Andersen, Jens Kirk

Hansen, Tina Beck

Møller, Cleide

During a 20-case-outbreak of verocytotoxin-producing Escherichia coli O26:H11 in 2007 in Denmark, two of the cases were also found to be infected with Yersinia enterocolitica. The source was an organic semi-dried fermented sausage and the question was: “Could Y. enterocolitica have survived, or even multiplied, during the production of the suspected sausage?” To elucidate this, the ComBase Predictor (CBP) and the Pathogen Modeling Program (PMP) were used as support tools. From information on the company’s website, it was calculated that the water phase salt changed from 4.6% to 8.0% during production and pH changed from 5.5 to 4.7. No nitrite was used. Predictions of growth/reduction of Y. enterocolitica and E. coli in a matrix covering these pH- and WPS-values were compared at 24°C mimicking fermentation temperature, at 16 and 5°C mimicking drying and storage temperatures, respectively. The results showed that Y. enterocolitica would be able to multiply during the first part of the production. Compared to E. coli growth of Y. enterocolitica was predicted to be slower in the beginning of the fermentation but faster in the end with CBP and faster during the whole fermentation with PMP. CBP predicted that an increase of one log-unit took approx. 50 h at conditions in the beginning of the drying period and approx. 100 h in the middle. During storage growth of Y. enterocolitica would only be expected in case of production failures, such as insufficient drying or addition of a too low amount of salt to the batter. A deterministic model was constructed in Microsoft Excel using information on the production of the implicated sausage. This model predicted the level of Y. enterocolitica to increase 2.3, 4.2 and 7.8 log-units during fermentation, drying and storage, respectively. At the point of release of the sausage for sale, 1 Y. enterocolitica could have increased to 106 and the sausage could, therefore, not be ruled out as the source of Y. enterocolitica found in two of the outbreak cases

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Downloaded from orbit.dtu.dk on: Dec 17, 2017Using ComBase Predictor and Pathogen Modeling Program as support tools inoutbreak investigation: an example from DenmarkMøller, Cleide Oliveira de Almeida; Hansen, Tina Beck; Andersen, Jens KirkPublished in:6th International Conference on Predictive Modeling In FoodsPublication date:2009Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Møller, C., Hansen, T. B., & Andersen, J. K. (2009). Using ComBase Predictor and Pathogen Modeling Programas support tools in outbreak investigation: an example from Denmark. In 6th International Conference onPredictive Modeling In Foods (pp. 263-266)Using ComBase Predictor and Pathogen Modeling Program as support tools in outbreak investigation: an example from Denmark T.B. Hansen, C.O.A. Møller and J.K. Andersen National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. (tibha@food.dtu.dk) Abstract During a 20-case-outbreak of verocytotoxin-producing Escherichia coli O26:H11 in 2007 in Denmark, two of the cases were also found to be infected with Yersinia enterocolitica. The source was an organic semi-dried fermented sausage and the question was: “Could Y. enterocolitica have survived, or even multiplied, during the production of the suspected sausage?” To elucidate this, the ComBase Predictor (CBP) and the Pathogen Modeling Program (PMP) were used as support tools. From information on the company’s website, it was calculated that the water phase salt changed from 4.6% to 8.0% during production and pH changed from 5.5 to 4.7. No nitrite was used. Predictions of growth/reduction of Y. enterocolitica and E. coli in a matrix covering these pH- and WPS-values were compared at 24°C mimicking fermentation temperature, at 16 and 5°C mimicking drying and storage temperatures, respectively. The results showed that Y. enterocolitica would be able to multiply during the first part of the production. Compared to E. coli growth of Y. enterocolitica was predicted to be slower in the beginning of the fermentation but faster in the end with CBP and faster during the whole fermentation with PMP. CBP predicted that an increase of one log-unit took approx. 