BLT Stages

Abstract Bachelor Project 1 FBT 2020-2021: Characterization of possible Bacillus cereus and Bacillus thuringiensis on fresh vegetables

Bacillus thuringiensis is a spore forming Bacillus, belongs to the Bacillus cereus group. The spores are used as biopesticides on various edible crops. B. thuringiensis, like B. cereus, can contain enterotoxin genes and as such are suggested to also have the ability to cause food borne toxi-infection when persons are exposed to foods which are contaminated with high numbers (usually more than 10⁵ CFU/g). B. thuringiensis has been found in foods linked to foodborne outbreaks that were reported to be caused by B. cereus. This is because B. thuringiensis is difficult to identify from B. cereus. The purpose of this thesis is the characterization of presumptive B. cereus (including thus also B. thuringiensis) on fresh produce. This study consists of four major parts. The first part is the Ultraviolet (UV)-C inactivation of the spore inoculum. The spore solution and the solutions of the commercial products are both tested. The inoculum is placed in the UV-C cabinet and stirred continuously. In the process, it is exposed to the UV-C light for a certain time. Then the inoculum is plated on tryptone soya agar (TSA). The second part is about the prevalence of presumptive B. cereus on vine tomatoes in the Flemish supermarket. In this process, the tomatoes are shaken in peptone physiological salt solution (pps) that simulates the washing procedure. After this, the inoculum is inoculated on mannitol egg yolk polymyxin agar (MYP-agar) and plate count agar (PCA). The last part discusses the biofilm formation of the spinach isolates. The inoculum is transferred to a polystyrene 96-well plate. After 48 hours of incubation, the wells are washed with pps, stained with crystal violet and destained with glacial acetic acid. After this, the resulting color will be measured with the Versamax at 595 nm. The results of the different methods are discussed below.

UV-C inactivation is effective at harvested B. thuringiensis spores. It provides a 4 log10 (CFU/ml) reduction. In contrast to the solution of the commercial B. thuringiensis products where a maximum of 1 log10 (CFU/ml) reduction occurred. It is important that the commercial products can be inactivated because they are used most frequently as on tomatoes.  Here, presumptive B. cereus was observed in a large amount of tomatoes. 97 % of the isolates of presumptive B. cereus taken were identified as B. thuringiensis. No exact results could be obtained on the dependence on the seasons because the months of March and April were also cold months in the year 2021. In addition to the tomatoes, also in a prior research study spinach was analysed for the presence of presumptive B. cereus. Most of the spinach isolates could form a biofilm. Those that could not form a biofilm were mainly isolates that were not identified as B. thuringiensis. The conclusion is that the spores as biopesticides are protected by several chemicals which means that inactivation with UV-C is not effective. Biopesticides are often used on different edible crops such as tomatoes to avoid insect pests. For acquiring more information on seasonality, a year around survey has to be performed. In addition to tomatoes, spinach plants are also sprayed with biopesticides. Various spinach isolates of presumptive B. cereus were also examined. Most B. thuringiensis were shown to be able to form biofilms but here replicate experiments are needed to assess reproducibility of results obtained.

Abstract Bachelor Project 2 FBT 2020-2021: The interaction of Bacillus cereus with a human epithelial Caco-2 cell line

Bacillus cereus is a bacteria that can cause gastrointestinal diseases after consuming contaminated food with enterotoxin producing strains or the preformed toxin cereulide. Bacillus cereus might cause many more cases of foodborne illness than is known, because the symptoms are relatively mild and brief. The first main aim is to estimate the effects of basic food-related stresses on enterotoxigenic Bacillus cereus pathogenic properties. The second main aim is to establish the reliable co-culture system between Bacillus cereus and the Caco-2 cells and select the adherence assay.

First of all the Bacillus cereus strains were characterized by detecting toxin genes via Real-Time Polymerase chain reaction. The enterotoxin production was also detected. Almost all strains encoded the nheA component. The cytotoxicity of Bacillus cereus strains on a Caco-2 cell line was observed using MTT. It showed that strains with similar main toxigenic properties differ in their cytotoxicity. The Bacillus cereus strains were also exposed to food-related stress to detect the effect of those stresses on the cytotoxicity of Bacillus cereus. In general, for all enterotoxin-producing strains there was a significant (p < 0,05) increase in cytotoxicity between cells stressed in severe stress conditions. Finally the multiplicity of infection and adherence of the strains on a Caco-2 cell line were performed after optimizing the protocol. The best results for the infection of the Caco-2 cell line were obtained at the MOI of where 90 % of the cells were found to be viable after 3 h incubation during which Bacillus cereus was able to produce enterotoxin. The results adherence showed that Bacillus cereus is very sensitive to saponin, triton X-100 and trypsin. Therefore, a mechanical method had to be used. The results showed that adherence properties are linked with pathogenic properties of the strains.

