety of cheese on the Canadian marketplace, as sampled under the two CFIA Food Microbial Surveillance Programs, resulted in 97.8% and 99.6% of cheese samples being assessed as satisfactory for all microbial tests. The frequencies of detection of Salmonella spp., E. coli O157:H7 and L. monocytogenes in these domestic and imported cheese samples are consistent with those observed internationally. In agreement with previous studies performed on the risks associated with raw-milk cheese (Choi et al., 2016; FSANZ, 2009; USFDA, 2016), microbial contamination was more frequently detected in raw-milk cheese samples. Contamination also appeared to be more frequent in some cheese classes but further studies are required to establish whether or not this is a significant association.
contaminated with L. monocytogenes supported the growth of the pathogen and thus represent a high nature of concern (Health Canada, 2011). Unsatisfactory levels of S. aureus were detected in 0.6% of NMMP samples and 0.1% of Targeted Survey samples. A survey performed in Norway for the presence of S. aureus at various points of the cheese making process in small-scale raw-milk cheese production found an initial prevalence of 47.2% in the bovine milk which fell to 24.7% in 30 day old cheese (Jakobsen et al., 2011). S. aureus may cause foodborne illness through production of preformed enterotoxins which are heat stable and could remain in the food product after the bacterial population which produced them is no longer detectable. Enterotoxins have been detected in cheese at S. aureus levels exceeding 105 CFU/g (Delbes et al., 2006). It has also been suggested that S. aureus levels exceeding 105 cells/g are required to produce the level of enterotoxin necessary to cause illness (Monville et al., 2012). Levels exceeding 105 CFU/g were found in 5 cheese samples analyzed under the NMMP and Targeted Survey. All 5 of these samples were raw-milk cheese. Therefore the presence of S. aureus at unsatisfactory levels in cheeses tested under the NMMP and Targeted Survey could be of public health concern in a small proportion of these cheese samples due to the potential presence of toxins. Unsatisfactory levels of generic E. coli were detected in 1.4% of NMMP samples but not in any of the Targeted Survey raw-milk cheese samples. This is lower than the recent U.S. study of raw-milk cheeses in which 5.4% of raw-milk cheeses were found to contain levels of generic E. coli that were considered violative (USFDA, 2016), however, an assessment criterion with lower levels was used (n = 5, c = 2, m = 10 CFU/g and M = 102 CFU/g). Unlike the other microorganisms in this study, generic E. coli are not generally associated with human illness. High levels of generic E. coli may indicate unsanitary practices and conditions under which pathogens could contaminate food products. Unsatisfactory levels of generic E. coli in cheeses analyzed under the NMMP suggests that a small proportion of cheese samples were exposed to sources of contamination that may have also included a pathogen. This is supported by the association (p < 0.001) between samples assessed as having unsatisfactory levels of generic E. coli and those containing a pathogen (S. aureus, L. monocytogenes and Salmonella spp.). Although the majority of both raw-milk cheeses and pasteurizedmilk cheeses tested under the NMMP and Targeted Surveys were assessed as satisfactory, statistical analysis indicated that raw-milk cheese samples tested under the NMMP were more likely to be assessed as being of unsatisfactory microbiological safety than pasteurized-milk cheeses. This association was also found for each of the individual microbial analyses performed, with the exception of E. coli O157:H7/ NM (which was not detected). In particular, a higher proportion of unsatisfactory analyses for unsatisfactory levels of S. aureus were found in raw-milk cheeses (2.69%) compared to pasteurized-milk cheeses (0.08%). This finding is consistent with studies which have identified S. aureus as ubiquitous in raw milk (primarily due to contamination from bovine mastitis, potentially increased by temperature abuse) and thus represents a concern regarding the production of raw-milk cheese (Jorgensen et al., 2005; Oliver et al., 2009; McMillan et al., 2016). Imported cheeses tested under the NMMP were also more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples. The relationship between import status and unsatisfactory assessment was statistically significant for generic E. coli, L.monocytogenes, and S. aureus. The higher occurrence of imported cheese samples of unsatisfactory microbiological safety likely reflects the high proportion of imported cheese (84.5%) within the raw-milk cheeses. Pasteurized-milk cheeses were fairly evenly distributed across all cheese classes, however, very few raw-milk cheeses belonged to classes A to C, which represent unripened cheeses and those with shorter ripening periods. The Canadian Food and Drugs Regulations restrict the sale of cheese made from unpasteurized-milk in Canada, unless they
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data statement The underlying data used in this study contains Confidential Business Information.
