Escuela Agrícola Panamericana, Zamorano Food Science and Technology Department Food Science and Technology Major Graduation Research Project Survival of E.coli O157:H7 in Sprouts produced in home-scale hydroponic system Presented by Brenda Fabiola Jovel González Advisors Ligia Luna M. Sc. Angela Shaw Ph.D Honduras, november 2023 2 Authorities SERGIO ANDRÉS RODRIGUEZ ROYO President ANA M. MAIER ACOSTA Vice President and Academic Dean ADELA M. ACOSTA MARCHETTI Head of Food Science and Technology Department HUGO ZAVALA MEMBREÑO General Secretary 3 Content List of Tables ........................................................................................................................................... 4 List of Figures .......................................................................................................................................... 5 List of Annex ............................................................................................................................................ 6 Resumen ................................................................................................................................................. 7 Abstract ................................................................................................................................................... 8 Materials and Methods ......................................................................................................................... 12 Location ................................................................................................................................................. 12 Bacterial Culture Preparation ............................................................................................................... 12 Inoculation ............................................................................................................................................ 13 Microbial Enumeration ......................................................................................................................... 14 Experimental Design ............................................................................................................................. 15 Results and Discussion .......................................................................................................................... 16 Conclusions ........................................................................................................................................... 22 Recommendations ................................................................................................................................ 23 References ............................................................................................................................................ 24 Annex .................................................................................................................................................... 27 4 List of Tables Table 1 Growth of E. coli O157:H7 on seeds/sprouts over seven days after inoculum and the differences between time periods ........................................................................................................ 17 Table 2 Growth of E. coli O157:H7 onto the surface of the jar after the first rinse over seven days .. 19 Table 3 Differences between growth of E. coli O157:H7 on seeds/sprouts and onto the surface of the jar over seven days ............................................................................................................................... 21 5 List of Figures Figure 1 Growth of E. coli O157:H7 on seeds/sprouts over seven days .............................................. 16 Figure 2 Growth of E. coli O157:H7 onto the surface of the jar over seven days ................................ 18 Figure 3 Growth of E. coli O157: H7 on seeds/sprouts and the jar surface over a seven days periods ...................................................................................................................................................... 20 6 List of Annex Annex A Statistical analysis seed ......................................................................................................... 27 Annex B Statistical analysis of surface .................................................................................................. 28 Annex C Significance of treatment........................................................................................................ 29 Annex D Counts of E. coli O157:H7 ....................................................................................................... 30 Annex E Test on the seed to verify that not have E. coli O157:H7 ....................................................... 38 Annex F Ampicillin requirement ........................................................................................................... 39 Annex G Results of bacterial culture preparetion ................................................................................ 