The study showed that enterotoxin-producing strains had a higher cytotoxicity when they were exposed to stresses. The best results for MOI were obtained where 90 % of the cells were viable. A mechanical method had to be used for adherence.

Abstract Bachelor Project 3 FBT 2020-2021: Identification and characterization of potential spoilage bacteria isolated from blue mussels (Mytilus edulis) stored under modified atmosphere packaging

The blue mussel is largely consumed in Belgium. Because this is a highly perishable food, mussels are sold alive. The mussels are most of the time packed with modified atmosphere packaging. This improves the shelf life. To get a more understanding of the shelf life of mussels, it is important to investigate the potential spoilage of this product. That’s why the following question is asked. What kind microbiological spoilage processes occur in mussels stored under gaseous atmospheres and how can it be analyzed under simulated conditions?

To get the answer to the question, a storage experiment is set up. This will tell how the microbiological growth is on the mussel in a certain condition. The bacteria obtained from that experiment were isolated. These isolates got characterized and identified with MALDI-TOF-MS. After this a simulation media was made to investigate the microbiological spoilage of the mussel in a more controlled way. Thanks to the simulation media, there is less natural variation that have an effect on the results.

The most bacteria that cause spoilage of the mussels, are best grow on the marine agar media. This media gives also the big variety of bacteria which is also shown with the characterization test and the identification by the MALDI-TOF-MS. For the identification of the bacteria, more investigating is necessary because not all the bacteria could be identified with the MALDI-TOF-MS. This could be done by 16s sequencing. The quality of the identification still needs to be checked with the SPeDE program by Ugent.

For experimenting in controlled conditions, a simulation media is made from mussels. This protocol is made and perfected. The media needs to meet following requirements: there is no contamination on the media and all the different kind of bacteria can grow on the media. This was successfully. But to know for sure that the simulation media is similar as the mussels, the bacteria also need to grow in the same way. This still needs to be investigated in the future.

Abstract Bachelor Project FBT 2019-2020: Prevalence of Bacillus thuringiensis and Bacillus cereus in fresh vegetables

Bacillus thuringiensis is a world-wide used biopesticide. It is a soil-dwelling bacterium and is closely related to the human pathogen Bacillus cereus. Bacillus thuringiensis produces parasporal crystals, which are toxic to insects. Although there has been a lot of research regarding safe use, more research has to be executed. Most food-borne outbreaks that were Bacillus cereus related, had a concentration above 5 log₁₀ CFU/g. It is not known to what extent Bacillus thuringiensis and Bacillus cereus are present on vegetables.

The main purpose of this study is to determine the prevalence and also behavior of Bacillus thuringiensis and Bacillus cereus on fresh vegetables. Some samples of spinach were plated on MYP agar to determine the natural occurrence of the Bacillus group members. Furthermore, different techniques were tested on these samples such as the phase-contrast light microscopy which can detect parasporal crystals. To test the behaviour of Bacillus thuringiensis on the spinach, inoculation of the spinach with Bacillus thuringiensis was executed and a number of presumptive Bacillus cereus were monitored during post-harvest storage.

Presumptive Bacillus cereus counts were found between 2.2 log₁₀ CFU/g and 1.6 log₁₀ CFU/g. Bacillus thuringiensis can be identified by detection of the parasporal crystals produced by the bacterium. Using this technique, 25 out of 33 isolates of presumptive Bacillus cereus could be identified as Bacillus thuringiensis. To further characterize these remaining isolates for their identity or pathogenic potential, more techniques have to be executed, this could include PCR to detect specific DNA sequence typical for  Bacillus cereus or Bacillus thuringiensis, DNA sequence for toxin genes or immunoassays to detect toxin production when the strains have grown to high numbers in lab media.

The result has shown that Bacillus thuringiensis spores do not increase after application on spinach leaves, nor increase during cold storage of the inoculated spinach leaves. Thus, mere prevalence of Bacillus thuringiensis spores does not seem to result in germination and outgrowth.

However, in practice this might be different. A decrease in the number of spores during pre-harvest growth can be expected. This is because in practice there are additional factors, such as the weather (UV-radiation and rainfall). UV-light is known to destroy bacteria, because of this, the concentration of spores might decrease. Rainfall can have a diluted effect on the spores, which will lower the concentration as well.