Declaration of competing interest None. 6
International Journal of Food Microbiology 321 (2020) 108521
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References
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Agriculture and Agri-Foods Canada (AAFC), 2017. Statistics of the Canadian Dairy Industry. Angelidis, A.S., Georgiadou, S.S., Zafeiropoulou, V., Velonakis, E.N., Papageorgiou, D.K., Vatopoulos, A., 2012. A survey of soft cheeses in Greek retail outlets highlights a low prevalence of Listeria spp. Dairy Sci. Technol. 92, 189–201. Choi, K.H., Lee, H., Lee, S., Kim, S., Yoon, Y., 2016. Cheese microbial risk assessments - a review. Asian-Australas J. Anim. Sci. 29, 307–314. Currie, A., Galanis, E., Chacon, P.A., Murray, R., Wilcott, L., Kirkby, P., Honish, L., Franklin, K., Farber, J., Parker, R., Shyng, S., Sharma, D., Tschetter, L., Hoang, L., Chui, L., Pacagnella, A., Wong, J., Pritchard, J., Kerr, A., Taylor, M., Victor, M.A.H., Flint, J., 2018. Outbreak of Escherichia coli O157:H7 infections linked to aged raw milk Gouda cheese, Canada, 2013. J. Food Prot. 81, 325–331. D’Aoust, J.Y., 1994. Salmonella and the international food trade. Int. J. Food Microbiol. 24, 11–31. Delbes, C., Alomar, J., Chougui, N., Martin, J.F., Montel, M.C., 2006. Staphylococcus aureus growth and enterotoxin production during the manufacture of uncooked, semihard cheese from cows’ raw milk. J. Food Prot. 69, 2161–2167. Dominguez, M., Jourdan-Da Silva, N., Vaillant, V., Pihier, N., Kermin, C., Weill, F.X., Delmas, G., Kerouanton, A., Brisabois, A., de Valk, H., 2009. Outbreak of Salmonella enterica serotype Montevideo infections in France linked to consumption of cheese made from raw milk. Foodborne Pathog. Dis. 6, 121–128. European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2017. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA J. 15 (12), 1–56 5077. Food Standards Australia New Zealand (FSANZ), 2009. Microbiological risk assessment of raw milk cheese. In: Risk Assessment Microbiology Section, pp. 296. Fox, P.F., McSweeney, P.L.H., 2004. Cheese: an overview. In: Cheese: Chemistry, Physics and Microbiology, pp. 1–18. Gaulin, C., Levac, E., Ramsay, D., Dion, R., Ismaïl, J., Gingras, S., Lacroix, C., 2012. Escherichia coli O157:H7 outbreak linked to raw milk cheese in Quebec, Canada: use of exact probability calculation and case-case study approaches to foodborne outbreak investigation. J. Food Prot. 75, 812–818. Gould, L.H., Mungai, E., Barton Behravesh, C., 2014. Outbreaks attributed to cheese: differences between outbreaks caused by unpasteurized and pasteurized dairy products, United States, 1998-2011. Foodborne Pathog. Dis. 11, 545–551. Government of Canada, 2018. Food and Drugs Act. Government of Canada, 2019. Food and Drug Regulations. Health Canada, 2008. Health Products and Food Branch Standards and Guidelines for the Microbiological Safety of Food - An Interpretive Summary. Health Canada, 2011. Policy on Listeria monocytogenes in Ready-to-Eat Foods. Health Canada, 2019. The Compendium of Analytical Methods. https://www.canada.ca/ en/health-canada/services/food-nutrition/research-programs-analytical-methods/ analytical-methods/compendium-methods.html.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbial safety of cheese in Canada
T
⁎
Kyle Ganz , Etsuko Yamamoto, Kate Hardie, Christine Hum, Hussein Hussein, Annie Locas, Marina Steele Food Safety Science Directorate, Science Branch, Canadian Food Inspection Agency, 1400 Merivale Road, Ottawa, ON, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Raw-milk Salmonella L. monocytogenes E. coli S. aureus Surveillance
A profile of the microbial safety of cheese in Canada was established based on the analysis of 2955 pasteurized and raw-milk cheeses tested under Canada's National Microbiological Monitoring Program (NMMP) and 2009 raw-milk cheeses tested under the Targeted Survey Program. 97.8% of NMMP and 99.6% of Targeted Survey cheese samples were assessed as being of satisfactory microbiological safety. Under the NMMP, Salmonella spp. was detected in 2 samples, Listeria monocytogenes was detected in 15 samples and no Escherichia coli O157/ H7:NM (non-motile) was detected. Cheese samples assessed as having unsatisfactory levels of S. aureus and generic E. coli were found in 18 and 41 samples, respectively. Under the Targeted Survey, L. monocytogenes was detected in 2 samples, while no Salmonella spp. or E. coli O157/H7:NM were detected. Cheese samples assessed as having investigative and unsatisfactory levels of S. aureus were found in 4 and 2 samples respectively. No samples were found to have investigative or unsatisfactory levels of generic E. coli. For cheese samples collected under the NMMP, logistic regression models indicated that contamination was more frequent in raw-milk cheeses compared to pasteurized-milk cheeses (OR = 5.0, 95% CI (3.0, 8.3)), and in imported cheeses compared to domestic cheeses (OR = 8.2, 95% CI (4.1, 16.1)). A statistically significant association was found between cheese samples assessed as having unsatisfactory levels of generic E. coli and detection of L. monocytogenes, Salmonella spp. or levels of S. aureus that were assessed as unsatisfactory (p < .001). These test results will help support risk analysis and inform food safety decisions.
