Progress report for GNE22-297
Project Information
Salmonella Enteritidis (SE) is a leading foodborne pathogen in the US, with many outbreaks traced back to eggs. Although SE can be vertically transmitted, once laid there are multiple routes by which the pathogen can contaminate eggs. Therefore, decontamination of eggs is critical to promote its food safety. One of the simplest on-farm approaches to reduce egg contamination is the prompt collection and refrigeration of eggs. Besides refrigeration, eggs are routinely washed in disinfectant containing water prior to shipping. Despite these strategies, there is a lack of decline in Salmonella outbreaks. Therefore, poultry producers are looking for effective strategies to promote egg safety. In this regard, probiotics are commonly used in poultry production for growth promotion and pre-harvest control of SE. In addition to probiotics, postbiotics (probiotic metabolites) also provide a novel antimicrobial alternative to the control of foodborne pathogens. However, to our knowledge, neither probiotics nor postbiotics have been studied for their ability to control SE on eggs. Hence, we propose the incorporation of probiotics and postbiotics in wash water to reduce SE contamination on eggs either as a conventional dip or spray application. We expect that results of the study will help identify different antimicrobial regimens and application modalities that can be tailored to specific production needs to promote the egg industry and public health. Overall, it is anticipated that these interventions could be used as part of a multi-hurdle on-farm approach to provide sustained antimicrobial activity against foodborne pathogens on eggs.
The overall objective of the proposed study is to determine the antimicrobial efficacy of select probiotics and postbiotics as an egg wash for controlling SE on eggs. As an alternative to washing eggs using the conventional dip method, our study will also investigate the spray application of probiotics/postbiotics to improve egg safety. Egg washing is a method to reduce pathogen contamination on eggs. However, specific requirements such as warm wash water and warm sanitizing rinse are required according to the USDA egg washing standards. In this regard, probiotic and postbiotic spray application avoids increases in egg temperature that can happen when using warm washes. This treatment would also be a more economical processing method since it would eliminate the cost to heat the wash water or decontaminate the wash water prior to its disposal. Further, probiotic/postbiotic spray has the potential to be an environmentally friendly option and can help reduce water usage on the farm19. Specifically, in this study, we will use an electrostatic spray applicator to ensure uniform product deposition on the egg surface20. Further, to ensure sustained probiotic viability in significant numbers, probiotic wash solutions will be prepared incorporating protectants namely gum acacia and inulin.
Our specific objectives for the proposed study are as follows:
- To determine the efficacy of incorporating probiotics in wash water to control SE on shell eggs when applied as an electrostatic spray and conventional dip solution.
- To determine the efficacy of incorporating postbiotics in wash water to control SE on shell eggs when applied as an electrostatic spray and conventional dip solution.
- To determine the effect of the different treatments and treatment modalities on egg quality and cuticle coverage.
The overall goal of this study is to develop a probiotic-based, natural and user-friendly strategy to reduce Salmonella contamination and improve egg safety.
Salmonella is a leading food pathogen in the US, with many outbreaks in humans attributed to eggs. In effect, source attribution studies have shown that 53% of all food-borne salmonellosis between 1985 and 2002 were attributed to contaminated eggs1. Further, approximately 35% of these Salmonella outbreaks involved the Northeast2. With specific reference to shell eggs, the outbreak in 2018, led to a recall of 206 million eggs3. Such incidents lead to huge losses for the egg industry. Thus, the poultry industry is under increasing consumer and regulatory pressure to guarantee food safety4. Among the different serovars of Salmonella enterica, Salmonella enterica serovar Enteritidis (SE) is the most frequently isolated Salmonella from layer flocks and is most associated with outbreaks traced back to the consumption of shell eggs4,5.