40 7 Resumen El consumo de germinados, sobre todo de alfalfa, es el más consumido en los Estados Unidos debido a su valor nutricional y a los beneficios que se les atribuyen para la salud. Sin embargo, los germinados son susceptibles a una contaminación microbiana, lo que provoca enfermedades transmitidas por los alimentos. El objetivo de este estudio fue examinar la supervivencia de E. coli O157:H7 en germinados cultivados mediante sistemas hidropónicos caseros y comparar su crecimiento en la superficie de los tarros. Se eligió microorganismo E. coli O157:H7, una bacteria patógena. El experimento se llevó a cabo en el laboratorio International Center for Food Industry Excellence (ICFIE) de Texas Tech University, y la preparación del cultivo bacteriano implicó varios pasos para establecer una población microbiana controlada y estandarizada. Se inocularon las semillas con E. coli O157:H7 y se tomaron muestras del día 1 al 7 para su análisis. Los resultados indicaron que E. coli O157:H7 estaba presente tanto en las semillas como en la superficie del tarro y durante el crecimiento de los brotes. Se utilizó un diseño de Bloques Completos al Azar (BCA) para evaluar los tratamientos. La bacteria proliferó más en las semillas, alcanzando concentraciones de 7.64 Log UFC/g y en la superficie 5.25 Log UFC/g al cabo de 24 horas. La fase de germinación es fuente potencial de contaminación en los germinados, debido a las condiciones favorables que ofrece para el crecimiento bacteriano. Los resultados muestran la necesidad de aplicar prácticas higiénicas estrictas en todo el proceso de producción de germinados para mitigar los riesgos de enfermedades de transmisión alimentaria asociados a su consumo. Palabras clave: Alfalfa, crecimiento, inoculo, patógeno. 8 Abstract The consumption of sprouts, particularly alfalfa sprouts, is the most consumed in the United States due to their nutritional value and perceived health benefits. However, sprouts are susceptible to microbial contamination, leading to foodborne illnesses. This study aimed to examine the survival of E. coli O157:H7, a pathogenic bacterium, in sprouts grown using home-scale hydroponic systems and to compare its growth on the surface of the jars. E. coli O157:H7, was chosen as a model microorganism. The experiment was conducted at the laboratory of The International Center for Food Industry Excellence at Texas Tech University, and bacterial culture preparation involved various steps to establish a controlled and standardized microbial population. Seeds were inoculated with E. coli O157:H7 at 4 Log CFU/g and samples were taken at periods 0, 3, 6, 12, and 24 hours, 2, 3, 4, 5, 6, 7 days for analysis. Results indicated that E. coli O157:H7 was present in both seeds/sprouts and on the surface of the jar during the entire experiment period. A Randomized Complete Block was used to evaluate the treatments. The bacterium proliferated on the sprouts, reaching concentrations of 7.64 Log CFU/g and onto the surface 5.25 Log/CFU/ml after 24 hours. The study highlighted the importance of the germination phase as a potential source of contamination in sprouts due to the favorable conditions it provides for bacterial growth. Overall, the research demonstrated the capability of E. coli O157:H7 to proliferate during the sprouting process and indicated that the hydroponic environment of sprout production might enhance bacterial growth. The findings underscore the need for stringent hygiene practices throughout the sprout production process to mitigate foodborne illness risks associated with sprout consumption. Keywords: Alfalfa, Growth, pathogen, inoculum 9 Introduction Sprouts are germinated seeds of herbaceous plants (Mir et al., 2021) and are sold for human consumption in salads and sandwiches. Alfalfa sprouts are the most common crop/seed used for sprout production and consumption in the U.S. (Belabre et al., 2022). Eighty-five percent of this production comes from California, Idaho, Oregon, Washington and Nevada. Consumption of sprouts has been on the rise in recent decades owing to their nutritional value, consumer perceived health benefits and ease and rapid production (Ikram et al., 2021). Raw sprouts have been identified to be an important risk food for the transmission of foodborne diseases (Barbosa et al., 2015). Microbial contamination of sprouts has been reported to occur due to the presence of pathogenic bacteria in seeds, contaminated water and the growth environment facilitating germination and the sprouting process provides optimal conditions for bacterial growth (Yang et al., 2013) . The contamination of sprouts has become a worldwide food safety concern. Between 2015 to 2020, 137 foodborne pathogen associated illnesses were reported resulting from contaminated sprouts in different states of the United States, 84 of which were caused by E.coli O157:H7 (Marler, 2022). Annually, almost 300 million illnesses and about 200,000 deaths are caused by diarrheagenic Escherichia coli (E. coli) worldwide and this varies by region. Approximately 265,000 illnesses and about 100 deaths are caused by the E. coli O157:H7 strain (North Carolina Public Health, 2019). E. coli species (including O157:H7) live in the intestine of mammals especially sheep, cattle and goats, but are not normally affected by this organism (Rovid Spickler, 2016). Many the foodborne reported outbreaks have been related to the ingestion of contaminated food with this pathogen (Centers for Disease Control and Prevention [CDC], 2014). Infectious with E. coli O157:H7 is associated with bloody diarrhea, nausea, vomiting, dehydration, asthenia and decreased diuresis (Ameer et al., 2023). The Food Safety Modernization Produce Safety Rule (FSMA PSR) stablished specific rules for produce growers, this focuses on the prevention of contamination before, during, and after the 10 production of fresh fruits and vegetables, typically eaten raw (Food and Drug Administration [FDA], 2015). The requirements in 21 CFR Part 11, Subpart M (Sprouts) apply to the growing, harvesting, packing and holding of all sprouts