1. Introduction Cheese is an important nutritional component to the Canadian diet. Approximately 466 million kilograms of cheese were produced domestically and approximately 27 million kilograms of cheese were imported into Canada in 2016 (AAFC, 2017). The average Canadian was reported to consume 15.07 kg of cheese in 2016 (AAFC, 2017). Since 1984, there have been approximately 12 documented Canadian foodborne outbreaks associated with cheeses (PHAC, 2018). Five of these outbreaks were attributed to Salmonella spp., four were attributed to Escherichia coli O157:H7 and three were attributed to Listeria monocytogenes. All four outbreaks attributed to E. coli O157:H7, and half of the total outbreaks, were linked to raw-milk cheeses. Worldwide, these pathogens, along with Staphylococcus aureus have also been linked to multiple cheese outbreaks (Dominguez et al., 2009; Gould et al., 2014; Johler et al., 2015). The European Food Safety Authority found that 4.8% of foodborne outbreaks which occurred in the European Union in 2016 were associated with cheeses (European Food Safety Authority EFSA and European Centre for Disease Prevention and Control ECDC,
⁎
2017). Overall, this indicates that cheese is a potential source of foodborne illness and that Salmonella spp., E. coli O157:H7, L. monocytogenes and S. aureus have been identified as causative agents. The Canadian Food Inspection Agency (CFIA) monitors the microbiological safety of imported and domestic cheeses in Canada under two different surveillance programs. The first of these programs is the National Microbiological Monitoring Program (NMMP), under which a wide variety of raw and pasteurized-milk cheeses were collected and tested between April 2012 and March 2017. The second program is the Targeted Surveys program. Targeted Surveys are studies of a finite period, performed to address specific data gaps and primarily focus on areas of highest health risk. Results from a microbiological Targeted Survey of raw-milk cheeses, sampled at retail establishments, between November 2014 and March 2018 were also included in this study. The objective of this study is to summarize results from these CFIA Food Microbial Surveillance Programs to provide a comprehensive profile of the microbial safety of cheese in Canada.
Corresponding author at: Floor 6, Room 244, 1400 Merivale Road, Tower 2, Ottawa, Ontario K1A 0Y9, Canada. E-mail address: [email protected] (K. Ganz).
https://doi.org/10.1016/j.ijfoodmicro.2020.108521 Received 20 June 2019; Received in revised form 27 September 2019; Accepted 12 January 2020 Available online 24 January 2020 0168-1605/ Crown Copyright © 2020 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
International Journal of Food Microbiology 321 (2020) 108521
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2. Materials and methods
Government of Canada's Food and Drug Act prohibits the sale of food containing any poisonous or harmful substance, therefore, detection of the pathogens Salmonella spp. or E. coli O157:H7/NM resulted in an unsatisfactory assessment. Samples in which the pathogen L. monocytogenes was detected were assessed in accordance with Health Canada's Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2011). According to this policy, no L. monocytogenes is permitted in Category 1 foods, which are defined as ready-to-eat foods in which growth of L. monocytogenes can occur throughout the stated shelf-life. Results of pH and water activity analyses were primarily used to make the Category determination. Assessment of the levels of generic E. coli and S. aureus in cheese samples was performed according to the assessment criteria for raw and pasteurized-milk cheeses outlined in the Health Products and Food Branch (HPFB) Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary (Health Canada, 2008). An investigative assessment was assigned to Targeted Survey samples where the levels of generic E.coli and S. aureus were found to be above the marginally acceptable level (m) but below the unacceptable level (M) (Table 1). A cheese sample was considered to be of satisfactory microbiological safety if all individual microbiological test results were considered satisfactory based on the assessment criteria outlined in Table 1, while a cheese sample was considered to be of unsatisfactory microbiological safety if one or more individual microbiological test results were unsatisfactory. A cheese sample was considered to be of investigative microbiological safety if an investigative assessment was given to one or more individual microbiological tests and all other analyses were satisfactory. All samples assessed as unsatisfactory and investigative were subject to appropriate follow-up actions by both industry and the CFIA.
2.1. Sample collection and analysis NMMP data from 595 raw-milk cheese samples (92 domestic and 503 imported) and 2360 pasteurized-milk cheese samples (1659 domestic and 701 imported) collected between April 2012 and March 2017 were assessed. NMMP cheese samples were collected at domestic cheese establishments (production facilities, packing facilities and warehouses) and import warehouses across all Canadian provinces by CFIA inspectors. Finished products in intact packaging was preferentially sampled, however, bulk products, such as cheese wheels, were occasionally sampled as well using aseptic techniques. Cheese samples were collected under sampling plans targeting a variety of cheese types. Each sample comprised five 200 g subunits that were randomly collected from each lot of cheese sampled. A random sample is one in which all elements in the lot have an equal and independent chance of being included in the sample. Targeted Survey data from 2009 raw-milk cheese samples (390 domestic and 1619 imported) collected between November 30, 2014 and March 31, 2018 were assessed. These cheese samples were collected from national retail chains and local/regional grocery stores located in 11 major cities across Canada. The number of samples collected from these cities was in proportion to the relative population of the respective areas. Each sample comprised either a single or multiple unit (s) of a single lot weighing a total of at least 250 g. Testing was performed in ISO 17025 accredited CFIA laboratories using methods validated in accordance with Health Canada's Compendium of Analytical Methods (Health Canada, 2019). These methods used for analysis are as follows: MFHPB-20, MFLP-29 and MFLP-40 for Salmonella spp.; MFHPB-10, MFLP-30 and MFLP-80 for E. coli O157:H7; MFHPB-30, MFLP-53, MFLP-74 and MFLP-77 for L. monocytogenes; MFHPB-19, MFHPB-34 and Compact Dry EC medium count plates for generic E. coli; and MFHPB-21 for S. aureus. Enumeration of generic E. coli using Compact Dry EC medium count plates (HyServe GmbH & Co. KG) was performed by diluting 1 part cheese sample with 10 parts buffered peptone water, mixing thoroughly, plating 1 ml of diluted sample and incubating at 42 °C for 24 ± 2 h. Both NMMP and Targeted Survey samples were tested for the presence of Salmonella spp., L. monocytogenes (all samples) and E. coli O157:H7/ NM (non-motile) (raw-milk cheese samples only). If L. monocytogenes was detected, levels of L. monocytogenes were enumerated as colony forming units per gram (CFU/g) and the pH and water activity of the cheese was measured to determine the potential of the cheese to support growth of L. monocytogenes, as described in Health Canada's Policy on L. monocytogenes in Ready-to-Eat Foods (Health Canada, 2011). Levels of generic E. coli were enumerated as CFU/g or most probable number per gram (MPN/g), depending on the methodology used, and levels of S. aureus were enumerated as CFU/g. Generally, the manner in which these methods were applied for the analysis of cheese provided limit of detections of 1.8 MPN/g for generic E. coli, 25 CFU/g for S. aureus and 5 CFU/g for L. monocytogenes. An equal portion (25 g) of each of the five sub-units collected under the NMMP, were aseptically combined into a single composite sample (125 g) for each analysis performed. For the Targeted Survey, 25 g of the sample was tested. Samples collected during the initial 4 month period of the Targeted Survey were only analyzed for one microorganism (E. coli O157:H7/ NM).