Salmonella contamination of eggs can occur via vertical and/or horizontal transmission. In the former instance SE colonization of the hen’s reproductive tract results in egg contamination during its formation5. Additionally, once laid, eggs can get contaminated by SE from various environmental sources. Therefore, decontamination of eggs is critical to promote its food safety6. Hence several physical and chemical methods were evaluated to help control SE on eggs. One of the commonly employed interventions involves egg washing in water containing chemical disinfectants7. However, chemicals tend to damage the cuticle layer of the eggshell which acts a barrier for pathogens thereby making the egg susceptible to subsequent SE contamination8. In addition, current antimicrobial interventions do not exert sustained protection against contamination that can occur later such as during egg handling, shipping and storage1. Moreover, as reported by the CDC, there is a lack of decline in Salmonella outbreaks associated with eggs3,4. Thus, there is a need for effective, user friendly and environmentally safe antimicrobial strategies that can be applied by the egg industry.
In this regard, probiotics including lactic acid bacteria (LAB) are ideal candidates to control SE since they occupy the same ecological niche, survive and thrive under similar environmental conditions and can competitively inhibit the pathogen9. Additionally, use of LAB would provide a protective buffer against contamination during the handling, processing, storage and distribution of eggs. In effect, probiotics are widely used in poultry industry to improve performance and reduce SE in birds10-12. Furthermore, the fermentates produced by probiotics (postbiotics) have been shown to exert antimicrobial properties13,14. However, little is known about the ability of probiotics and postbiotics to inhibit pathogens on shell eggs. Therefore, we propose the application of select LAB strains and postbiotics as an electrospray or dip wash to control SE on shell eggs. Overall, we expect that incorporation of probiotics and postbiotics in wash water will help provide a practical and user-friendly solution to the control of SE on eggs.
2023 Project Update
Salmonella is a leading food pathogen in the US, with many outbreaks in humans attributed to eggs. For instance, the 2018 outbreak related to shell eggs led to a recall of 206 million eggs. Such occurrences cause the egg business to suffer significant losses. As a result, the poultry industry is under increasing consumer and regulatory pressure to guarantee food safety. Eggs can get contaminated with salmonella through horizontal or vertical transfer. Eggs can also become contaminated with Salmonella Enteritidis (SE) from a variety of environmental sources. Decontamination of eggs is, therefore, essential to promoting food safety. Therefore, we propose the application of selected probiotic strains and postbiotics as an electrospray or dip wash to control SE on shell eggs.
Research
Power analysis: A power analysis was conducted to determine the number of samples required to detect a statistically significant difference in pathogen populations between treatments and controls using the PROC-POWER procedure of SAS version 9.3.
Salmonella cultures: A four-strain mix of SE consisting of SE-12 (chicken liver isolates), SE-21 (chicken intestine isolate), SE-28 (chicken ovary isolate) and SE-31 (chicken gut isolate) will be used in the study. To distinguish these strains from any naturally occurring strains, they will be pre-induced for resistance to 50µg/ml of Nalidixic acid (NA) using standard protocols. Each strain will be cultured in 10 mL of tryptic soy broth (TSB, Difco) with 50 µg/mL NA (TSB-NA) for 24 h at 37°C.The SE cultures will be washed twice (centrifugation at 3,600 X g for 15 mins) in sterile phosphate buffered saline (PBS, pH 7). The pellet will be finally resuspended in PBS. To make the four-strain cocktail (inoculum; 6 log CFU/egg), equal amounts of washed bacterial culture from each strain will be combined. The bacterial population in the cocktail will be determined by plating appropriate dilutions on Xylose Lysine Dextrose agar with NA (XLD-NA) followed by incubation at 37°C for 24 - 48 h21.
Probiotic cultures: Lactobacillus rhamnosus NRRL-B-442 (LR; USDA ARS), L. paracasei DUP 13076 (LP; PI’s collection) and Hafnia alvei (HA; Lallemand) were selected based on our previous studies demonstrating their anti-Salmonella properties21. and preliminary experiments (See supplementary information). Briefly, each strain will be cultured separately in de Mann Rogosa Sharpe broth (MRS, Difco), incubated at 37°C for 24 h, and washed twice (centrifugation at 3,600 X g for 15 mins) in sterile phosphate buffered saline (PBS, pH 7) to obtain the bacterial pellet.