except sprouts that are grown in soil or non-soil substrates (e.g., mats, perlite, or other growth media) and that are harvested above the soil or substrate line without their roots (§112.141) (FDA, 2019). The FSMA PSR specifies that sprout growers must take measures reasonably necessary to prevent the introduction of known or reasonably foreseeable hazards into seeds or beans that will use for sprouting. They must visually examine seeds and beans, and the packaging used to ship seeds or beans, for signs or potential contamination with known or reasonably foreseeable hazards and use only seeds that have been treated with scientifically valid method to reduce microorganisms of public health significance and handle the seeds in a manner to minimize potential contamination. They must grow, harvest, pack, and hold the sprouts in a fully enclosed building, and food contact surfaces you use to grow, harvest, pack or hold sprouts must be cleaned and sanitized before contact with sprouts or seeds or bean used to grow sprouts. They must take samples of spent sprout irrigation water or sprouts and test for E. coli O157:H7, Salmonella species, and any pathogens and have a written corrective action plan if any of these pathogens are positive (CFR, 2016). For E. coli, the focus is on the water use during growth (called agricultural water). The requirement for agricultural water used during cultivation activities is a microbial water quality profile (MWQP). There are two values that are included within the MWQP, Geometric Mean (GM) which specifies that there must be an average of less than 126 CFU of E. coli per 100ml of water Statistical Threshold Value (STV), which tells that the maximum is 416 CFU of E. coli per 100ml of water. The requirement for agricultural water used during and after harvest is not detectable, generic E. coli in 100 mL of water (CFR, 2016). Similar to other fresh vegetables, sprout production has embraced soilless production in controlled environment agricultural facilities such as hydroponics (Al-Kodmany, 2018). These facilities have been reported to have lower food safety risks in comparison to soil-based 11 production (Wang et al., 2020). Despite of this, recent reports have indicated food borne pathogen contamination in hydroponic produced vegetables (Sela Saldinger et al., 2023). The fact that hydroponic production of vegetables and sprouts is being adopted at home-scale level in urban centers where control is limited, poses a bigger food safety risk, which creates the need to examine the survival dynamics of the major foodborne pathogens in sprouts in similar homes units, especially where enteric pathogens such as E. coli can be introduced and persist in plants for longer periods compared to soil-grown crops (Xu y Warriner, 2005). This study was aimed to: First, to determine the differences between the two treatments growth in sprouts and the surface of the jar in terms of survival. Second, to examine the survival of E. coli O157:H7 in sprouts, produced in home-scale hydroponic systems. Third, to determine the survival of E. coli O157:H7 on the surface of the jar. 12 Materials and Methods Location The experiment was carried out at the International Center for Food Industry Excellence (ICFIE) in the Experimental Sciences Building at Texas Tech University in Lubbock, Texas, United States of America. Bacterial Culture Preparation For this study E. coli O157:H7 ATCC strain A4 was used, this strain was obtained from feces of a patient with hemolytic uremic syndrome. The first day, one loop of the isolated was taken and transferred to 10ml of Brain Heart Infusion (BHI), this was repeated in two more tubes. The culture was labeled and incubated for 24 hours at 37 °C. The next day, one tube was selected and conducted through serial dilutions, which were spread plated onto MacConkey selective agar and incubated for 24 hours at 37 °C, to determine the concentration of bacteria on the culture (Log CFU/ml). The third day, one colony was selected from the plates and spread plated to be put in 10ml BHI tubes with ampicillin antibiotic with different concentrations (10μg /ml, 25 μg /ml, and 50μg/ml). The fourth day, 1ml was transferred from the 10ml BHI tubes with different concentrations (10μg /ml, 25 μg /ml, and 50μg/m) to 9ml tubes of BHI without ampicillin antibiotic and were incubated for 24 hours at 37 °C. The fifth day, 1ml taken from all the 10 ml BHI tubes without ampicillin antibiotic was put into two new 9ml tubes of BHI with a concentration of 10 μg /ml ampicillin antibiotic to stabilize the strain and were incubated for 24 hours at 37 °C. On the day six, 1ml from the 10 ml BHI with a concentration of 10μg/ml tubes with the strain stabilized was put into new 9ml tubes of BHI with a concentration 25μg/ml ampicillin antibiotic to generate resistance, the tubes were incubated for 24 hours at 37 °C. There were made serial dilutions from the tube of concentration 10 μg/ml BHI plated onto MacConkey Agar with a concentration of 10 μg/ml ampicillin antibiotic to determine the log concentration of the bacteria and incubated for 24 hours at 37 °C. On the day 7, the CFU present in a concentration of 10 μg/ml ampicillin antibiotic was counted and 1ml was transferred from the 10ml BHI tubes with a 13 concentration of 25 μg/ml to a concentration of 50 μg/ml to be incubated for 24 hours at 37 °C to generate resistance. Also, were made serial dilutions from the tube concentration 25 μg/ml BHI and plated the inoculum onto MacConkey agar with 25 μg /ml of antibiotic to determine the log concentration of the