This project showed that none of the presumptive Bacillus cereus samples had a natural contamination higher then 5 log₁₀ CFU/g. However, the only used vegetable was spinach and only few samples could be analysed due to corona-crisis, so further research has to be conducted.

Abstract bachelor project 1 2017-2018: Inactivation of L. innocua and E. coli by short-wave ultraviolet light (UVC) on raspberry agar

Because of the growing demand of fresh produce, the incidence of food-borne outbreaks are on the rise. Although the food industry already implements certain prevention measures like the hazard analysis critical control point (HACCP), many producers search for emerging techniques to decrease the contamination of minimally processed products. Ultraviolet light is already widely used for it’s germicidal properties in the decontamination of surfaces and drinking water.

The purpose of this study was to investigate the inactivation of Listeria innocua and Escherichia coli on raspberry agar (RA) by short-wave UV and assess the impact of the UVC dose and the freezing on the inactivation. Also the role of the pH in inactivation of E. coli and L. innocua in raspberry agar was determined.

L. innocua and E. coli were innoculated on three different types of agar: nutrient agar with a pH of 7 (NA7), nutrient agar with a pH of 3 (NA3) and raspberry agar. A set of controls and samples were made for each agar. The samples were treated with UVC inside a UV chamber. To measure the effect of the dose on the UV treatment, the samples were treated for different treatment times and at different lengths away from the UV lamp. To measure the impact of the freezing, the samples were frozen to -40 °C for 60 min before the treatment. By comparing the values of the controls, the effect of the pH on L. innocua and E. coli could be determined.

The results showed that the pH had no significant effect on the growth of E. coli or the inactivation by the UVC light. However, the pH had an effect on the initial contamination of L. innocua. The presence of acidic pH in the NA3 resulted in a 0,13 and 0,42 log CFU/g reduction of the 9 and 7 log CFU/ml cocktail concentration, respectively. However, when comparing NA7 with raspberry agar the reduction was higher: 0,58 and 1,28 log CFU/g for cocktail concentrations of 9 and 7 CFU/ml, respectively. This showed that the pH is not the only factor to explain the reduction of L. innocua on RA. The freezing resulted in a 0,89 ± 0,55 log CFU/g reduction for E. coli and a 0,73 ± 0,09 log CFU/g reduction for L. innocua on raspberry agar, but it made the UVC treatment less effective. While the fresh plates often showed total inactivation, with a minimum reduction of 3,26 log CFU/g for L. innocua and 3,77 ± 0,10 log CFU/g reduction for E. coli, frozen plates only showed 1,17 ± 0,58 and 1,97 ± 0,58 log CFU/g reduction for L. innocua and E. coli, respectively. The treatment time as well as the distance had a significant influence on the dose of UVC. At a treatment time of 10 min, E. coli was totally inactivated (reduction > 7,0 CFU/g) below the detection limit of 1 CFU/g, while L. innocua showed total inactivation on the RA as well (>5,7 CFU/g). When the treatment time was lowered, the variation of the results increased with reductions between 0,7 to 5,5 log CFU/g for L. innocua and for E. coli a reduction of 3,2 ± 1,3 log CFU/g with a treatment time of 1 minute.

The treatment time and distance had a big influence on the efficiency of the UVC treatment with 10 minutes at 60 mm showing almost total inactivation. The freezing slightly reduced the initial contamination level, but combining freezing with UVC treatment was not efficient enough to completely get rid of the contamination.

Abstract bachelor project 2 2017-2018: The effect of decontamination on sprouts

In recent years, there have been numerous food outbreaks associated with sprouted seeds. The bacterial pathogens most frequently associated with illness on sprouted seeds are Salmonella and E. coli O157.

To prevent future food outbreaks, research was done to reduce the outgrowth of pathogenic bacteria during the germination process of the sprouts.

This was investigated on the basis of three types of disinfectants (sodium hypochlorite, hydrogen peroxide and peracetic acid) and a commercial bacteriophages mix.