2.3. Determination of cheese pasteurization status, origin and class Sample information on the pasteurization status, country of origin and product type was recorded at the time of sample collection. For NMMP samples this information was recorded as part of the sample description by CFIA inspectors, whereas for Targeted Surveys this information was derived from information on the product labels. Based on this recorded information, the cheese samples were classified as rawmilk or pasteurized-milk cheeses, and of domestic or imported origin and were assigned to one of seven cheese classes (Classes A to G) using the cheese classification scheme developed by Ottogalli (Ottogalli, 1998, 2000) (Table 2). The Ottogalli cheese classification scheme was selected because it is based on the microbiological aspects of cheese and cheese production and because it includes an extensive list of preclassified cheeses that were used to classify the cheese samples. The preclassified list classified some cheese types (e.g., Gouda, Edam, etc.) into two different classes due to large variations in their physical properties (e.g., moisture content and pH) and ripening times (e.g., 3–6 months for Gouda). As this type of detailed information on cheese samples was not captured, it was not possible to classify some of these cheese types, therefore they were placed in the unclassified group. A small number of additional cheese samples did not match the exact name of a cheese on the pre-classified list but were classified based on their similarity to a pre-classified cheese on this list (Table 2, footnotes 1–3). Overall, a total of 67.2% of cheeses within the NMMP dataset and 80.8% of the cheeses within the Targeted Survey dataset were assigned a cheese class. 2.4. Statistical analyses
2.2. Sample assessment Due to the differences in the design of the NMMP and Targeted Survey programs, results from these two programs were analyzed separately. Due to the small number of unsatisfactory samples found in the Targeted Survey data set, comparative statistical analyses of relationships between variables were not performed. Analyses were performed using Stata/SE version 15 (StataCorp, College Station, TX). For
NMMP and Targeted Survey test results were assessed as being of satisfactory, unsatisfactory, or investigative microbiological safety using assessment criteria outlined in Table 1, which are based on Canadian Acts, Regulations and policies as described below (Government of Canada, 2018, 2019; Health Canada, 2011). Section 4(1)a of the 2
International Journal of Food Microbiology 321 (2020) 108521
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Table 1 Microbiological assessment criteria used to assess the microbiological safety of cheese analyzed under the National Microbiological Monitoring Program and Targeted Survey Program. na
ca
ma
Ma
Satisfactory
Investigative
Unsatisfactory
Assessment criteria for Salmonella spp., E. coli O157:H7 and L. monocytogenes – Not detected Salmonella spp. 5/1b 0 0 E. coli O157:H7 5/1b 0 0 – Not detected L. monocytogenesc 5/1b 0 0 – Not detected
n/a n/a n/a
Detected Detected Detected
Assessment criteria for generic E. coli NMMP raw-milk cheeses 5
2
5 × 102
2 × 103
n/a
NMMP pasteurized-milk cheeses
5
2
102
2 × 103
Targeted Survey raw-milk cheeses
1
–
5 × 102
2 × 103
≤M in any subunit and if > m then in ≤c subunits ≤M in any subunit and if > m then in ≤c subunits ≤m
> M in any subunit or > m in > c subunits > M in any subunit or > m in > c subunits >M
Assessment criteria for S. aureus NMMP raw-milk cheeses
5
2
103
104
n/a
NMMP pasteurized-milk cheeses
5
2
102
104
Targeted Survey raw milk cheeses
1
–
103
104
≤M in any subunit and if > m then in ≤c subunits ≤M in any subunit and if > m then in ≤c subunits ≤m
n/a > m to ≤M
n/a > m to ≤M
> M in any subunit or > m in > c subunits > M in any subunit or > m in > c subunits >M
a
As described in Health Canada's Health Products and Food Branch Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary (Health Canada, 2008). b 5 subunits of 25 g were tested for NMMP samples and 1 unit of 25 g was tested for Targeted Survey samples. c All NMMP and Targeted Survey cheese samples in which L. monocytogenes was detected were category 1 foods for which there is no acceptable level of L. monocytogenes.