50 h at conditions in the beginning of the drying period and approx. 100 h in the middle. During storage growth of Y. enterocolitica would only be expected in case of production failures, such as insufficient drying or addition of a too low amount of salt to the batter. A deterministic model was constructed in Microsoft Excel using information on the production of the implicated sausage. This model predicted the level of Y. enterocolitica to increase 2.3, 4.2 and 7.8 log-units during fermentation, drying and storage, respectively. At the point of release of the sausage for sale, 1 Y. enterocolitica could have increased to 106 and the sausage could, therefore, not be ruled out as the source of Y. enterocolitica found in two of the outbreak cases. Keywords Outbreak, Yersinia enterocolitica, Escherichia coli, VTEC, semi-dried fermented sausage. Introduction In February 2007, an outbreak of verocytotoxin-producing Escherichia coli O26:H11 was realized in Denmark. The outbreak involved 20 cases primarily children in the age from 2 to 3 years. Using a credit card traceability method, a possible source was traced to be an organic semi-dried fermented sausage. The source was later confirmed when the outbreak strain was recovered from raw material used in the sausage production. Besides E. coli O26:H11, Yersinia enterocolitica was also found in two of the cases and during the outbreak investigation the following question arose: “Could Y. enterocolitica have survived, or even multiplied, during the production of the suspected sausage?” To elucidate this, the ComBase Predictor (CBP) and the Pathogen Modeling Program (PMP) were used as support tools. Methods Environmental conditions of sausage To determine intrinsic characteristics, such as concentration of water phase NaCl, concentration of NaNO2 and pH, of the suspected semi-dried sausage the following information was collected from the company’s website and from the label. To produce 100 g of the organic sausage 127 g meat, 4 g salt (NaCl), 2 g spices and 0.5 g dextrose were used. The ready-to-eat sausages contained 9 g fat, 22 g protein and 2 g carbohydrates per 100 g.    The weight reduction (WR) was calculated on the basis of the recipe of the used batter  WR = 100 * ((M + Nb + S + D) – 100) / (M + Nb + S + D)                                              (1)  where WR is the weight reduction in %, M is gram of meat, Nb is gram of NaCl, S is gram of spices and D is gram of dextrose used for the production of 100 g of sausage.  The NaCl content, measured in gram, in the ready-to-eat sausage, (Ns) was calculated on the basis of amount of NaCl in the batter (Nb) as well as WR  Ns = 100 * Nb / (100 – WR)                                                                                               (2)    The water content in the ready-to-eat sausage (Ws) was calculated on the basis of the labelled content of fat, protein and carbohydrates as well as the calculated Ns  Ws = 100 – (F + P + C + Ns)                                                                                              (3)  where Ws is water content in %, F is gram of fat, P is gram of protein and C is gram of carbohydrate in 100 g of ready-to-eat sausage.  The % water in the batter (Wb) was calculated using formulae (1) and (3)  Wb = 100 * Ws / (100 – WR)                                                                                             (4)   The water phase NaCl concentrations in batter (WPSb) was calculated on the basis Nb from recipe and formula (4)  WPSb = 100 * Nb / (Wb + Nb)                                                                                           (5)  The water phase NaCl concentrations in the ready-to-eat sausage (WPSs) was calculated on the basis of formula (2) and (3)  WPSs = 100 * Ns / (Ws + Ns)                                                                                            (6)  Both WPSb and WPSs are measured in %.  