1

Risk posed by STEC and other pathogenic

bacteria in seeds and sprouted seeds

Opinion of the BIOHAZ Panel

EFSA-Q-2011-00877

Maria Teresa da Silva Felício

On behalf of the BIOHAZ Unit

2

• EFSA scientific report with a comprehensive overview of experiences during the 2011 outbreaks of STEC 0104:H4 in Germany and France (3 October 2011)

• Report from the EFSA Task Force on Tracing seeds, in particular fenugreek seeds, in relation to the 2011 STEC O104:H4 outbreaks in Germany and France (5 July 2011)

• Joint rapid risk assessment of the outbreak in France focusing on four key areas: food source identification, collaborative trace-back investigations, awareness-raising amongst clinical practitioners, and public health advice (29 June 2011)

• Joint EFSA/ECDC public health advice on prevention of diarrheal illness with special focus on STEC (11 June 2011)

• Fast track risk assessment on consumer exposure to STEC through the consumption of raw vegetables and advice on options to mitigate the risks of possible food contamination and human infection (9 June 2011)

• Joint technical report with ECDC on the prevalence and incidence of STEC in humans, food and animals based on data supplied by EU Member States and data from outbreak investigation (9 June 2011)

EFSA activities in relation to the recent STEC O104 outbreak

3

• Reg. (EC) 852/2004 on the hygiene of foodstuffs lays down general hygiene requirements to be respected by food businesses at all stages of the food chain.

• In addition to the general hygiene rules, microbiological criteria have been laid down in Reg. (EC) 2073/2005

• Recent developments indicate that it may be necessary to consider more specific measures for certain food commodities (e.g. sprouted seeds)

Background

4

Terms of Reference

1. To assess the Public Health risk caused by pathogenic bacteria (STEC and others) that contaminate seeds (only those used for sprouting) and sprouted seeds intended for human consumption.

2. To the extent possible, to identify risk factors contributing to the contamination of seeds and sprouted seeds by bacterial pathogens.

3. To recommend specific risk mitigation options. To assess their efficacy in reducing the risk posed by bacterial pathogens. These should cover all parts of the food chain (from seed production to consumption or sprouted seeds).

4. To recommend, if relevant, microbiological criteria for seeds and sprouted seeds, water, and other material that may contribute to bacterial contamination during the production chain.

Sprouted seeds

5

Sprouts

ShootsCress

6

Description of the EU sprouted seeds sector and production processes (I)

• The seed sprout production chain has 3 parts:

• Seed production involves field preparation, planting, growth, irrigation, fertilization, pollination, swathing, field drying, seed harvest, storage and transport.

• Seed processing mainly consists in the elimination of extraneous material (soil, debris).

•Sprouted seeds production consists of receipt of seeds and storage, an optional decontamination of seeds, germination and growth, harvest, packing, storage, transportation and distribution.

•The shelf life of sprouts is 3-10 days. Storage conditions vary: sprouts and shoots at refrigeration temperatures, whereas cress, normally at ambient temperature.

Description of the EU sprouted seeds sector and production processes (II)

7

• Seeds are produced for several end-uses (e.g. edible, feed, oil production, horticulture etc) and not specifically for sprout production. Sprouted seeds producers may not know the origin and primary purpose of the seeds they use.

• Seed processing, shipping and selling practices often involve mixing multiple lots of different origins, which can be accompanied by a varietal mixing, complicating traceback and providing an opportunity for cross contamination.

•The varieties of sprouted seeds most commonly available on the EU market are: (i) Mung Bean sprouts, (ii) alfalfa sprouts, (iii) radish sprouts, (iv) peas shoots/cress, (v) sunflower shoots/cress. New varieties are introduced every year.

• The sprout sector in Europe: market value of 500 m €. Approximately 100 companies in EU, most of them small. The top producing countries are Netherlands, UK, Denmark and France.

• Sprouted seeds may also be produced at home by consumers.

8

Sprouted-seeds associated outbreaks (OBs) to date

• Most OBs reported in USA, some also in Canada, Germany, Sweden, Finland, Denmark, Germany, France, UK, Japan and Australia. For most the seed was suspected to be the origin of contamination, although poor production practices have also been implicated.

• Some OBs linked to sprouts for which seed decontamination was undertaken. Seed decontamination in isolation may not reliably prevent cases of foodborne illness.