Objective 1: To determine the efficacy of incorporating probiotics in wash water to control SE on shell eggs when applied as an electrostatic spray and conventional dip solution.
Probiotic wash treatments: To prepare probiotic wash treatments, washed probiotic cultures will be resuspended in sterile potable water containing either 5% v/v gum acacia (G) or inulin (I). Although our preliminary data demonstrated significant antimicrobial effect for the probiotic cultures, we also observed decrease in their survival on the eggshell. Hence, to improve their viability we will include protectants in the wash treatments. These ingredients have been previously reported to promote probiotic viability and resistance to environmental stress including low temperature18,22. Briefly, the probiotic pellets will be reconstituted in sterile potable water (~ 9 log CFU/ml) containing G or I for application as a spray or dip wash.
Egg inoculation: Freshly laid, unwashed eggs will be procured from the UConn commercial poultry farm. The eggs will be spot inoculated with 200 µl of SE cocktail (~ 7 log CFU/egg)23. The inoculum will be spotted evenly on the egg surface and air dried for 2 h at 23°C in a biosafety cabinet. Ten eggs will be randomly sampled after air drying to determine the efficiency of inoculation.
Wash treatments: The different treatments will consist of control (just water, no probiotic, no protectant), water with 5% v/v gum acacia (WG), water with 5% v/v inulin (WI), chlorine (wash water with 200 ppm chlorine), LR with G (GLR), LR with I (ILR), LP with G (GLP), LP with I (ILP), HA with G (GHA) and HA with I (IHA). The use of chlorine in the study will help evaluate our treatments against a commonly employed disinfectant in the egg industry7. Wash water temperature will be held at 32°C, the minimum water temperature requirement for egg washing as recommended by USDA24,25. The egg wash experiments will be conducted in consultation with Co-PD Dr. Upadhyaya to ensure accurate replication of industry practices.
Electrostatic spray application of wash treatments: Different probiotic wash treatments will be prepared as described above. Inoculated eggs will be subjected to spraying using an electrostatic sprayer in a biosafety cabinet20,26. Briefly, eggs will be sprayed with the treatment solution (200 µl/egg) and sampled for microbiological analysis and/or stored at 4°C. Salmonella and probiotic populations will be enumerated on day 0 (immediately after spray treatment) and days 3, 7, 14 and 21 of refrigerated storage. Three eggs from each treatment will be sampled at each timepoint (15 eggs/treatment/trial) and the study will be repeated three times. A total of 450 eggs will be used for the whole trial.
Dipping: Inoculated eggs will be washed in the different wash treatments as previously described27. Briefly, each inoculated egg will be placed in a stomacher bag containing 50 ml of wash treatment and placed on a shaker water bath held at 32°C for 3 min27. Following the wash, eggs will either be sampled immediately or placed in an egg carton and stored at 4°C for 21 days. Salmonella and probiotic populations will be enumerated on day 0 (immediately after wash) and days 3, 7, 14 and 21 of storage. Three eggs from each treatment will be sampled at each timepoint (15 eggs/treatment/trial) and the study will be repeated three times. A total of 450 eggs will be used for the whole trial.
Microbiological analysis: Following treatment, each egg will be transferred to a stomacher bag containing 30 mL of neutralizing broth and gently rubbed by hand for 1 min. SE population on eggs will be determined by plating the samples on XLD+NA plates. The plates will be incubated at 37°C for 48 h before enumeration. For SE enrichment, 10 ml of neutralizing broth will be added to 90 ml of selenite cystine broth and enriched at 37°C for 48 h. The enriched sample will then be streaked on XLD agar and incubated at 37°C for 48 h. Representative SE colonies will be confirmed using the Salmonella agglutination assay. Wash water from the dipping experiments will be processed as previously described to enumerate SE and probiotic populations27. For detection of SE in egg contents the eggshell will be sanitized with 75% ethanol, dried and cracked open28,29. The egg contents (yolk and albumen) and the shell will be placed into separate sterile stomacher bags containing 10 ml of selenite cysteine broth. The samples will then be homogenized for 1min in a stomacher and incubated at 37°C for 48 h. The bacterial colonies will be detected as described above. Similarly, samples will also be plated on MRS agar to enumerate probiotic populations. The probiotic colonies will be confirmed using the API 50 CH test kit and API CHL medium30.