bacteria. On the last day, there were made serial dilutions from the 10ml BHI tubes with a concentration of 25 μg/ml and plate it in MacConkey agar with 50 μg/ml of ampicillin (antibiotic) to establish resistant strains. There were resistant strains of E. coli because of the high concentration of background flora found on the seeds. Then, 1ml with a concentration of 25 μg/ml from the 10ml BHI tubes was transferred to a concentration of 50μg/ml BHI ampicillin and incubated 8 hours at 37 °C, until attaining 108 CFU/ml. 1ml of the concentrations that contains 50 μg/ml was put into 100ml BHI and incubated. After this inoculum was prepared, it was divided into five 20ml aliquots in centrifuge tubes. The samples were centrifuged for 10 minutes at 4,000 x g at room temperature, the supernatant was decanted carefully, to not lose the pellet. 10ml of BHI with 10% of glycerol were added and then the pellets were mixed well by shaking and suspended into one 50ml tube to bring the volume up to 50ml with BHI broth with 10% glycerol. The biological safety hood was used to make the strains, all the materials were autoclaved before starting to pipette 1ml of the cocktail into sterile 2ml cryogenic tubes. Finally, the cryogenic tubes were labeled and put into a labeled freezer box at -80 °C. Inoculation One vial of the ampicillin resistant E. coli O157:H7 was taken from the -80 °C freezer, one loop into 10ml of BHI and was incubated at 37 °C overnight. After the incubation, the 10ml of ampicillin resistant E. coli were put into sterile 30ml high-strength centrifuge tubes. The sample was centrifugated for 10 minutes at 4,000 x g at room temperature, and it was added 10ml of sterile distilled water and mixed well by shaking and suspended into 200ml of sterile distilled water and then 40ml were divided into five beakers. 14 Alfalfa seeds were purchased from a commercial provider. The suppliers of the seeds provided a certification of analysis that the seeds were heat treated and negative for E. coli and Salmonella. Each batch (20g) will be immersed in 40ml of E. coli O157:7 inoculum (original concentration 108 CFU/ml), gently swirling for 5 minutes at room temperature, to achieve the targeted 104 CFU/g level of inoculation on the seed. Excess bacterial suspension was drained, and the inoculated seed was spread over sterile absorbent sheets and air-dried overnight for 16 to 18 hours, under a biological safety hood. The inoculated seeds were soaked in sterile tap water and were left to soak at 25 °C for 8 hours, after which the excess will be drained. The seeds were sprouted by aseptically transferring them to a new sterile glass jar containing a small amount of sterile tap water in the base to maintain uniform moisture. Sprouting jars were inverted and kept at an angle of 45° in a tray to ensure proper drainage during the incubation. The sprouting jars were covered with a light blocking material at room temperature 25 °C with a relative humidity of 70 ± 5%. Germinating seeds were rinsed with 200 mL of sterile distilled water twice daily. The sprouts were exposed to light on day 6 and day 7 to the development of chlorophyll, and the leaves turn into a green color. The trial was done in 5 replicates. Microbial Enumeration Before inoculation, samples of the jar surface and sprout seeds were taken, to establish that no foodborne pathogens were present prior to the experiment. After inoculating the ampicillin resistant E. coli O157:H7, samples of the seed or sprouts were collected at the time 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 4 days, 5 days 6 days and 7 days from the inner surface of the lid (5 cm2) (S), and sprouts (SP). For each sample time, 2 g of sprouts were taken for microbial analysis. The sample sprouts were aseptically placed within a pre-weighed stomacher bag, and the mass was determined. The bags were transferred immediately to the lab where 18ml of Buffer Peptone Water (BPW) was added and the mixture was pummeled for 2 minutes with a stomacher (Lab Blender 400, Seward Laboratory, Worthington, UK). Plant tissues were further mashed manually by pressing with a thumb through the bag to facilitate the release of potentially internalized bacteria. The lid surface was 15 swabbed on the inside (5 square cm) and then placed in a pre-weighed stomacher bag with BPW. Samples were then directly plated on MacConkey Agar amended with 50 µg/ml ampicillin. Experimental Design Two treatments were evaluated, five replicates were established in the experiment for a total of ten experimental units. A Randomize Complete Block Design was used to compare the growth of E. coli O157:H7 over time (0 hours to 7 days) An ANOVA and a DUNCAN mean separation were performed to estimate significant differences between treatments (P < 0.05). The Statistical Analysis System® (SAS version 9.4) was used. 16 Results and Discussion According to Hamilton y Vanderstoep (1979) the germination step is the main source of contamination in sprouts as bacteria present in the seeds may become internalized during sprouting. As Robertson et al. (2002) mentioned, seeds can harbor high levels of bacteria ranging from 3.00 to 6.00 Log CFU/g. For this reason, this step focuses not only on the sources of contamination in the sprouts but also on the seeds (Figure 1). The seeds of alfalfa were germinated at (70 ± 5%) of moisture and warmth temperatures around 21 °C to 26 °C (Benincasa et al., 2019), these factors are ideal for pathogen growth onto the sprouts. According to Peñas et al. (2009) during the germination of the seeds, macromolecules such as