• Reported foodborne OB data in the EU from 2004 to 2010:

• 9 Salmonella OBs: Denmark (2007), Estonia (2009), Finland (2007 and 2009), Norway (2007), Sweden (2006 and 2007) and UK (2 in 2010)

• 1 S. aureus OB in Denmark in 2008.

• On May 2011, Germany reported an ongoing OB due to STEC O104:H4. On June 2011, France reported a cluster of HUS cases (confirmed infection with STEC O104:H4). Trace back investigations support the hypothesis that both OBs were linked to fenugreek seeds used for sprouting, which became contaminated at some point prior to leaving the importer.

9

EU-monitoring data on occurrence of pathogenic bacteria in seeds and sprouted seeds

From 2004 to 2010:

• No information was found on the occurrence of foodborne pathogenic bacteria on dry seeds used for sprouting.

•Some data on the occurrence of STEC, Salmonella and L. monocytogenes in samples of sprouts are available:

• No STEC-contaminated sprouts samples were reported.

• For L. monocytogenes: 4 of 14 investigations resulted in the detection of contaminated sprouts.

• For Salmonella: 7 of 43 investigations resulted in the detection of contaminatedsprouts.

• Overall the number of investigations in sprouts is very small, with usually a low number of units tested and small sample sizes analysed.

10

• 11 food safety events notified regarding seeds used for sprouting or sprouted seeds from 2001 to 2011.

•Four out of these were related to seeds as follows:

• Germany (2008)• Finland (2009) • Sweden (2007)• France (2011)

• The remaining notifications regarded sprouted seeds and were distributed as follows:

• Denmark on alfalfa (2007) and beetroot (2010) sprouts • UK on mixed sprouts (2004)• Germany on sprouts mixture, and beetroot sprouts (2011).

RASFF notifications on sprouted seeds

11

EFSA Comprehensive European Food Consumption Database consumption data for sprouted seeds

• Large variation in consumption patterns across different MS, age groups and gender both in the percentage of the consumers that declaredconsumption, and in the average daily amount of product consumed.

•This would make the task of an EU risk assessment very complex.

• Sprouted seeds are mainly consumed within composite dishes without being the main ingredient, therefore the consumption figures in this database are an underestimation. Some consumers may not be aware that they had consumed sprouts in their food.

12

Survival and growth of pathogens on seeds and sprouted seeds

• Pathogenic bacteria may survive for extended time periods on or in seeds.

• Cracks or openings in the seed coat increase the opportunity for bacterial attachment and ingress.

• Information on survival of human pathogenic bacteria on seeds is only available from lab models. Naturally occurring, non pathogenic bacteria can persist for long periods on seeds.

• Pathogen growth in sprouted seeds has mostly been quantified in lab models, which probably overestimates the extent of multiplication and dissemination.

• Sprouted seeds are usually packaged in conditions maintaining high relative humidity, and bacterial growth will depend on storage temperature during shelf life.

• For those packaged in polymeric films, respiration may deplete O2 levels to potentiallypermit development of strict anaerobic bacteria (e.g. C. botulinum).

• Some sprouts may be lightly heat treated before consumption (e.g. stir-fried). The impact of these heat treatments on bacterial pathogens is not well documented.

13

The BIOHAZ Panel made the following conclusions and recommendations :

14

On the assessment of the public health risk (I)

•Sprouted seeds are ready-to-eat foods.

•They present a particular food safety concern due to:

(i) potential for pathogenic bacteria to contaminate seeds and to growduring sprouting, and

(ii) to their consumption patterns (raw or minimally processed).

•The various types of sprouted seeds (sprouts, shoots, cress) may not represent the same risk. There is mostly data for sprouts (scarce for shoots and cress, for which production is more comparable to that of fresh-cut leafy vegetables)

•Alfalfa and mung bean sprouts have been the most commonly consumed, and most frequently implicated products in OBs.

15

•Salmonella and pathogenic E. coli (including STEC) most commonly reported pathogens causing sprout-associated OBs. Very low contamination levels* of dry seeds can cause sprout associated-OBs.

•Other pathogens (e.g. B. cereus, S. aureus, L. monocytogenes and Y. enterocolitica) have also been implicated with OBs, although rarely.

•Epidemiological data shows that a single contaminated seed lot may be used by several sprouting plants, even in different countries, causing widespread OBs.

•Due to limitations in available data, it is not possible to carry out a QMRA to estimate the proportion of infections attributable to the consumption of sprouted seeds.