Objective 2: To determine the efficacy of incorporating postbiotics in wash water to control SE on shell eggs when applied as an electrostatic spray and conventional dip solution.
Preparation of postbiotics and treatment solutions: Each probiotic strain will be cultured as described in objective 1. The overnight cultures will be centrifuged at 3,600 X g for 15 mins. The supernatant will then be filtered sterilized using a 0.22µm Millipore filter to obtain the postbiotic31. To prepare the wash treatments, gum acacia or inulin will be added to the postbiotic at 5% v/v. With objective 2, the wash treatments will include control, chlorine, WG, WI, GLR, ILR, GLP, ILP, GHA and IHA.
Electrostatic spray application of postbiotics: Briefly, inoculated eggs will be sprayed with 200 µl of wash water, chlorine, WG, WI or wash water containing 40% of postbiotic treatments31 (GLP, GLR, GHA, ILP, ILR or IHA) as described under objective 1. This level of incorporation (40%) is based on previous studies on the application of postbiotic wash to control Listeria on hot dogs and our previous work on attenuating Salmonella virulence21,32. Following spray application, eggs will be sampled for microbiological analysis and/or stored at 4°C. Salmonella populations will be enumerated on day 0 (start of experiment) and days 3, 7, 14 and 21 of storage. Three eggs from each treatment will be sampled at each timepoint (15 eggs/treatment/trial) and the study will be repeated three times. A total of 450 eggs will be used for the whole trial. Microbial analysis will be performed as described above.
Dipping: Inoculated eggs will be washed in the different treatment solutions as previously described. Briefly, each egg will be washed in 50 ml of the control or postbiotic dip solution31 and sampled to enumerate Salmonella and probiotic populations as in objective 1. Beyond initial sampling, washed eggs will be stored at 4°C for 21 days to evaluate sustained antimicrobial effect under egg storage conditions33,34. Three eggs from each treatment will be sampled at each timepoint (15 eggs/treatment/trial) and the study will be repeated three times. A total of 450 eggs will be used for the whole trial.
Objective 3: To determine the effect of the different treatments and treatment modalities on egg quality and cuticle coverage.
Egg quality determination: Based on the results of objective 1 and 2, the two most effective postbiotic and probiotic wash water treatments for spray and dip application will be identified. Uninoculated eggs will be subject to these selected treatments and compared against control and chlorine treated eggs. Three eggs from each treatment will be sampled at each timepoint (240 eggs/treatment/trial) and study will be repeated three times. Weight loss (%) of eggs during storage will be calculated using the formula [{initial egg weight (g) – egg weight after storage (g)}/initial egg weight (g)] X 100. Haugh unit will be calculated using the formula HU=100×log (H+7.57–1.7W0.37), where H is the albumen height (mm) and W is the egg weight (g)35,36. Shell thickness will be measured at the large end, equatorial region and small end. Briefly, an intact egg will be first weighed, and the yolk will be separated to determine yolk weight. The eggshell will be carefully rinsed with intact shell membrane, allowed to dry at room temperature for 2 days, final weight recorded, and the albumin weight derived by difference37.
Evaluation of cuticle coverage: For this assay, 1 cm2 eggshell pieces around the equator of the egg will be cut and mounted on a 9 mm diameter aluminum stub, sputter coated, and examined under a scanning electron microscope. Scoring of the cuticle will be done using the following criteria: cuticle score 1 = 90 to 100% cuticle cover, score 2 = 60 to 90% cuticle cover, score 3 = 20 to 60% cuticle cover and score 4 = 0 to 10% cuticle cover38. This assay will be done to evaluate if these treatments and application modalities can help extend the viability of the cuticle and thus maintain this protective barrier on eggs39.