3. Results
the NMMP data set, chi-square tests were used to determine if there were associations between the assessment status of the samples (i.e. satisfactory or unsatisfactory) and sample variables (i.e., pasteurization status, origin and cheese class). The chi-squared test was also used to determine if there was an association between an unsatisfactory assessment due to levels of generic E. coli that exceed the assessment criteria and detection of pathogens. For the purpose of this analysis, pvalues < 0.05 were considered to be statistically significant. Regression modelling, using an unsatisfactory assessment as the dependent variable, was performed to further explore these relationships and generate odds ratios (OR). A multivariable logistic regression model was built using pasteurization status and origin as predictor variables, and variable significance was tested using Wald tests.
3.1. Sample assessment Under the NMMP, 2955 cheese samples were collected. Of these cheese samples, 97.8% (n = 2891) were assessed as being of satisfactory microbiological safety. In contrast, 2.2% (n = 64) of samples were assessed as being of unsatisfactory microbiological safety. Chi-square analysis indicated that there was no statistically significant relationship between the year of sampling and the frequency of unsatisfactory samples in an individual year (p = 0.45). The prevalence of Salmonella spp. and L. monocytogenes in cheeses sampled under the NMMP were found to be 0.1% (n = 2) and 0.5% (n = 15), respectively (Table 3). All 15 L. monocytogenes positive cheese samples were determined by pH and water activity to belong to
Table 2 Classification of cheese samples. Class
Common types
A: Fresh soft cheeses, practically amicrobial Rarely ripened, rennet-free, and amicrobial due to rapid coagulation by organic acids. B: Fresh soft cheeses, rich in lactic acid bacteria Unripened, rennet coagulated, and use biological acidification.
Ricotta Panir/Paneer Queso Frescoa Mozzarella Goat Bocconcinib Burratab Feta Halloumi Brie Camembert Blue Cheese Roquefort Raclette Monterey Cheddar Swissc
C: Short ripened soft cheeses Soft or semi-soft consistency that are usually rindless or have a thin rind. D: Soft surface ripened cheeses Surface microflora ripen the cheese from the outside surface towards the center. E: Blue cheeses Soft or semi-soft, blue veined due to the growth of mycelia and spores of Penicillia. F: Semi-hard cheeses Semi-hard cheeses with medium ripening time G: Hard cheeses Hard consistency cheeses with long ripening time Unclassified Total samples a b c
NMMP cheese samples Pasteurized
Raw
79
0
0
440
0
5
180
2
0
194
102
2
107
68
136
89
47
18
518
159
1463
753 2955
217
385 2009
13 Queso Fresco samples were classified as class A based on similarity to Queso Blanco. 20 Burrata and 6 Bocconcini cheese samples were classified as class B based on similarity to Mozzarella. 31 Swiss cheese samples were classified as class G based on similarity to Emmental cheese. 3
Targeted Survey raw-milk cheese samples
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Table 3 Proportion of NMMP and Targeted Survey cheese samples assessed as being of unsatisfactory microbiological safety by analysis. National Microbiological Monitoring Program
Targeted Survey on raw-milk cheese
Analyte
Unsatisfactory
n
Occurrence
Unsatisfactory
n
Occurrence
E. coli O157:H7/NM Salmonella spp. L. monocytogenes Unsatisfactory levelsa of S. aureus Unsatisfactory levelsa of generic E. coli
0 2 15 18 41
600 2953 2954 2948 2953
0% 0.1% 0.5% 0.6% 1.4%
0 0 2 2 0
2009 1723 1723 1723 1723
0% 0% 0.1% 0.1% 0%
a
As described in Table 1 and in Health Canada's Health Products and Food Branch Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary.
safety on the basis of one or more microbial analyses. Statistical analysis indicated that raw-milk cheese samples are more likely to be of unsatisfactory microbiological safety compared to pasteurized-milk cheese samples. The 29 pasteurized-milk NMMP cheese samples assessed as being of unsatisfactory microbiological safety were based on the results of 32 unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 21), while the remaining were due to the presence of L. monocytogenes (n = 9) and levels of S. aureus (n = 2). The 35 raw-milk NMMP cheese samples of unsatisfactory microbiological safety were based on the results of 44 unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 20) and levels of S. aureus (n = 16) which exceeded the levels specified in the assessment criteria, followed by the presence of L. monocytogenes (n = 6) and Salmonella spp. (n = 2). When stratified by microorganism, the relationship between pasteurization status and unsatisfactory assessment remained statistically significant.
category 1 as per Health Canada's Policy on Listeria monocytogenes in Ready-to-Eat Foods (Health Canada, 2011), i.e. supporting the growth of L. monocytogenes. Enumeration of L. monocytogenes in these samples revealed levels ranging from < 5 to 630 CFU/g with 9 of the 15 samples containing levels below the limit of detection. E. coli O157:H7/NM was not detected in any of the samples. Unsatisfactory levels of generic E. coli and S. aureus were observed in 41 and 18 of the samples, respectively. Levels of generic E. coli ranging from < 1.8 MPN/g to 6.2 × 104 CFU/g and levels of S. aureus ranging from < 23 to 4.3 × 105 CFU/g were observed in all cheese samples tested under the NMMP. Of the 41 cheese samples assessed as having unsatisfactory levels for generic E. coli, a pathogen was also detected in 11 of these samples. A chi-squared test demonstrated an association between samples assessed as having unsatisfactory levels of generic E. coli and those containing a pathogen (p < 0.001). This association was also observed when raw-milk and pasteurized-milk cheeses were analyzed separately (p < 0.001). Under the Targeted Survey, 2009 raw-milk cheese samples were collected. Of the cheese samples analyzed, 99.6% (n = 2001) were assessed as being of satisfactory microbiological safety. Four (0.2%) samples were assessed as being of unsatisfactory microbiological safety (Table 3) and four samples (0.2%) with levels of S. aureus between 103 and 104 CFU/g were assessed as investigative. Of the four samples assessed as unsatisfactory, two samples (0.1%) were positive for L. monocytogenes with levels < 5 CFU/g and two samples (0.1%) had unsatisfactory levels of S. aureus > 104 CFU/g. Levels of S. aureus ranging from < 25 to 8.7 × 105 CFU/g and levels of generic E. coli ranging from < 1.8 to < 50 CFU/g were observed in all cheese samples tested under the Targeted Survey.