lipids and proteins are broken down to form nutrients that are more easily digested and absorbed for E. coli O157:H7. For this reason, the pathogen grows during the sprouting process (Figure 1). Figure 1 Growth of E. coli O157:H7 on seeds/sprouts over seven days 0 1 2 3 4 5 6 7 8 9 0hr 3hr 6hr 12hr 24hr 48hr 72hr 4 days 5 days 6 days 7 days E. co li O 15 7: H7 (L og C FU /g ) Time E.coli O157:H7 Log CFU/g sprouts in seven days D C D G E A AB F C BC BC 17 Data also shows differences between treatment and periods of time. E.coli O157:H7 can reach concentrations of 6.1 Log CFU/g in seeds around 24hr, this can occur at 3 Log CFU/g initial concentration (Shaw et al., 2016). Similarly, in this study E. Coli O157:H7 could proliferate from 4.00 Log CFU/g to 7.6482 Log CFU/g in 24 hours (Table 1). Table 1 provides data on the growth of E. coli O157:H7 with measured given in hours (seed) and days (sprouts). There are significant differences between growth of E. coli O157:H7 and time periods. At the beginning 0 hours (group G), sprouts had a relatively low bacterial contamination of 4.00 log CFU/g. That indicates the initial level of contamination. Between 3 hours and 12 hours (groups D), there is a significant increase in the bacterial population in the sprouts. Suggests that E. coli O157:H7 adapted and multiplied rapidly during this period. Comparing the groups with different treatments (A, AB, BC, and C). It can observe the duration of exposure to E. coli O157:H7 significantly influences the bacterial populations in sprouts. Longer exposure times generally result in higher bacterial loads. Table 1 Growth of E. coli O157:H7 on seeds/sprouts over seven days after inoculum and the differences between time periods Group E. coli O157:H7 seed/sprout Log CFU/g Time G 4.0000 0hr E 6.2308 3hr D 6.9860 6hr D 6.8944 12hr BC 7.6482 1 day A 8.2634 2 days AB 8.0572 3 days BC 7.7484 4 days F 5.7244 5 days C 7.4924 6 days C 7.4244 7 days CV (%) 4.46 Note. A-G: Different letters represent differences between treatment and time. C-C: Same letters represent that there are not differences. AB: Letters in pairs do not represent differences with any of the periods that contains one of the letter of the group. Hr: Hours. 18 The way E. coli O157:H7 interacts in the soil and hydroponic systems is different. The hydroponic system provides a more favorable condition for bacterial growth because it has more moisture and water. E. coli O157:H7 is more movable in hydroponic environment according to Erickson et al. (2010) for this reason the pathogen could proliferate in the surface of the jar and attached to wet surfaces once the jar drained. According to Redondo et al. (2019) E. coli O157:H7 can survive for months in waters containers . E. coli O157:H7 exhibited significant growth, on the surface, from 5.00 Log CFU/ml to 8.00 Log CFU/ml (Figure 2). Therefore, on day five of germination, alfalfa sprouts have a higher nutrient content compared to the beginning of the germination process, minerals and nutrients increase in a 70% more and the minerals could be dispersed around the jar according to López Hernández et al. (1990). The sprouts were already in direct contact with the surface of the jar and the microorganism was able to again more strength on day five too. Figure 2 Growth of E. coli O157:H7 onto the surface of the jar over seven days 0 1 2 3 4 5 6 7 8 9 10 0hr 3hr 6hr 12hr 24hr 48hr 72hr 4 days 5 days 6 days 7 days E. co li O 15 7: H7 (L og C FU /m l) Time E.coli O157:H7 Log CFU/g surface over seven days E D BC D BC BC B BC A CD BC 19 Table 2 presents the growth of E. coli O157:H7 on the surface of the jar, with measurements given by hours and days. At the beginning 0 hours (group E), there is no detectable bacterial contamination on the surface of the jar. Indicates that the surface is initially clean and uncontaminated. Between 3 hours and 12 hours (groups D, BC), there is a significant increase in the bacterial population on the surface of the jar. E. coli O147:H7 adapted rapidly during this period, leading to a substantial increase bacterial load. Comparing the groups (A, BC, CD and D). It can be observed the duration of exposure to E. coli O157:H7 significantly influences the bacterial population on the jar. Table 2 Growth of E. coli O157:H7 onto the surface of the jar after the first rinse over seven days Group E. coli O157:H7 surface Log CFU/ml Time E 0.00 0hr D 5.2982 3hr CD 5.9682 6hr BC 6.5992 12hr D 5.2544 1 day BC 6.4508 2 days BC 6.2298 3 days BC 6.6492 4 days A 8.973 5 days B 6.8218 6 days BC 6.5434 7 days CV (%) 9.7 Note. A-B: Different letters represent differences between treatment and time. D-D: Same letters represent that there is no differences. BC: Letters in pairs do not represent differences with any of the periods that contains one of the letter of the group. Hr: Hours. The counts of E. coli O157:H7 in the two treatments increased from the initial inoculum level by about 2 Log CFU/ml in 3 hours into the seed and 5 Log CFU/ml onto the surface after the inoculum (Figure 3). 