Statistical analysis: Three samples for each treatment at each sampling time will be included in each experiment and three independent trials will be conducted. For all objectives, completely randomized design will be followed. Pooled data will be analyzed using PROC-MIXED procedure of SAS 9.3. Differences among means will be detected at p ≤ 0.05 using Fisher’s least significance difference test with appropriate corrections for multiple comparisons.
Expected outcomes: Successful completion of the study is expected to identify potential probiotic and postbiotic wash treatments for use as an antimicrobial hurdle to control SE on eggs. In this regard, initial trials have demonstrated >4 log reduction in SE populations on probiotic-treated eggs unlike the untreated eggs. We also expect to help demonstrate the use of electrospray as an alternative to dip washing. In summary, we expect that results of the study will help identify different antimicrobial regimens and application modalities that can be tailored to specific production needs thereby promoting egg safety. Overall, these interventions are expected to be used as part of a multi-hurdle approach to maintain antimicrobial activity against foodborne pathogens in eggs.
Pitfalls/Limitations: Although we do not expect markedly different results from our preliminary data, if supplementation of these cultures coated with protectants is not effective in reducing significant pathogen populations during storage, we will increase LP, LR and HA doses on eggs. Further, additional LAB strains in the PIs collection that have been previously characterized for their antimicrobial activity against SE will be evaluated for their potential application on eggs. In addition, if needed, postbiotic treatments will be tested at higher concentrations to improve their antimicrobial efficacy.
2023 Update:
Bacterial cultures: A four-strain mix of Salmonella Enteritidis (SE) consisting of SE-12, SE-21, SE-28, and SE-31 was pre-induced for resistance to 50µg/ml of Nalidixic acid (NA) using standard protocols. Each strain was cultured in 10 mL of tryptic soy broth (TSB, Difco) with 50 µg/mL NA (TSB-NA) for 24 h at 37°C. To make the four-strain cocktail (inoculum; 8 log CFU/egg), equal amounts of washed bacterial culture from each strain were combined. The bacterial population in the cocktail was determined by plating appropriate dilutions on Xylose Lysine Dextrose agar with NA (XLD-NA) followed by incubation at 37°C for 24h.
Preparation of postbiotics and treatment solutions: Lactobacillus rhamnosus NRRL-B-442 (LR; USDA ARS), L. paracasei DUP 13076 (LP; PI’s collection) were cultured separately in de Mann Rogosa Sharpe broth (MRS, Difco), incubated at 37°C for 24h. The overnight cultures were centrifuged at 4,500 X g for 15 mins. The supernatant was filtered sterilized using a 0.22µm Millipore filter to obtain the postbiotics. The wash treatments will include control, chlorine, postbiotics LP, and LR.
Egg inoculation: Freshly laid eggs were procured from the UConn commercial poultry farm. The eggs were spot inoculated with 200 µl of SE cocktail (~ 8 log CFU/egg). The inoculum was placed evenly on the egg surface and air-dried for 2 h at 23°C in a biosafety cabinet. Ten eggs were randomly sampled after air drying to determine the efficiency of inoculation.
Dipping: Inoculated eggs were washed in the different treatment solutions. Each egg was washed in 30 ml of the control or postbiotic dip solution and sampled to enumerate. Salmonella was enumerated on day 0 (immediately after wash) and days 3, 7, 14, and 21 of storage. Three eggs from each treatment were sampled at each time point (15 eggs/treatment/trial), and the study was repeated three times. A total of 180 eggs were used for the whole trial.