3.3. Associations between assessment status and origin Of the 2955 samples analyzed under the NMMP, 59.3% (n = 1751) cheeses were of domestic origin and 40.7% (n = 1204) were imported. A total of 10 domestic cheese samples, representing 0.6% of the domestic total (95% CI, 0.3–1.1%), and 54 imported cheese samples, representing 4.5% of the imported total (95% CI, 3.4–5.8%), were assessed as being of unsatisfactory microbiological safety (Table 5). Statistical analysis indicated that imported cheese samples are more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples. The 10 domestic NMMP cheese samples of unsatisfactory microbiological safety were assessed as unsatisfactory based on the results of 10 unsatisfactory analyses. The majority of these analyses were unsatisfactory due to levels of generic E. coli (n = 6), while the remaining were unsatisfactory due to the presence of L. monocytogenes (n = 2) or levels S. aureus (n = 2). The 54 imported NMMP cheese samples of unsatisfactory microbiological safety were based on the results of 66
3.2. Associations between assessment status and pasteurization status of the milk Of the 2955 samples analyzed under the NMMP, 2360 cheeses were made with pasteurized milk and 595 cheeses were made with raw-milk. As shown in Table 4, 29 pasteurized-milk cheese samples, representing 1.2% (95% CI, 0.8–1.8%) of the total pasteurized cheese samples tested under the NMMP, and 35 raw-milk cheese samples, representing 5.9% (95% CI, 4.1–8.1%) of the total raw-milk cheese samples tested under the NMMP, were assessed as being of unsatisfactory microbiological
Table 5 Unsatisfactory cheese samples tested under the National Microbiological Monitoring Program by the origin of the cheese. Origin
Table 4 Cheese samples assessed as being of unsatisfactory microbiological safety tested under the National Microbiological Monitoring Program by the pasteurization status of the milk. Pasteurization status
Unsatisfactory
n
Proportion
95% CI
Pasteurized-milk cheese Raw-milk cheese
29 35
2360 595
1.2% 5.9%a
0.8–1.8% 4.1–8.1%
Domestic pasteurized-milk cheese Domestic raw-milk cheese All domestic cheese Imported pasteurized-milk cheese Imported raw-milk cheese All imported cheese
a A logistic regression model indicated that raw-milk cheese samples were more likely to be of unsatisfactory microbiological safety compared to pasteurized-milk cheese samples (OR = 5.0, 95% CI (3.0, 8.3)).
Unsatisfactory
n
Proportion
95% CI
8
1659
0.5%
0.2–0.9%
2 10 21
92 1751 701
2.2% 0.6% 3.0%
0.6–7.6% 0.3–1.1% 1.9–4.5%
33 54
503 1204
6.6% 4.5%a
4.6–9.1% 3.4–5.8%
a A logistic regression model indicated that imported cheese samples were more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples (OR = 8.18, 95% CI (4.1, 16.1)).
4
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unsatisfactory NMMP samples by class was not performed due to the large proportion of NMMP cheese samples which could not be classified into Ottogalli cheese classes.
unsatisfactory analyses. The majority of these unsatisfactory analyses were due to levels of generic E. coli (n = 35) and levels S. aureus (n = 16) which exceeded the levels specified in the assessment criteria, followed by the presence of L. monocytogenes (n = 13) and Salmonella spp. (n = 2). When stratified by microorganism, the relationship between import status and unsatisfactory assessment remained statistically significant for generic E. coli, L.monocytogenes, and S. aureus. Analyses indicated that samples of unsatisfactory microbiological safety collected under the NMMP were more frequently associated with raw-milk cheeses and imported cheeses. Since a higher proportion of raw-milk cheeses were imported (84.5%) compared to being produced domestically (15.5%), additional chi-square tests were performed to determine if an association between unsatisfactory assessment and rawmilk cheeses was still observed when the origin of the cheeses was controlled. It was observed that within domestic cheeses, raw-milk cheeses were still more frequently associated with samples of unsatisfactory microbiological safety, and this was shown to be statistically significant (p = 0.04). Within imported cheeses, raw-milk cheeses were also more frequently associated with samples of unsatisfactory microbiological safety, and this was shown to be statistically significant (p = 0.003). A logistic regression model was built to explore the associations when both the origin and pasteurization status as predictors are considered. Both variables remain significant in the model when controlling for the other, with an OR of 5.55 for origin status (p < 0.001, 95% CI 2.67–11.52) and an OR of 2.43 for pasteurization status (p = 0.001, 95% CI 1.42–4.18). Of the 2009 raw-milk cheese samples analyzed under the Targeted Survey, 80.6% (n = 1619) of samples were of imported origin and 19.4% (n = 390) were of domestic origin. The 4 samples assessed as being of unsatisfactory microbiological safety were of imported origin.