First there have been done pre-experiments. The stability of the disinfectants was tested, the influence of the disinfectants on the length of sprouts was tested, the influence on the germination percentage after contact with the disinfectants was tested as well and the removal of the active component of the disinfectants was investigated. Only sodium hypochlorite (NaOCl) and hydrogen peroxide (H₂O₂) showed stability of the concentration of active component. NaOCl did not have any effect on the length of the sprouts, but the other three disinfectants did.  Peracetic acid with pH 3.5 and pH 6.5 had a negative effect on the germination percentage of the sprouts, NaOCl and H₂O₂ did not. The active component of all three disinfectants disappeared after one hour interacting with the seeds. After the pre-experiments, the disinfectants could be tested in the drums. A concentration of 80 ppm NaOCl pH 6.5 and a concentration of 100 ppm H₂O₂ had no effect on the reduction of the bacteria during the germination of the seeds. Peracetic acid was not tested in the drums, due to not getting the 40 ppm concentration and the pH 6.5 correct at the same time in large quantity. The bacteriophages in a concentration of log 12 PVE/ml eliminated all the Salmonella colonies (CFU/g) on the seeds, even in the enrichment were no Salmonella colonies (CFU/g) detected.

Taking all results in count, the tested concentration of the three disinfectants cannot be used in the industry during the germination of seeds. On the other hand, the bacteriophages were a successful disinfectant to disinfect leek seeds during the soak step.

Abstract bachelorproef 1 2016-2017: Bepaling van de temperatuur cardinaal parameters voor Listeria monocytogenes en Salmonella enterica en validatie in salade en spinazie extract

Salmonella enterica and Listeria monocytogenes are two important pathogens in the food industry. Due to their growth potential in food they are also important to determine the ultimate date of (safe) consumption. Current methods to determine growth potential of these pathogens in foods are laborious and time-consuming, thus the use of predictive microbiology is preferred. But to be able to predict the growth there’s a need to know the exact growth characteristics of these pathogenic bacteria. Recently, a study claimed that Salmonella growth was enhanced by the use of salad and spinach extract and that even growth at temperatures of 4 °C could occur.

The determination of the temperature cardinal parameters for different Listeria monocytogenes and Salmonella enterica strains.

To confirm if L. monocytogenes and S. enterica can grow in salad and spinach extract at low temperatures and to validate the results from the cardinal experiment.

The bacterial suspensions of some selected L. monocytogenes and S. enterica strains were diluted in microtiter plates and the optical density was spectrophotometrically measured during incubation at various temperatures. The result are a set of growth curves which can be used to determine the generation time at the different temperatures. Those can be used to make a graph and to fit a model and so determine the cardinal parameters (minimum, optimum, maximum growth temperature).

For the salad and spinach, the different extracts were diluted and inoculated with the selected strains and kept at 4 °C, 7 °C, 12 °C. After 5, 9 and 14 days of storage the pathogens’ numbers were determined by plating on appropriate media. These counts are compared to the initial inoculum of day zero to see if there was any growth during incubation.

All Salmonella strains started to grow at 9 °C while all Listeria strains grew already at 2 °C. Salmonella Enteritidis did not yet reach its maximum growth speed at 42°C within the monitored temperature range. It should be noted that due to the nature of the strain, the Listeria strain 101 isolated from fish did not generate useful results for calculation of cardinal values.

Growth in salad or spinach extracts was confirmed at all temperatures for L. monocytogenes but only at 12 °C for Salmonella enterica strains. This is in accordance with the primary results from the cardinal experiment.

Extra measurements at higher temperatures should be performed for S. Enteritidis. A new experiment needs to be performed for Listeria strain 101. All other results should be linked to a model to determine cardinal values. Salmonella was shown not to grow at refrigeration temperatures (< 8°C) but could grow, although slower than L. monocytogenes, at 12°C.

Abstract bachelorproef 2 2016-2017: Optimization of a heat treatment for Bacillus cereus endospores

Bacillus cereus is a pathogen which produces endospores and toxins, these toxins are an important cause of food intoxications. Thus, it is important to inactivate the bacteria/ endospores before they can form any of these toxins.

The goal of this experiment is to optimize a heat treatment for B. cereus endospores. The spores used were from different strains of B. cereus (the strains used were LMG18989, ATCC14579 and NVH391 - 98).

The heat treatment and heat activation step was performed by two different methods. In the first method, the spores were treated in a hot water bath in capillary whereas they were treated in a PCR - device with thin - walled PCR tubes in the second method. Also, a variety of media and incubation times were used to differentiate the damaged bacteria from the undamaged ones.

The results obtained from the heat activation showed a change in the number of spores that germinated before and after the heat activation. Only one strain showed different results. This was the mesophilic strain, with this strain the number of germinated spores was lower after the heat activation than before.

Due to clumping of the spores and perhaps in combination of them sticking to the side of the plastic tubes no useful results were obtained for the inactivation. However, the results were used for the other goal. There were no indications of damaged spores between the 24 - hour and the 72 - hour incubation, almost no change in Log (CFU/ml) was recorded. Even after 7 days, no change in Log (CFU/ml) was seen. The difference between media was clear. The number of spores counted on the TSA + 1,5% NaCl were always lower than the number of spores counted on the normal TSA. This is due to the harder growing conditions in the TSA + 1,5% NaCl.