•Large OBs involving these products illustrate the potential to cause major Public Health emergencies affecting previously healthy people, and not limited to vulnerable populations.

On the assessment of the public health risk (II)

*Data for Salmonella

16

On the identification of risk factors (I)

• Pathogenic bacteria can be carried and transmitted by animals, humansand the environment, and they may contaminate seeds in the field and sprouted seeds throughout the production chain.

• Seeds may be contaminated via diverse routes. The most relevant risk factors are associated with agricultural practices on seed production, storage and distribution. Potential sources of contamination are: irrigation water and/or manure, presence of birds and rodents in storage facilities, dust and soil particles.

• Processing conditions (e.g. temperature, humidity) during sprouting of seeds favour the growth and dissemination of pathogenic bacteria and should be considered as major risk factors.

• The widespread distribution of seed lots is a risk factor that may increase the size and geographical spread of outbreaks.

17

• Poor traceability of the seed lots may delay the action taken by health authorities to control OBs. Poor traceability also presents difficulties for the sprouted seed producers to assess the hygienic quality of lots of seeds.

• Microbiological methods to detect emerging pathogens in seeds and sprouted seeds may not yet exist or be applied, posing an additional challenge in the identification of the source of outbreaks.

• There is limited information on risk factors during the production of shoots and cress.

• Contamination by asymptomatic carriers of pathogenic bacteria should not be excluded as a potential risk factor.

• Considering all the above, it has been difficult to control the hygiene of the production process of sprouted seeds.

On the identification of risk factors (II)

18

Regarding possible specific mitigation options (I)

• The Objective: Food safety management based on HACCP principles, including GAP, GHP and GMP along the whole chain (seed production to the final sprouted product).

• HA should include risk classification of commodities, regions of origin, and seed suppliers. In seed (and to some extent sprout) production, it can be difficult to define CCPs (including hazard control measures, critical limits and monitoring).

• Preventing seed contamination is particularly important because of the long survival of bacterial pathogens on seeds, and their multiplication during sprouting.

• GAP and GHP in primary production (inc. harvest and storage) should be applied with a high level of stringency, similar to that applied for the primary production of fresh-produce.

• Example measures include:

(i) identifying seed crops intended for sprout production; (ii) safe use of fertilizers and water;(iii) minimizing seed contamination with soil during harvest; (iv) workers harvesting/handling seeds to follow hygiene and health requirements; (v) seeds transported, processed and stored under conditions minimizing contamination; (vi) removing damaged seeds, from which it is more difficult to remove pathogens; (vii) improving traceability and minimizing mixing of seed lots.

19

• Washing of seeds to remove dirt before sprouting.

• Measures to prevent introduction of pathogens in sprouted seeds production very important. During sprouting, GMP, GHP and HACCP principles should be applied as for other ready-to-eat foods.

• Use of potable water is necessary during sprouting.

• Decontamination of seeds prior to sprouting, is currently practiced in some EU MS as an additional risk mitigation measure (part of a combined intervention strategy).

• No method of decontamination is available to ensure elimination of pathogens in all types of seeds without affecting seed germination or sprout yield. Decontamination of seeds would need to be optimised for each type of seed.

Regarding possible specific mitigation options (II)

20

•The safety and efficacy of different seed decontamination treatments (e.g. chemical, heat treatment, irradiation, alone or in combination) should be evaluated in a harmonised way at EU level.

• The consequence of any decontamination treatment on the background microflora, and its potential impact on the pathogenic bacteria during sprouting, should be taken into account.

• A chill chain for sprouts and shoots from end of production to consumption is necessary to limit growth of bacterial pathogens.

• Stakeholders, including consumers and also those practicing home-sprouting, at all parts of the production chain, should be informed of the food safety risk posed by sprouted seeds.

Regarding possible specific mitigation options (III)

21

Regarding microbiological criteria (I)

• Sprouted seeds are ready-to-eat foods. Finding pathogenic bacteria in seeds (for sprouting) or in sprouted seeds indicates a Public Health risk.

• Testing alone may convey a false sense of security. A negative sample result does not ensure absence of the pathogen in the tested lot, particularly where it is present at low or heterogeneous prevalence (statistical limitation of sampling plans).

• Currently not possible to evaluate the extent of Public Health protection provided by specific microbiological criteria for seeds/sprouted seeds. Need for data collection to conduct QMRA.