20 Figure 3 Growth of E. coli O157: H7 on seeds/sprouts and the jar surface over a seven days periods There was a higher growth of E. coli O157:H7 in the sprouts compared to the surface of the jars. Over time, the growth of E. coli O157:H7 on the seeds increased in both treatments. Even though, on day five there was a lower count in the sprouts due to the germination process, which is completed between day 3-8 according to Jiménez Pérez (2004), that is when they are ready to be harvested and are directly touching the surface of the jar which makes the pathogens more likely to stay on the surface on the jar instead of the sprouts. According to Macarisin et al. (2013) the pathogens are able to attach and even proliferate on plants and abiotic surfaces. As was expected, due to the sprouts provides favorable nutritional conditions not only for its growth but also for other organisms that associate with it, seeking food and survival opportunities like pathogens (Arriagada Ríos, 2000). The growth of E. coli O157:H7 in the sprouts shows a higher concentration compared to the surface. 0 1 2 3 4 5 6 7 8 9 10 0hr 3hr 6hr 12hr 24hr 48hr 72hr 4 days 5 days 6 days 7 days E. c ol i O 15 7: H7 (L og C FU /g ) Time E.coli O157:H7 over seven days Seed/Sprouts Surface a a a a a a a a a a a b b b a b b b b b b b 21 In this study, we identified that E. coli O157:H7 was capable to proliferate by at least 4.00 Log CFU/g to 8.2634 Log CFU/g during sprouting and 0.00 Log CFU/ml to 8.9739 onto the surface in a short time of period generating a risk for the consumer from the beginning of germination process and from the begging of the first rinsed (Table 3). The results showed significant differences in the treatments (P<0.05) except at 12 hours (P>0.05), the bacteria could stabilize in both treatments (Table 3). Table 3 Differences between growth of E. coli O157:H7 on seeds/sprouts and onto the surface of the jar over seven days Time E. coli O157:H7 seed/sprout Log CFU/g ± SD E. coli O157:H7 surface Log CFU/ml ± SD CV (%) 0hr 4.0000 ±0.00a 0.00b 0.0 3hr 6.2308± 0.32a 5.2982 ± 0.70b 7.09 6hr 6.9860 ± 0.31a 5.9682 ± 0.56b 3.25 12hr 6.8944 ± 0.39a 6.5992 ± 0.11a 4.11 1 day 7.6482 ± 0.29a 5.2544 ± 1.61b 17.49 2 days 8.2634 ± 0.54a 6.4508 ± 0.20b 4.47 3 days 8.0572 ± 0.33a 6.2298 ± 0.22b 1.78 4 days 7.7484 ± 0.39a 6.6492 ± 0.21b 3.66 5 days 5.7244 ± 0.15a 8.9730 ± 0.24b 2.64 6 days 7.4924 ± 0.24a 6.8218 ± 0.16b 3.27 7 days 7.4244 ± 0.08a 6.5434 ± 0.37b 3.97 Note. CV (%): Coefficient of variation. CFU: Colony Forming Units. Log: Logarithms. g: Gram. ml: Milliliter SD: Standard Deviation. abDifferent letters in each column denote significant differences between treatments (P<0.05) . aaSame letters do not denote differences between treatments. Hr: Hours. 22 Conclusions E. coli O157:H7 is able to increase by 4-7 log Colony Forming Units (CFU) from 0 hours to 7 days onto the seeds/sprouts. E. coli O157:H7 can migrate from sprouts to the surface of the jar once the jar is rinsed and maintain viability for a period of seven days. E. coli O157:H7 can persist and proliferate in both environments sprouts and surfaces, this characteristic represents a risk, since it could lead an easy spread of the bacteria in hydroponic systems. 23 Recommendations Take samples of the water that is used for the germination and sprouting process. Ensure that samples are taken at different time intervals to obtain a comprehensive understanding of E. coli O157:H7 can growth on water. Evaluate the cotyledons of sprouts or the short stem to pinpoint the regions with the greatest pathogen presence. This will help identify if one part of the sprout is more susceptible to contamination than others. Evaluate the reduction of bacterial load using a commercial disinfectant available at home. Perform efficacy test with the disinfectant in a series of controlled experiments to assess its ability to reduce bacterial contamination in sprouts. Conduct a comparative analysis of the bacterial contamination levels between E. coli O157:H7 and L. Monocytogens in sprout samples. This assessment will provide insights into the relative prevalence and potential risks associated with these two pathogens, aiding in the development of targeted mitigation strategies for sprout safety. 24 References Al-Kodmany, K. (2018). The Vertical Farm: A Review of Developments and Implications for the Vertical City. Buildings, 8(2), 24. https://doi.org/10.3390/buildings8020024 Ameer, M. 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Horticulturae, 6(1), 1. https://doi.org/10.3390/horticulturae6010001 26 Xu, J. y Warriner, K. (2005). Coliphage as an indicator of fecal contamination in hydroponic cucumber (Cucumis sativus L) greenhouses. Journal of the Science of Food and Agriculture, 85(14), 2397– 2400. https://doi.org/10.1002/jsfa.2249 Yang, Y., Meier, F., Ann Lo, J., Yuan, W., Lee Pei Sze, V., Chung, H.‑J. y Yuk, H.‑G. (2013). Overview of Recent Events in the Microbiological Safety of Sprouts and New Intervention Technologies. Comprehensive Reviews in Food Science and Food Safety, 12(3), 265–280. https://doi.org/10.1111/1541-4337.12010 27 Annex Annex A Statistical analysis seed R-Square Coeff Var Root MSE Surv Mean 0.985847 4.707581 0.310142 6.588145 28 Annex B Statistical analysis of surface R-Square Coeff Var Root MSE Surv Mean 0.948496 9.711664 0.571999 5.889818 29 Annex C Significance of treatment Significance of treatment Hour 3 TRT Pr>F 0.0227 Hour 6 TRT Pr>F 0.0016 Hour 12 TRT Pr>F 0.1683 Hour 24 TRT Pr>F 0.0285 Hour 48 TRT Pr>F 0.001 Hour 72 TRT Pr>F <.0001 Day 4 TRT Pr>F 0.0027 Day 5 TRT Pr>F <.0001 Day 6 TRT Pr>F 0.0107 Day 7 TRT Pr>F 0.0074 Note. TRT: Treatment. 30 Annex D Counts of E. coli O157:H7 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 3 Seed J1 94 75 84.5 10000 845000 5.927 Seed 17 12 14.5 100000 1450000 6.161 Seed J2 TMTC TMTC TMTC 100 TMTC TMTC Seed 77 84 80.5 10000 805000 5.906 Seed 13 9 11 100000 1100000 6.041 Seed J3 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 114 11 62.5 100000 6250000 6.796 Seed J4 TMTC TMTC TMTC 100 TMTC TMTC Seed 98 65 81.5 10000 815000 5.911 Seed 5 45 25 100000 2500000 6.398 Seed J5 TMTC TMTC TMTC 100 TMTC TMTC Seed 97 157 127 10000 1270000 6.104 Seed 28 9 18.5 100000 1850000 6.267 Note. TMTC: Too many to count Numbers To Count. CFU: Colony Forming Units. Log: Logarithms. mL: Milliliters. Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 6h Seed J1 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 61 70 65.5 100000 6550000 6.816 Seed J2 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 39 44 41.5 100000 4150000 6.618 Seed J3 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 249 234 241.5 100000 24150000 7.383 Seed J4 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 250 89 169.5 100000 16950000 7.229 Seed J5 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 79 74 76.5 100000 7650000 6.884 31 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 12h Seed J1 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 187 212 199.5 100000 19950000 7.300 Seed J2 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 151 201 176 100000 17600000 7.246 Seed J3 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 19 26 22.5 100000 2250000 6.352 Seed J4 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 6 9 7.5 1000000 7500000 6.875 Seed J5 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 7 3 5 1000000 5000000 6.699 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 24h Seed J1 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 64 61 62.5 1000000 62500000 7.796 Seed J2 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 49 29 39 1000000 39000000 7.591 Seed J3 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 110 128 119 1000000 119000000 8.076 Seed J4 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 12 48 30 1000000 30000000 7.477 Seed J5 TMTC TMTC TMTC 100 TMTC TMTC Seed TMTC TMTC TMTC 10000 TMTC TMTC Seed 19 21 20 1000000 20000000 7.301 32 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 48 Seed J1 226 200 213 1000000 213000000 8.328 Seed 62 35 48.5 10000000 485000000 8.686 Seed J2 206 126 166 1000000 166000000 8.220 Seed 18 53 35.5 10000000 355000000 8.550 Seed J3 31 19 25 1000000 25000000 7.398 Seed 4 8 6 10000000 60000000 7.778 Seed J4 TMTC TMTC TMTC 1000000 TMTC TMTC Seed 101 89 95 10000000 950000000 8.978 Seed J5 48 75 61.5 1000000 61500000 7.789 Seed 11 6 8.5 10000000 85000000 7.929 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 72 Seed J1 239 140 189.5 1000000 189500000 8.278 Seed 33 13 23 10000000 230000000 8.362 Seed J2 129 80 104.5 1000000 104500000 8.019 Seed 20 15 17.5 10000000 175000000 8.243 Seed J3 32 67 49.5 1000000 49500000 7.695 Seed 8 4 6 10000000 60000000 7.778 Seed J4 243 246 244.5 1000000 244500000 8.388 Seed 28 30 29 10000000 290000000 8.462 Seed J5 43 56 49.5 1000000 49500000 7.695 Seed 4 5 4.5 10000000 45000000 7.653 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 4 Seed J1 82 108 95 1000000 95000000 7.978 Seed 4 17 10.5 10000000 105000000 8.021 Seed J2 107 100 103.5 1000000 103500000 8.015 Seed 12 16 14 10000000 140000000 8.146 Seed J3 53 70 61.5 1000000 61500000 7.789 Seed 6 6 6 10000000 60000000 7.778 Seed J4 65 56 60.5 1000000 60500000 7.782 Seed 9 4 6.5 10000000 65000000 7.813 Seed J5 12 17 14.5 1000000 14500000 7.161 Seed 2 0 1 10000000 10000000 7.000 33 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 5 Seed J1 52 77 64.5 10000 645000 5.810 Seed 3 4 3.5 100000 350000 5.544 Seed J2 79 82 80.5 10000 805000 5.906 Seed 7 4 5.5 100000 550000 5.740 Seed J3 81 41 61 10000 610000 5.785 Seed 12 5 8.5 100000 850000 5.929 Seed J4 48 40 44 10000 440000 5.643 Seed 1 3 2 100000 200000 5.301 Seed J5 36 45 40.5 10000 405000 5.607 Seed 12 7 9.5 100000 950000 5.978 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 6 Seed J1 TMTC TMTC TMTC 100000 TMTC TMTC Seed 52 60 56 1000000 56000000 7.748 Seed J2 TMTC TMTC TMTC 100000 TMTC TMTC Seed 29 32 30.5 1000000 30500000 7.484 Seed J3 36 25 30.5 1000000 30500000 7.484 Seed 1 0 0.5 10000000 5000000 6.699 Seed J4 31 28 29.5 1000000 29500000 7.470 Seed 5 4 4.5 10000000 45000000 7.653 Seed J5 37 34 35.5 1000000 35500000 7.550 Seed 6 2 4 10000000 40000000 7.602 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 7 Seed J1 198 214 206 100000 20600000 7.314 Seed 69 55 62 1000000 62000000 7.792 Seed J2 222 185 203.5 100000 20350000 7.309 Seed 44 38 41 1000000 41000000 7.613 Seed J3 121 155 138 100000 13800000 7.140 Seed 44 35 39.5 1000000 39500000 7.597 Seed J4 156 151 153.5 100000 15350000 7.186 Seed 32 26 29 1000000 29000000 7.462 Seed J5 157 182 169.5 100000 16950000 7.229 Seed 36 44 40 1000000 40000000 7.602 34 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 3 Surface J1 TMTC TMTC TMTC 100 TMTC TMTC Surface 8 1 4.5 10000 45000 4.653 Surface 0 1 0.5 100000 50000 4.699 Surface J2 152 73 112.5 100 11250 4.051 Surface 11 3 7 10000 70000 4.845 Surface 0 0 0 100000 0 TMTC Surface J3 TMTC TMTC TMTC 100 TMTC TMTC Surface 82 38 60 10000 600000 5.778 Surface 6 7 6.5 100000 650000 5.813 Surface J4 TMTC TMTC TMTC 100 TMTC TMTC Surface 89 81 85 10000 850000 5.929 Surface 18 16 17 100000 1700000 6.230 Surface J5 TMTC TMTC TMTC 100 TMTC TMTC Surface 32 32 32 10000 320000 5.505 Surface 3 3 3 100000 300000 5.477 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 6h Surface J1 TMTC TMTC TMTC 100 TMTC TMTC Surface 33 76 54.5 10000 545000 5.736 Surface 8 2 5 100000 500000 5.699 Surface J2 TMTC TMTC TMTC 100 TMTC TMTC Surface 13 4 8.5 10000 85000 4.929 Surface 4 0 2 100000 200000 5.301 Surface J3 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 54 14 34 100000 3400000 6.531 Surface J4 TMTC TMTC TMTC 100 TMTC TMTC Surface 162 146 154 10000 1540000 6.188 