Optimization of the heat treatment of the three bacillus cereus strains is still ongoing. For future research, Tween80 should be used in PPS, plastic needs to be eliminated as much as possible, sonication could help to dissolve the clumps but may damage the spores and beads could be added to the test tubes with PPS also to dissolve the clumps with the same risk of damaging the spores. For the different incubation times it can be stated that it doesn’t make a big enough difference to distinguish the damaged from the non – damaged bacteria. In contrary to the different media where it was clear that TSA + 1,5% NaCl had less growth than the normal TSA as expected.

Abstract bachelorproef 1 2015-2016: Evaluation of chemical treatments to enhance the safety of alfalfa and leek sprouts

Sprouted seeds are young seedlings obtained from the germination of seeds. They are considered as ready to eat foods ,however, there is a high risk factor for sprouts because they are eaten raw and up to now there is no tested and working bactericidal step. This can cause outbreaks if the sprouts are infected with pathogens like Salmonella and E. coli O157:H7. In this study the survival of pathogens on three alfalfa and leek seed batches in storage conditions will be tested and the effect of decontamination treatments on the microbiota of the seeds. The consequences on the germination from these decontamination treatments will also be taken into account.

The amount of bacteria are measured using the plate count method. Different media are used to determine the microbial composition on the seeds. The mini-most probable number method is used when the amount of bacteria is too low for the plate count method. Furthermore, tests to determine the germination efficiency of the seeds are performed.

The pathogens survived on the seeds for at least 250 days under 15 °C and 0,43 aw. The best surviving Salmonella strain is Salmonella Typhimurium. After 264 days the highest E.coli O157:H7 log CFU/g reduction is 2,32 coming from batch A1 with an inoculation of 2,83 ± 0,18. The lowest was a mere 0,63 log CFU/g reduction coming from batch A3 with an 3,70 ± 0,74 log CFU/g. The highest reduction for Salmonella is batch A1 from Salmonella Thompson with 1,78 log CFU/g reduction coming from 3,36 ± 0,12 and the lowest is from Salmonella Typhimurium batch L1 with 0,23 log CFU/g coming from 4,01 ± 0,33 log CFU/g

There are generally less bacteria on the alfalfa seeds than on the leek seeds, probably because the alfalfa seeds have an extra shell. The most prominent bacteria are anaerobic bacteria and pseudomonas species. On the RCA are approximately 2,65 log CFU/g for alfalfa seeds and 5,70

log CFU/g for leek seeds. On the PAF are approximately 1,17 log CFU/g for alfalfa and 3,88 log CFU/g for leek seeds. The Enterobacteriaceae are also quite high with alfalfa having an average of 1,07 log CFU/g and leeks having an average of 2,78 log CFU/g.

The decontamination treatments tested are washing with water, washing with 200 ppm NaOCl solution and washing with 1% H₂O₂ solution. The water was tested to see how much bacteria were rinsed away. Surprisingly, this treatment had an adverse effect causing an increase in log CFU/g and as expected no effect on the germination percentage. An additional test showed that the analysis method with stomaching removed the most bacteria but that during 30 minutes other bacteria came loose during soaking and so a small increase of log CFU/g in function of time could be seen. This increase is smaller on PCA with about 0,35 log CFU/g increase compared to VRBG with about 0,68 log CFU/g increase after 30 min.

The 200 ppm NaOCl treatment had the best results with the highest reduction being 1,68 log CFU/g on the mesophilic bacteria and having almost no effect on the germination percentage. The H₂O₂ treatment caused an increase in Enterobacteriaceae and also a decrease up to 22,3 % in germination percentage.

These results were still too low to be considered a good antimicrobial step. A reduction of 5,0 log CFU/g is preferred. With the pathogens surviving for at least 250 days a good antimicrobial step is needed to consider the sprouts “safe”.

• De literaire achtergrond rond de gebruikte toepassingen en de onderzochte bacterie ‘Listeria monocytogenes’ in de loop van het onderzoek;
• Bespreking van het eerste experiment: de validatie van een groei/niet-groei model voor Listeria monocytogenes in surimisalade en de invloed van gewijzigde omgevingscondities;
• Bespreking van het tweede experiment: de validatie van een groei/niet-groei model voor elf verschillende Listeria monocytogenes stammen in negen verschillende media met aangepaste omgevingscondities.