4. Discussion Under the NMMP, 94.1% of raw-milk cheeses and 98.8% of pasteurized-milk cheeses were assessed as being of satisfactory microbiological safety and under the Targeted Survey, 99.6% of the raw-milk cheese samples were assessed as being of satisfactory microbiological safety. These results suggest that the vast majority of cheeses on the Canadian marketplace are of good microbiological safety. Cheese contamination has previously been found to occur at various points in the cheese making process such as from contaminated input ingredients, introduced during unsanitary manufacturing processes or during postprocessing (Fox and McSweeney, 2004). E. coli O157:H7/NM was not found in any of the raw-milk cheeses sampled under the two programs examined in this study. This is consistent with a recent study of 1606 domestic and imported raw-milk cheeses collected at manufacturers, distribution centers and retail stores in the United States (USFDA, 2016), for which no E. coli O157:H7 was detected. However, several international (Gould et al., 2014) and Canadian (Currie et al., 2018; Gaulin et al., 2012; Honish et al., 2005) foodborne outbreaks of E. coli O157:H7 have been associated with raw-milk cheeses. Considering the ready-to-eat nature of cheese, the relatively low infectious dose (Teunis et al., 2008), and the severity of illness (Pennington, 2010), the presence of any level of E. coli O157:H7 in cheese remains a concern to public health. Salmonella spp. was not detected in any of the Targeted Survey samples and was detected in 0.1% of the NMMP samples. These two cheese samples were raw-milk cheeses. This is consistent with the recent study of raw-milk cheeses in the United States (USFDA, 2016) where Salmonella spp. was detected in 0.19% of samples. Considering the ready-to-eat nature of cheese, the relatively low infectious dose of certain serovars of Salmonella spp. (D'Aoust, 1994) and the severity of illness (Thomas et al., 2013), the presence of any level of Salmonella spp. in cheese remains a concern to public health. L. monocytogenes was detected in 0.5% of NMMP samples and 0.1% of Targeted Survey samples. This is consistent with recent international studies on the prevalence of L. monocytogenes in cheeses at retail, which revealed prevalence rates of 0% in soft cheese in Greece (Angelidis et al., 2012), 0.4% in soft and semi-soft cheeses in Sweden (Lambertz et al., 2012), 1.6% in pasteurized-milk cheeses in Portugal (Mena et al., 2004), and 2.7% in hard cheeses in the United Kingdom (Little et al., 2009). The recent study on raw-milk cheeses in the United States (USFDA, 2016) found a prevalence of L. monocytogenes of 0.62%. Results of pH and water activity analyses indicated that the cheeses
3.4. Distribution of samples assessed as unsatisfactory by type of cheese class The proportion of cheese samples assessed as being of unsatisfactory microbiological safety in each class for the NMMP samples is shown in Fig. 1. Class D: soft surfaced ripened cheeses, and Class F: semi-hard cheeses, contained the highest proportion of cheese samples of unsatisfactory microbiological safety at 5.1% (95% CI, 2.9–8.2%) and 6.6% (95% CI, 3.1–12.2%), respectively. Class G: hard cheeses, had the lowest proportion of cheese samples of unsatisfactory microbiological safety with 0.6% (95% CI, 0.1–1.5%). Of the 4 samples assessed as being of unsatisfactory microbiological safety under the Targeted Survey, two were from Class G and two were unclassified. The vast majority of the Targeted survey samples were Class G (72.8%) or unclassified (19.2%) (Table 2). Comparative analysis on the proportion of 7.0
n=136
Unsatisfactory Samples (%)
6.0 n=296 5.0 4.0 3.0
n=79
n=440
n=182 n=970
n=175
2.0 1.0
n=677
0.0 A: Fresh soft cheeses, amicrobial
B: Fresh soft cheeses, lactic acid bacteria
C: Short ripened soft cheeses
D: Soft surface ripened cheeses
E: Blue cheeses
F: Semi-hard cheeses
G: Hard cheeses
Unclassified
Fig. 1. Cheese samples assessed as being of unsatisfactory microbiological safety under the National Microbiological Monitoring Program by Class (n = total number of samples tested per class). 5
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have been aged for 60 days, which likely accounts for this observed bias against unripened raw-milk cheeses. The frequency of samples of unsatisfactory microbiological safety in each class was compared. For the NMMP samples, Class D, the soft surfaced ripened cheeses, and Class F, the semi-hard cheeses, were observed to contain higher proportions of samples of unsatisfactory microbiological safety (Fig. 1). Class G, the hard cheeses, contained the fewest samples of unsatisfactory microbiological safety. In contrast to the NMMP samples, the majority of Targeted Survey samples belonged to Class G (72.8%) and the unclassified group (19.2%) (Table 2). This may explain why fewer samples of unsatisfactory or investigative microbiological safety were detected in the Targeted Survey when compared to the NMMP. The Targeted Survey samples assessed as of either unsatisfactory or investigative microbiological safety were distributed between Class G and the unclassified group. These results suggest that some classes of cheeses may be more susceptible to microbial contamination and/or more supportive of bacterial growth. The Ottogalli cheese classes were based on the microbiological aspects of cheese and cheese production, thus these differences likely reflect differences in how these cheeses were produced and the different inherent microbial barriers to growth. Lastly, we observed that the same cheese types and brands were often represented by multiple samples in the dataset. As samples were collected as part of our randomly sampled NMMP and Targeted sampling plan by different samplers over several years, we would expect that more widely available cheese types and brands may be more represented at higher levels than less widely available products. Of the 64 NMMP samples that were assessed as unsatisfactory, 24 samples were of the same cheese type and/or brand as at least one other unsatisfactory cheese sample. None of these samples were of the same lot of cheese. Of the 8 Targeted Survey cheese samples that were assessed as either unsatisfactory or investigative, 5 were of the same cheese type and of these 5, 4 were of the same brand and of these 4, 3 were of the same lot. Without detailed knowledge of the market share that these cheese types represent in Canada, it was not possible to assess if these cheese types and brands were over-represented in the dataset. These cheese samples were assessed as unsatisfactory or investigative due to levels of generic E. coli and/or S. aureus and L. monocytogenes. In summary, the microbiological safety of cheese on the Canadian marketplace, as sampled under the two CFIA Food Microbial Surveillance Programs, resulted in 97.8% and 99.6% of cheese samples being assessed as satisfactory for all microbial tests. The frequencies of detection of Salmonella spp., E. coli O157:H7 and L. monocytogenes in these domestic and imported cheese samples are consistent with those observed internationally. In agreement with previous studies performed on the risks associated with raw-milk cheese (Choi et al., 2016; FSANZ, 2009; USFDA, 2016), microbial contamination was more frequently detected in raw-milk cheese samples. Contamination also appeared to be more frequent in some cheese classes but further studies are required to establish whether or not this is a significant association.
