Prevalence, behavior and risk assessment of Salmonella spp. and Shiga toxin-producing Escherichia coli on basil leaves and strawberries

Consumption of fresh fruits and vegetables (F&V) is important for a healthy and balanced diet due to the presence of vitamins, minerals, fibres and other dietary components. However, F&V, in particular leafy green vegetables, that are consumed raw, have been associated with some notable outbreaks of microbial foodborne disease. Within this context and due the lack of data relating to the prevalence and behaviour of pathogens on F&V, the EU FP7 Veg-i-Trade project was developed. This PhD research deals with the microbiological analysis and exposure assessment of particular case studies of F&V being Salmonella and STEC in strawberries and basil. The objective of the PhD research was (i) to collect information about the prevalence of Salmonella and STEC on basil and strawberries and to investigate the behaviour of these pathogens during the storage of these commodities at different reasonably foreseen temperatures and (ii) subsequently use the obtained data as input in the comparative exposure assessment model for a quantitative risk ranking. To do so, first detection methods (i.e. the GeneDisc multiplex real-time PCR kit and culture methods for detection of Salmonella and STEC) needed some evaluation for their appropriateness in use for these specific food products (iii). In addition, exposure to pathogens due to consumption of basil and strawberries were compared to exposure to already prior well studied lettuce as a leafy green

Risk ranking of microbiological hazards Salmonella spp. and Shiga toxin-producing E. coli (STEC) on strawberry, butterhead lettuce and fresh herbs

Risk ranking is a useful tool to set priorities in controlling or monitoring risks. A comparative retail to fork exposure assessment model was built to show the effect of storage mode, period and washing practices by Belgian and Spanish consumers on the concentration of Salmonella and E. coli O157 on basil, strawberries and butterhead lettuce. Taking the measured prevalence of the pathogens into account, a risk ranking is made aimed at comparing pathogens, commodities or regions within Europe. Data relating to the survival of Salmonella and E. coli O157 on basil, strawberries and butterhead lettuce and the prevalence of both pathogens Salmonella and STEC on these commodities were collected by lab experiments during the Veg-i-Trade project. In addition information about the removal of pathogens and E. coli from leafy greens and strawberries and consumer behavior relating to storage mode and period and washing practices (Belgian versus Spanish consumers) were collected from literature and by a consumer behavior questionnaire, respectively. The exposure assessment model was built in excel and run via Monte Carlo simulation in the add-in function @Risk (100 000 iterations). Simulating the model resulted in the concentration and prevalence after storage and washing at consumers house and a distribution of the number (and fraction) of contaminated portions in one year of consumption. The concentration of pathogens after storage was generally higher for the Belgian consumers compared to the Spanish consumers due to less frequent storage in refrigerator and shorter storage periods applied by Belgian consumers. A higher concentration on basil and butterhead lettuce was found compared to strawberries mainly due to the higher die off of the pathogens on strawberries compared to lettuce and basil. Taking washing practices in account similar ranking was obtained. In addition, a small decrease was noticed in prevalence, e.g. prevalence of Salmonella on strawberries at retail was max. 1,2% versus max. 0,8% after storage and washing practices for 95% of the Belgian consumers. By including consumption frequency, a distribution of the number of contaminated portions in one year was calculated. The highest number of contaminated portions was found in case of basil, followed by butterhead lettuce and strawberries, due to less die-off of the pathogens compared to strawberries, less storage in refrigerator, less washing and higher prevalence on basil. In addition, a higher number was found for basil in case of Salmonella compared to STEC, whereas both pathogens were comparable for strawberries and butterhead lettuce, due to difference in survival and prevalence of Salmonella and STEC on basil. A higher number of contaminated portions for strawberries was found when consumed by Belgian consumers, whereas for lettuce when consumed by Spanish consumers. This can be explained by the more frequent consumption of strawberries by Belgian consumers in contrast to the more frequent consumption of lettuce by the Spanish consumers in one year

GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil

Introduction: VTEC have been increasingly recognized as food borne pathogen, also in fresh produce. Detection methods are primarily focused on E. coli O157:H7, but there is an increasing attention for non-O157 VTEC (1). Adequate detection methods are lacking for non-O157 VTEC, in particular for fresh produce with high levels of competing flora (2). Multiplex PCR methods are recommended for detection of VTEC virulence factors and have potential to detect multiple enteric pathogens or emerging virulence factors (e.g. aggR) in one run. Materials and methods: The aim of the present study is to validate an appropriate approach for high throughput multiscreening for VTEC and Salmonella in fresh produce focusing on the case studies of strawberries, lettuce and basil. Sample replicates were inoculated (1000, 100, 10 cfu/25 g) and analysed after 1 and 5 days storage at 7°C using in parallel GeneDisc Multiplex PCR (stx2, stx1, eae, aggR and Salmonella) and IMS-chromogenic media (CT-SMAC (O157), MAC (O26) and Brilliance Salmonella (Salmonella spp.)). Results and Discussion: Both methods enabled detection of the E. coli O157 and O26 and Salmonella Thompson strain at all inoculums if analysed after 24h although, in particular for basil, GeneDisc needed ½ dilution of DNA extract to overcome inhibition and competitive flora troubled reading of chromogenic media. After 5 days cold storage, pathogens were recovered for basil (stored at 10°C) whereas lower inoculums levels were more difficult to detect for strawberries (13/18 for VTEC and 10/18 for Salmonella) and lettuce (16/18 for VTEC and 18/18 for Salmonella). GeneDisc Multiplex PCR was shown to be a suitable screening method for simultaneous sensitive detection of VTEC and Salmonella in fresh produce although overcoming PCR inhibition by DNA dilution was needed in particular after 5 days storage and for the leafy vegetables. IMS combined with chromogenic media was shown to be a cumbersome method in particular for VTEC to differentiate from competing flora in leafy vegetables. When using CHROMagar STEC as chromogenic media (without IMS) better and clearer results were obtained

Survival of Salmonella and Escherichia coli O157:H7 on strawberries, basil and other leafy greens during storage

The survival of Salmonella and Escherichia coli O157:H7 on strawberries, basil leaves, and other leafy greens (spinach leaves, lamb and butterhead lettuce leaves, baby leaves, and fresh-cut iceberg lettuce) was assessed at cold (<7 degrees C) and ambient temperatures. All commodities were spot inoculated with E. coli O157:H7 or Salmonella to obtain an initial inoculum of 5 to 6 log and 4 to 5 log CFU/g for strawberries and leafy greens, respectively. Samples were air packed. Strawberries were stored at 4, 10, 15, and 22 degrees C and basil leaves and other leafy greens at 7, 15, and 22 degrees C for up to 7 days (or less if spoiled before). Both Salmonella and E. coli O157:H7 showed a gradual decrease in numbers if inoculated on strawberries, with a similar reduction observed at 4, 10, and 15 degrees C (2 to 3 log after 5 days). However, at 15 degrees C (and 10 degrees C for E. coli O157:H7), the survival experiment stopped before day 7, as die-off of both pathogens below the lower limit of detection was achieved or spoilage occurred. At 22 degrees C, strawberries were moldy after 2 or 4 days. At that time, a 1- to 2-log reduction of both pathogens had occurred. A restricted dieoff (on average 1.0 log) and increase (on average <0.5 log) of both pathogens on basil leaves occurred after 7 days of storage at 7 and 22 degrees C, respectively. On leafy greens, a comparable decrease as on basil was observed after 3 days at 7 degrees C. At 22 degrees C, both pathogens increased to higher numbers on fresh-cut iceberg and butterhead lettuce leaves (on average 1.0 log), probably due to the presence of exudates. However, by using spot inoculation, the increase was rather limited, probably due to minimized contact between the inoculum and cell exudates. Avoiding contamination, in particular, at cultivation (and harvest or postharvest) is important, as both pathogens survive during storage, and strawberries, basil, and other leafy green leaves are consumed without inactivation treatment

GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil

Introduction: VTEC have been increasingly recognized as food borne pathogens in fresh produce. Detection methods are primarily focused on E. coli O157:H7, but there is an increasing attention for non-O157 VTEC. Adequate detection methods are lacking for non-O157 VTEC, in particular for fresh produce with high levels of competing flora. Multiplex PCR methods are recommended for detection of VTEC virulence factors and have potential to detect multiple enteric pathogens or emerging virulence factors (e.g. aggR) in one run. Purpose: The aim of the present study is to validate an appropriate approach for high throughput multiscreening for VTEC and Salmonella in fresh produce focusing on the case studies of strawberries, lettuce and basil. Methods: Sample replicates were inoculated (1000, 100, 10 cfu/25 g) and analysed after 1 and 5 days storage at 7°C using in parallel GeneDisc Multiplex PCR (stx2, stx1, eae, aggR and Salmonella) and IMS-chromogenic media. Results: Both methods enabled detection of the E. coli O157 and O26 and Salmonella Thompson strain at all inoculums if analysed after 24h. For basil, GeneDisc needed ½ dilution of DNA extract to overcome inhibition and competitive flora troubled reading of chromogenic media. After 5 days cold storage, pathogens were recovered for basil (stored at 10°C) whereas lower inoculums levels were more difficult to detect for strawberries (13/18 for VTEC and 10/18 for Salmonella) and lettuce (16/18 for VTEC and 18/18 for Salmonella). Significance: GeneDisc Multiplex PCR was shown to be a suitable screening method for simultaneous sensitive detection of VTEC and Salmonella in fresh produce although overcoming PCR inhibition by DNA dilution was needed in particular after 5 days storage and for the leafy vegetables. IMS combined with chromogenic media was shown to be a cumbersome method in particular for VTEC to differentiate from competing flora in leafy vegetables

Survival of norovirus, murine norovirus 1, MS2 phage and E. coli in various types of water used for irrigation of fresh produce

Introduction: Viral food borne outbreaks, in particular Norovirus, have been associated with the consumption of fresh produce. Crops can be contaminated pre-harvest via irrigation water, (manured) soil or wild life, or post-harvest via contact with infected food handlers, contaminated transport or washing water or equipment. The use of surrogate viruses can be used to model survival of Norovirus in the environment. Purpose: The present study is focused on the survival and detection of NoV, Murine Norovirus 1 (MNV-1), MS2 phage and E. coli in different types of irrigation water (rain water, bore hole water, river water and open well water). Methods: These types of water were inoculated with human pathogenic NoV GI.3 and GII.4, surrogate viruses MNV-1 and MS2 and E. coli as the bacterial fecal indicator and stored at 10°C and 22°C. Monitoring of (non-cultivable) NoV was performed using real-time RT-qPCR, of (cultivable) MNV-1 by RT-qPCR and plaque assay (cell lines), of MS2 phage by plaque assay and E. coli by plating on Rapid’E. coli 2 (Bio-Rad). Results: All inoculated microorganisms (E. coli, MS2, MNV-1 (plaques and RT-qPCR) and NoV) showed a significant faster reduction at 22°C than at 10°C in all different types of water. The type of water had an impact on survival and highest reductions were observed in river water (≥ 2 log after 7, 14, 28, 35 and ≥ 62 days respectively at 10°C) and in open well water (≥ 2 log after 7, 14, 21, 62 and > 35 days respectively at 10°C). As expected, RT-qPCR signals for MNV-1 (and NoV) were detected for a prolonged period time but does not necessarily relate to infectivity, for which plaque assay are needed. At 22°C MNV-1 could be detected up to 35 and 62 days by RT-qPCR unlike only up to 10 days by means of plaque assay in river water and open well water, respectively. Significance: The stability of the microorganisms depends on the temperature and the type of water. The longest survival was noted at (commonly encountered) low temperature (10°C) and bore hole water or rain water. In particular NoV and MNV-1 show highest stability when monitoring using RT-qPCR, although this lacks information on the infectivity of micro-organism thus detected