Microbiological analysis: Following treatment, each egg was individually transferred to a sterile stomacher bag containing 10 ml of neutralizing buffer and will be hand-rubbed for 1 minute. Appropriate dilutions of the buffer were plated on XLD+NA to enumerate the surviving Salmonella populations on the outer shell surface. If colonies are not detected by direct plating, the sample was enriched in selenite cysteine broth for 48 h at 37°C, followed by streaking on XLD-NA plates. For the egg contents, the eggs that were washed in the neutralizing broth were disinfected with 70% ethanol for 30 seconds, dried, and cracked open aseptically, and the broken shell and egg contents (yolk + albumen) were collected into separate stomacher bags containing selenite cysteine broth with NA. The bags with the egg contents or shells will be homogenized for 1 min in a stomacher and then incubated at 37°C for 24 to 48 h to detect surviving SE. Following egg wash, the wash water will also be processed in the dipping experiments to enumerate surviving SE and probiotic populations.
2023 Update:
When eggs were dipped in the probiotic wash water, a reduction in SE on the outer shell surface was observed. On day 3, the control and chlorine-treated groups had ~6 log CFU/egg and 5 log CFU/egg of SE, while the postbiotic treatment groups were only enrichment positive (P<0.05). By day 7, the postbiotic-treated eggs (LP and LR) showed SE enrichment positive, unlike the control and chlorine groups, where the SE recovered ~ 4 log CFU/egg and ~3 log CFU/egg, respectively (P<0.05). On days 14 and 21, SE counts in the LP and LR groups were enrichment negative, while control and chlorine groups were still SE positive. These findings suggest that dipping eggs with postbiotic wash water effectively decreases SE contamination on the outer shell surface.
Salmonella Enteritidis counts were evaluated on the outer and inner shell surfaces and the internal contents (yolk and albumen). In the dip method, although below detection limits were noticed on the outer shell surface, a higher amount of SE was detected on the inner shell surface. However, postbiotic-treated groups effectively reduced SE in the inner shell surface (P<0.05). By day 7, SE counts were below detection limits but enrichment positive in the LP and LR groups, where there was still a recovery of ~4 log CFU/egg in control and chlorine(P<0.05). On day 21, the control and chlorine groups were still enrichment positive for SE, whereas the LP and LR groups were enriched negatively.
Regarding the internal contents, the control group consistently showed higher levels of SE than the probiotic treatment groups. Apart from this, analysis of wash water revealed that ~3.5 log CFU/mL (control treatments) of SE was recovered after egg treatment, but there was no SE recovery in the postbiotics group (P < 0.05).
Current/Future research plans by the next reporting period (July 2024)
Standardize the protocol for the electrostatic application of probiotics and postbiotics on SE-inoculated eggs. Plan to finish the experiments of electrostatic spray application and conventional dipping of probiotics and postbiotics on SE inoculated eggs (Objective 1 & 2). Complete the data analysis of the trials. Also, plan to finish the experiments on egg quality analysis and cuticle evaluation experiments (Objective 3).
Education & Outreach Activities and Participation Summary
Participation Summary:
The graduate student will work with Dr. Indu Upadhyaya in conducting the outreach component of the study. The target audience for the outreach objective will be poultry producers, researchers, scientists, and poultry extension professionals. We will educate the poultry producers on the use of probiotics and postbiotics to control SE on eggs. Further, we will also discuss the advantage of spray vs dip washing of eggs. In addition, we will communicate the results of our study to poultry producers through extension conferences and meetings such as the Connecticut Poultry Association, Massachusetts Poultry Growers Association, Pennsylvania Poultry Sales and Service Conference and Northeast Conference on Avian Diseases. We will include our results on the University of Connecticut Poultry Pages website, Naturally@UConn website and the Connecticut Poultry Association website to provide more access to producers. Further, we will present our results at the Microbiology Symposium at the University of Connecticut, as well as in meetings from the Poultry Science Association. The data obtained from the project will also be published in peer-reviewed journals.
2023 Update
The outreach activities include sharing findings at the Pennsylvania Poultry Sales and Service Conference and presenting the findings at the Connecticut Poultry Association meeting.