Production of semi-dried sausages can be split into three major steps, fermentation, drying and storage. As there was no information available on the website concerning specific processing temperatures, a standard procedure was adopted. Thus, the widely used fermentation temperature of 24°C was chosen to mimic the fermentation step and 16°C to mimic the drying step. Storage temperature was set to 5°C as indicated on the label. Predictions from CBP and PMP Growth/reduction rates of Y. enterocolitica and E. coli were predicted using both ComBase Predictor (CBP) as well as Pathogen Modeling Program (PMP). The growth and reduction rates were defined as the time in hours to obtain 1 log-increase and 1 log-reduction of the microorganisms, respectively. In all predictions, lag was excluded. In PMP, the procedure for predicting time to 1 log-increase was to set the difference between ‘level of concern’ and ‘initial level’ equal to 1 log CFU. With regard to time to 1 log-reduction in PMP, time to obtain 5 log-reductions was predicted and divided by 5. In CBP, time to 1 log-increase was estimated from the predicted doubling time by division by 0.301 (ln(2) / ln(10)). No non-thermal inactivation models were available for Y. enterocolitica and E. coli in CBP.  Spreadsheet model A deterministic model was constructed in Microsoft Excel for evaluating the fate of Y. enterocolitica and E. coli during production conditions similar to the sausage implicated in the outbreak. Interviewing the company collected the information on specific production conditions. Fermentation was carried out at 16°C for 48 h, drying from 16 to 13°C in 8 d, reducing the weight gradually, and chilled storage at 5°C for up to 4 months. Intrinsic characteristics of the ready-to-eat sausage were measured to be pH = 5.1 and aw = 0.96. By conversion of the aw to WPSs = 6.6% as described by (Resnik & Chirife, 1988) and using the formulae (1) to (6), WPSb was estimated to be 4%. The company informed that pH of the batter usually was around 5.5. Prediction of growth/survival was performed with CBP for Y. enterocolitica and PMP for E. coli. In all predictions, lag was excluded. Results and discussion Production of semi-dried sausages is a dynamic process where pH, temperature and WPS change with ongoing processing. From the website and label information, WPSb was calculated to be 4.6% and WPSs to be 8%. As there is no drying going on during the fermentation step, WPS will be equal to the WPSb during this processing step. WPS in the product after the drying step will be equal to WPSs, as the sausage is vacuum packaged during the storage period and no further loss of water can occur. There was no information on  Table 1: Time (h) to obtain 1 log-increase (bold) or 1 log-reduction (italic) at 24°C and 16°C of Yersinia enterocolitica and Escherichia coli. Yersinia enterocolitica Escherichia coli 24°C  16°C 24°C  16°C pH NaCl (% w/v) CBP PMP  CBP PMP CBP PMP  CBP PMP 6.0 4.5 8.7 4.4  19 9.1  7.2 6.0  22 17  5.0 11 4.6  23 9.6  8.3 6.7  24 18  6.0 16 o.m.  36 o.m.  11 181  32 455  6.5 20 o.m.  45 o.m.  13 180  36 454  7.0 25 o.m.  56 o.m.  o.m. 179  o.m. 453  8.0 o.m. o.m.  o.m. o.m.  o.m. 176  o.m. 451              5.0 4.5 14 4.6  32 9.3  14 11.7  41 31  5.0 17 4.7  39 9.6  15 13.0  46 34  6.0 25 o.m.  59 o.m.  21 94  59 235  6.5 32 o.m.  75 o.m.  24 94  67 238  7.0 40 o.m.  94 o.m.  o.m. 95  o.m. 241  8.0 o.m. o.m.  o.m. o.m.  o.m. 96  o.m. 247              4.5 4.5 21 5.0  48 9.9  23 18.0  70 47  5.0 25 5.0  59 10  27 20.6  79 53  6.0 38 o.m.  91 o.m.  35 59  102 147  6.5 47 o.m.  114 o.m.  41 59  116 150  7.0 59 o.m.  146 o.m.  o.m. 60  o.m. 153  8.0 o.m. o.m.  o.m. o.m.  o.m. 62  o.m. 159 CBP: ComBase Predictor, PMP: Pathogen Modeling Program, o.m.: outside model.  