Surface 13 53 33 100000 3300000 6.519 Surface J5 TMTC TMTC TMTC 100 TMTC TMTC Surface 80 117 98.5 10000 985000 5.993 Surface 21 15 18 100000 1800000 6.255 35 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 12h Surface J1 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 34 47 40.5 100000 4050000 6.607 Surface J2 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 52 22 37 100000 3700000 6.568 Surface J3 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 48 27 37.5 100000 3750000 6.574 Surface J4 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 58 64 61 100000 6100000 6.785 Surface J5 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 39 19 29 100000 2900000 6.462 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 24h Surface J1 TMTC TMTC TMTC 100 TMTC TMTC Surface 43 45 44 10000 440000 5.643 Surface 0 0 0 1000000 0 1.000 Surface J2 TMTC TTC TMTC 100 TMTC TMTC Surface 135 130 132.5 10000 1325000 6.122 Surface 2 2 2 1000000 2000000 6.301 Surface J3 TMTC TMTC TMTC 100 TMTC TMTC Surface 158 171 164.5 10000 1645000 6.216 Surface 1 2 1.5 1000000 1500000 6.176 Surface J4 TMTC TMTC TMTC 100 TMTC TMTC Surface TMTC TMTC TMTC 10000 TMTC TMTC Surface 3 11 7 1000000 7000000 6.845 Surface J5 TMTC TMTC TMTC 100 TMTC TMTC Surface 249 247 248 10000 2480000 6.394 Surface 0 0 0 1000000 0 1.000 36 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 48 Surface J1 199 202 200.5 10000 2005000 6.302 Surface 12 4 8 1000000 8000000 6.903 Surface J2 169 151 160 10000 1600000 6.204 Surface 6 0 3 1000000 3000000 6.477 Surface J3 159 132 145.5 10000 1455000 6.163 Surface 11 3 7 1000000 7000000 6.845 Surface J4 247 227 237 10000 2370000 6.375 Surface 15 2 8.5 1000000 8500000 6.929 Surface J5 140 131 135.5 10000 1355000 6.132 Surface 1 2 1.5 1000000 1500000 6.176 Time (hr) Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 72 Surface J1 27 25 26 100000 2600000 6.415 Surface 5 2 3.5 1000000 3500000 6.544 Surface J2 17 13 15 100000 1500000 6.176 Surface 3 3 3 1000000 3000000 6.477 Surface J3 15 13 14 100000 1400000 6.146 Surface 1 0 0.5 1000000 500000 5.699 Surface J4 22 24 23 100000 2300000 6.362 Surface 3 1 2 1000000 2000000 6.301 Surface J5 11 19 15 100000 1500000 6.176 Surface 1 1 1 1000000 1000000 6.000 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 4 Surface J1 172 278 225 10000 2250000 6.352 Surface 33 26 29.5 100000 2950000 6.470 Surface J2 TMTC TMTC TMTC 10000 TMTC TMTC Surface 69 68 68.5 100000 6850000 6.836 Surface J3 TMTC TMTC TMTC 10000 TMTC TMTC Surface 82 81 81.5 100000 8150000 6.911 Surface J4 TMTC TMTC TMTC 10000 TMTC TMTC Surface 52 31 41.5 100000 4150000 6.618 Surface J5 TMTC TMTC TMTC 10000 TMTC TMTC Surface 25 34 29.5 100000 2950000 6.470 37 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 5 Surface J1 TMTC TMTC TMTC 1000000 TMTC TMTC Surface 148 159 153.5 10000000 1.54E+09 9.186 Surface J2 TMTC TMTC TMTC 1000000 TMTC TMTC Surface 65 57 61 10000000 6.1E+08 8.785 Surface J3 TMTC TMTC TMTC 1000000 TMTC TMTC Surface 213 162 187.5 10000000 1.88E+09 9.273 Surface J4 TMTC TMTC TMTC 1000000 TMTC TMTC Surface 71 84 77.5 10000000 7.75E+08 8.889 Surface J5 TMTC TMTC TMTC 1000000 TMTC TMTC Surface 58 50 54 10000000 5.4E+08 8.732 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 6 Surface J1 60 50 55 100000 5500000 6.740 Surface 3 3 3 1000000 3000000 6.477 Surface J2 64 70 70 100000 7000000 6.845 Surface 12 5 5 1000000 5000000 6.699 Surface J3 29 50 39.5 100000 3950000 6.597 Surface 15 11 13 1000000 13000000 7.114 Surface J4 55 73 64 100000 6400000 6.806 Surface 4 9 6.5 1000000 6500000 6.813 Surface J5 126 141 133.5 100000 13350000 7.125 Surface 5 15 10 1000000 10000000 7.000 Day Type Jar Plate 1 Plate 2 Average Dilution CFU/mL Log CFU/mL 7 Surface J1 52 63 57.5 100000 5750000 6.760 Surface 1 0 0.5 1000000 500000 5.699 Surface J2 61 62 61.5 100000 6150000 6.789 Surface 8 2 5 1000000 5000000 6.699 Surface J3 123 114 118.5 10000 1185000 6.074 Surface 21 18 19.5 100000 1950000 6.290 Surface J4 144 132 138 10000 1380000 6.140 Surface 68 65 66.5 100000 6650000 6.823 Surface J5 TMTC TMTC TMTC 10000 TMTC TMTC Surface 112 129 120.5 100000 12050000 7.081 38 Annex E Test on the seed to verify that not have E. coli O157:H7 Seed at hour 0 Dilutions Plate 1 Plate 2 Average Average*Dil 10^1 0 0 0 0 1 0 0 10^2 0 0 0 0 1 39 Annex F Ampicillin requirement Ampicillin requirement Stock Concentration ampicillin 50μg/ml 4mg/ml using sterile distilled water and stored at 4 °C. Was added 12.5ml of stock to 1 L sterile distilled water to achieve final concentration of 50μg/ml. 50μg/ml of ampicillin in 1 L of sterile distilled water 4𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 × 12.5𝑚𝑚𝑚𝑚 = 50μg/ml 25 μg /ml of ampicillin in 1 L of sterile distilled water 12.5𝑚𝑚𝑚𝑚 × 25μg 𝑚𝑚𝑚𝑚 × 𝑚𝑚𝑚𝑚 50μg = 6.5𝑚𝑚𝑚𝑚 10 μg /ml of ampicillin in 1 L of sterile distilled water 12.5𝑚𝑚𝑚𝑚 𝑥𝑥 10 μg 𝑚𝑚𝑚𝑚 × 𝑚𝑚𝑚𝑚 50μg = 2.5𝑚𝑚𝑚𝑚 40 Annex G Results of bacterial culture preparetion Day three of culture Strain A4 10^7 10^8 10^9 Plate 1 23 9 1 Plate 2 46 14 2 Counts 8.537819095 9.060698 9.176091 Day six of culture (10µg/ml) Strain A4 10^7 10^8 10^9 Plate 1 46 3 1 Plate 2 43 7 0 8.648360011 8.69897 8.69897 Day seven of culture (25 µg/ml) Strain A4 10^7 Plate 1 14 Plate 2 19 Counts 8.217484 Day eight of culture (50 µg/ml) Strain A4 10^6 10^8 Plate 1 50 1 Plate 2 52 0 Counts 7.707570176 7.69897 List of Tables List of Figures List of Annex Resumen Abstract Materials and Methods Location Bacterial Culture Preparation Inoculation Microbial Enumeration Experimental Design Results and Discussion Conclusions Recommendations References Annex