• Microbiological criteria, including the design of sampling plans, should be considered as one of the components of the food safety management system for the sprouted seed production chain.

• Existing criteria (Reg. 2073/2005) for sprouted seeds/seeds are: food safety criteria for Salmonella and L. monocytogenes, and a total E. coli process hygiene criterion.

• Consideration should be given to the development of new, or revision of the existingcriteria for pathogens most frequently associated with OBs (Salmonella and pathogenic E. coli).

22

• Currently, there are no criteria for pathogenic E. coli. If criteria were to be proposed, serotypes associated with severe human disease should be considered.

• Microbiological criteria for Salmonella, pathogenic E. coli and L. monocytogenes could be considered for seeds, during sprouting and in the final product, to this respect:

(i)detection and mitigation of a contamination problem earlier in seeds would avoid contamination being amplified during the sprouting process;(ii)testing seeds alone does not permit to detect contamination at a later stage. Therefore microbiological criteria could be useful during the sprouting process and/or for the final product;(iii) for the final sprouted seeds, the time required for the detection methods for pathogenic bacteria combined with the short shelf-life may not allow to withdraw the product in the event of a non-compliance, and(iv)an additional value of testing would be to build up knowledge about the hygienic performance of seed/sprouted seeds producers.

• When targets for seeds are considered, it should be noted that low levels of Salmonella (4 MPN/kg) have been sufficient to cause OBs.

Regarding microbiological criteria (II)

23

• The 2-class sampling plan in Reg. 2073/2005 (“absence in 25g”, n=5; c=0)will not give sufficient confidence to demonstrate absence of pathogen at low levels in seeds. In order to increase probability of rejection of a positive lot, it would be necessary to analyze kg quantities of the sample.

• Pooling strategies can be applied to reduce the number of analytical samples when testing seeds. Different approaches have been proposed but they need to be validated and standardized.

• Seeds intended for home sprouting should be subjected to a sampling protocol having at least the same level of stringency as for seeds intended for commercial production.

• The inspection lot on which the sampling plan is applied should represent only one seed production lot.

Regarding microbiological criteria (III)

24

•Testing especially important for new seed commodities, or when seeds are sourced from new suppliers.

•Dust/debris from seed storage areas may be regularly tested for Salmonella and STEC. Total E. coli counts may be included to provide evidence of faecal contamination.

•During sprouting, testing spent irrigation water proposed as an alternative strategy to the analysis of a large number of sprout samples. There are some uncertainties regarding the sensitivity.

•Sampling could be conducted on production environments. It could be applied for pathogenic bacteria (such as L. monocytogenes ), as well as indicator bacteria.

•There are currently no indicator organisms that can effectively substitute for the testing of pathogens in seeds, sprouted seeds or irrigation water.

•Testing for E. coli, Enterobacteriaceae and Listeria spp. can inform process hygiene control. Further work may be required to assess the value of tests.

• Important to use standard methods for testing (EN/ISO) especially developed for seeds, spent water and sprouts.

• Rapid (e.g. molecular) methods for detection and/or typing of pathogens are important to obtaintimely information, these should be validated according to EN/ISO methods.

Regarding microbiological criteria (IV)

25

Current work (I):

• Commission is discussing drafts for new regulation regarding:

• traceability requirements for seeds intended for sprouts production and sprouts,• hygiene requirements for the production of sprouts and• microbiological criteria for sprouts.

• EFSA has recently received a new mandate from Commission requesting a scientific opinion on the risk posed by pathogens in food of non-animal origin with the following Terms of Reference (ToR):

1. To compare the incidence of food-borne human cases linked to food of non-animal origin and food-borne cases linked to food of animal origin. This ToR should provide an indication of the proportionality between these two groups as regard humans cases and, if possible, human burden.

26

Current work (II):

2. To identify and rank specific food/pathogen combinations mostoften linked to food-borne human cases originating from food of non-animal origin in the EU.

3. To identify the main risk factors for the specific food/pathogen combinations identified under ToR 2, including agricultural production systems, origin and further processing.

4. To recommend possible specific mitigating options and to assess their effectiveness and efficiency to reduce the risk for humansposed by food/pathogen combinations identified under ToR 2.

5. To recommend, if considered relevant, microbiological criteria for the identified specific food/pathogen combinations throughout the production chain.

27

Thank you for your attention!

Thanks to the BIOHAZ Panel and experts members of this WG.