the fermentation or pH conditions of the sausage on the website, but it was stated that dextrose was added to boost the fermentation. This information was used to estimate the final pH of the sausage to be around 4.7. Initial pH was assumed to be similar to pH of raw meat, i.e. around 5.5 – 6.0. As the suspected sausage was organic, no nitrite was added. It was decided to compare growth and survival of Y. enterocolitica and E. coli in a matrix covering pH-values from 4.5 to 6.0 and WPS from 4.5% to 8.0%. Subsequently, the growth/reduction rates of Y. enterocolitica and E. coli in this matrix were predicted, at 24°C mimicking fermentation, at 16°C mimicking drying and at 5°C mimicking storage. Both CBP as well as PMP were used for the predictions (Table 1). The results showed that Y. enterocolitica would be able to multiply during the fermentation at 24°C as well as during the first part of the drying at 16°C (Table 1). Compared to E. coli growth of Y. enterocolitica was predicted to be slower in the beginning of the fermentation (24°C) but faster in the end with CBP and faster during the whole fermentation with PMP (Table 1). During drying and chilled storage only CBP covered WPS concentrations higher than 5% in the case of Y. enterocolitica. CBP predicted that an increase of one log-unit took approx. 50 h at conditions in the beginning of the drying period and approx. 100 h in the middle (Table 1). As multiplication of Y. enterocolitica is unlikely at WPS above 7% (ICMSF, 1996), no further growth during the rest of drying as well as storage would be expected. However, in case of a drying failure or addition of a too low amount of NaCl in the batter, growth of Y. enterocolitica could occur. In a semi-dried sausage with WPS of 7 %, CBP predicted a one log-increase of Y. enterocolitica in about 1 month at 5°C (results not shown). -20246810121416Start fermentation(16°C, 48 h)End fermentation& start drying (16to 13°C in 8 d)Mid drying (WPS= 5.0%)End drying & startchilled storage(5°C)End chilledstorage (120 d)Count (log10 CFU)Y. enterocolitica - outbreakE. coli - outbreakY. enterocolitica - normalE. coli - normal Figure 1: Predicted behaviour of Yersinia enterocolitica and Escherichia coli under normal production conditions and conditions similar to the semi-dried sausage implicated in the Escherichia coli O26:H11 outbreak in Denmark 2007. When the outbreak source was finally confirmed, a model was constructed in Microsoft Excel that calculated the expected behaviour of Y. enterocolitica and E. coli during production of the implicated sausage. As shown in Figure 1, the model predicted the level of Y. enterocolitica to increase 2.3, 4.2 and 7.8 log-units during fermentation, drying and storage under outbreak production conditions. The corresponding numbers for E. coli were 2.2, 1.9 and –3.1 log-units. As can be seen from Figure 1, E. coli levels were expected to be highest in the beginning of the shelf-life period. This corresponded well with the fact that most of the cases occurred in the first month after release of the sausage for sale. Conclusions  CBP and PMP were successfully used as support tools to evaluate whether two cases of infection with Y. enterocolitica could have originated from the same sausage that caused an outbreak of Escherichia coli O26:H11. Results showed that this was very likely because, at the point of release of the sausage for sale, production failures had led to an extra 3 log-increase of Y. enterocolitica as compared to normal production conditions. References ICMSF (1996) Microorganisms in foods 5. Characteristics of microbial pathogens, Blackie Acedemic & Professional, London, UK (ISBN 0412 47350 X) Resnik, S. and Chirife, J. (1988) Proposed theoretical water activity values at various temperatures for selected solutions to be used as reference sources in the range of microbial growth. Journal of Food Protection 51 (5), 419-423. 