contaminated with L. monocytogenes supported the growth of the pathogen and thus represent a high nature of concern (Health Canada, 2011). Unsatisfactory levels of S. aureus were detected in 0.6% of NMMP samples and 0.1% of Targeted Survey samples. A survey performed in Norway for the presence of S. aureus at various points of the cheese making process in small-scale raw-milk cheese production found an initial prevalence of 47.2% in the bovine milk which fell to 24.7% in 30 day old cheese (Jakobsen et al., 2011). S. aureus may cause foodborne illness through production of preformed enterotoxins which are heat stable and could remain in the food product after the bacterial population which produced them is no longer detectable. Enterotoxins have been detected in cheese at S. aureus levels exceeding 105 CFU/g (Delbes et al., 2006). It has also been suggested that S. aureus levels exceeding 105 cells/g are required to produce the level of enterotoxin necessary to cause illness (Monville et al., 2012). Levels exceeding 105 CFU/g were found in 5 cheese samples analyzed under the NMMP and Targeted Survey. All 5 of these samples were raw-milk cheese. Therefore the presence of S. aureus at unsatisfactory levels in cheeses tested under the NMMP and Targeted Survey could be of public health concern in a small proportion of these cheese samples due to the potential presence of toxins. Unsatisfactory levels of generic E. coli were detected in 1.4% of NMMP samples but not in any of the Targeted Survey raw-milk cheese samples. This is lower than the recent U.S. study of raw-milk cheeses in which 5.4% of raw-milk cheeses were found to contain levels of generic E. coli that were considered violative (USFDA, 2016), however, an assessment criterion with lower levels was used (n = 5, c = 2, m = 10 CFU/g and M = 102 CFU/g). Unlike the other microorganisms in this study, generic E. coli are not generally associated with human illness. High levels of generic E. coli may indicate unsanitary practices and conditions under which pathogens could contaminate food products. Unsatisfactory levels of generic E. coli in cheeses analyzed under the NMMP suggests that a small proportion of cheese samples were exposed to sources of contamination that may have also included a pathogen. This is supported by the association (p < 0.001) between samples assessed as having unsatisfactory levels of generic E. coli and those containing a pathogen (S. aureus, L. monocytogenes and Salmonella spp.). Although the majority of both raw-milk cheeses and pasteurizedmilk cheeses tested under the NMMP and Targeted Surveys were assessed as satisfactory, statistical analysis indicated that raw-milk cheese samples tested under the NMMP were more likely to be assessed as being of unsatisfactory microbiological safety than pasteurized-milk cheeses. This association was also found for each of the individual microbial analyses performed, with the exception of E. coli O157:H7/ NM (which was not detected). In particular, a higher proportion of unsatisfactory analyses for unsatisfactory levels of S. aureus were found in raw-milk cheeses (2.69%) compared to pasteurized-milk cheeses (0.08%). This finding is consistent with studies which have identified S. aureus as ubiquitous in raw milk (primarily due to contamination from bovine mastitis, potentially increased by temperature abuse) and thus represents a concern regarding the production of raw-milk cheese (Jorgensen et al., 2005; Oliver et al., 2009; McMillan et al., 2016). Imported cheeses tested under the NMMP were also more likely to be of unsatisfactory microbiological safety compared to domestic cheese samples. The relationship between import status and unsatisfactory assessment was statistically significant for generic E. coli, L.monocytogenes, and S. aureus. The higher occurrence of imported cheese samples of unsatisfactory microbiological safety likely reflects the high proportion of imported cheese (84.5%) within the raw-milk cheeses. Pasteurized-milk cheeses were fairly evenly distributed across all cheese classes, however, very few raw-milk cheeses belonged to classes A to C, which represent unripened cheeses and those with shorter ripening periods. The Canadian Food and Drugs Regulations restrict the sale of cheese made from unpasteurized-milk in Canada, unless they
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data statement The underlying data used in this study contains Confidential Business Information.
Declaration of competing interest None. 6
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