Using ComBase Predictor and Pathogen Modeling Program as support tools in outbreak investigation: an example from Denmark

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 17, 2017Using ComBase Predictor and Pathogen Modeling Program as support tools inoutbreak investigation: an example from DenmarkMøller, Cleide Oliveira de Almeida; Hansen, Tina Beck; Andersen, Jens KirkPublication date:2009Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Møller, C., Hansen, T. B., & Andersen, J. K. (2009). Using ComBase Predictor and Pathogen Modeling Programas support tools in outbreak investigation: an example from Denmark. Poster session presented at 6thInternational Conference on Predictive Modelling in Foods, Washington, DC, United States.Using ComBase Predictor and Pathogen Modeling Program as support tools in outbreak investigation: an example from Denmark       Tina Beck Hansen, Cleide O.A. Møller and Jens Kirk AndersenNational Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, DenmarkQUESTION:Could the Yersinia enterocolitica, found in children suffering from ANSWER:YES! Yersinia enterocolitica can grow in the sausage – accordingMETHOD:Step 1. Collecting information on intrinsic factors of sausageStep 2 Comparing growth & reductionVTEC O26:H11 infection, also have come from the outbreak sausage?     to ComBase Predictor and Pathogen Modeling Program. .     rates using CBP & PMPStep 3. Predicting growth & reduction during sausage production161820)Target values for sausage productionValues of outbreak sausage• Growth of both Y. enterocolitica as well as VTECwas possible during the fermentation and the firstpart of dryingStep 3. Results101214ount (log 10 CFU)Weight 220 g 220 gpH 4.8 – 4.9 5.1 – 5.2aw 0.95 – 0.96 0.95 – 0.96  • Further growth of Y. enterocolitica was possible inthe end of drying Yersinia enterocolitica2468Predicted co • Slow reduction of VTEC but growth of Y. enterocoliticawas possible during storage• Fermentation error could have led to an additionall f b h l llVTEC0Start fermentation(16°C, 48 h)End fermentation& start drying(16-13°C, 8 d)Mid drying(WPS=5.0 %)End drying & startchilled storage(5°C)End chilledstorage (120 d)1 og increase o  ot  Y. enteroco itica as we  asVTEC at the point of release of the outbreaksausageGrowth / reduction rates during sausage processingReduction in PMP 7.0Step 2. Results     Step in processing pHWPS(w/v %)Temperature(°C)Time to 1 log change (without lag)Yersinia enterocolitica VTECFrom PMPTime to 1 log decrease =time to 5 log reduction / 5From CBP  Start fermentation 5.5 3.4 24 7.1 h 6.3 hEnd fermentation 4.8 3.4 24 11 h 10 hStart drying 4.8 3.4 16 25 h 27 hGrowth in CBP & PMP 7.0Time to 1 log increase =doubling time · ln(10) / ln(2)Mid drying 4.8 5.0 16 45 h 40 hEnd drying 4.8 6.0 16 3 d 8 dChilled storage 4.8 6.0 5 15 d 26 dpHBatter pH =Water and salt contents in batter and ready-to-eat sausage  pH of raw meat pH = 5.5Sausage pH =Dextrose added to boost the fermentation pH = 4.8Step 1. ResultsCalculation of Formula ResultWeight reduction (%) WR = 100 · ((M+N+S+D) - 100) / (M+N+S+D) 25 %S lt t t i b tt ( / %) Nb 100 N / (M N S D) 3 0 / %NutrientsPer 100 g sausage:Fat 9 g (F)a  con en  n a er w w  =  ·   + + + .  w w Water content in batter (%) Wb = 100 · Ws / (100 – WR) 84 %Water phase salt in batter (w/v %) WPSb = 100 · Nb / (Wb + Nb) 3.4 w/v %Salt content in ready-to-eat sausage (w/w %) Ns = 100 · N / Y 4 0 w/w %Protein 22 g (P)Carbohydrates 2 g (C)List of ingredients             .   Water content in ready-to-eat sausage (%) Ws = 100 – (F+P+C+Ns) 63 %Water phase salt in ready-to-eat sausage (w/v %) WPSs = 100 · Ns / (Ws + Ns) 6.0 w/v %For 100 g (Y) sausage:127 g meat (M), 4 g salt (N), 2 g spices (S), 0.5 g dextrose (D), no nitrite.CBP: http://www.combase.cc/predictor.htmlPMP: http://ars.usda.gov/Services/docs.htm?docid=11550

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Using ComBase Predictor and Pathogen Modeling Program as support tools in outbreak investigation: an example from Denmark

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