Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Our projecttargeted a diverse audience, including scientific communities, industry professionals, governmental agencies, and public health stakeholders. This multi-faceted approach aimed to promote awareness and encourage collaboration on food safety and antimicrobial resistance, particularly focusing on Campylobacter reduction and the development of antibiotic alternatives. - Our primary audience consisted of researchers, academics, and students specializing in microbiology, food safety, and antimicrobial resistance. Through conference presentations, peer-reviewed publications, and collaboration in multi-institutional research projects, we engaged with scientists working on similar issues. For example, the presentation of our findings at international microbiology conferences (e.g., ASM Microbe) attracted attention from global experts. Additionally, the research training provided to graduate and postdoctoral researchers fostered an understanding of innovative methodologies in pathogen control and bioactive molecule characterization. - Our project also focused on professionals within the poultry production sector, which is directly impacted by Campylobacter outbreaks. Industry stakeholders were reached through workshops, seminars, and direct collaboration with poultry farms and veterinary services. These events emphasized the significance of bioactive molecules and alternative antimicrobial strategies to reduce reliance on traditional antibiotics, ensuring long-term sustainability in poultry production. - We alsoengage public health officials, given their role in implementing regulations and overseeing food safety standards. This audience was reached through policy briefings, webinars, and discussions with representatives from agencies such as the FDA and USDA. These interactions helped raise awareness about the growing threat of antibiotic-resistant Campylobacter and the need for novel solutions. We also submitted technical reports and recommendations based on our research findings to inform future policy development. ? Changes/Problems:In this reporting period, we encountered some challenges in our approach, impacting the timeline and requiring modifications to specific areas of the project: A significant delay occurred due to supply chain disruptions, which affected the procurement of critical reagents and materials. This delay has impacted the timely execution of experimental phases, particularly those involving the isolation and characterization of bioactive molecules. As a result, adjustments to the research timeline were made, and alternate suppliers were identified. We alsoencountered delays in obtaining the proteomics and metabolic results. These analyses are crucial for identifying the bioactive molecule(s) responsible for the antimicrobial activity observed in EcN supernatants. The delay stemmed from technical issues in processing the samples and the extended time required for data acquisition and analysis. As a result, the timeline for completing this aspect of the project has been extended. We are working closely with our collaborators to expedite the data analysis and expect to resume normal progress in the coming months. During the bioactive molecule isolation process, achieving high-purity fractions proved more challenging than anticipated. Preliminary proteomic analysis revealed a complex mixture of peptides and small molecules requiring additional steps for purification. To overcome this challenge, we have employed additional fractionation techniques, which, although effective, have extended the timeframe for this phase of the project. We expect this will lead to a better understanding of the bioactive compounds. An unexpected outcome was observed during the biofilm assays. While we anticipated moderate biofilm inhibition, EcN supernatant showed a higher-than-expected inhibition rate (up to 85%) againstCampylobacter jejunibiofilms. This led to an unplanned but necessary series of follow-up experiments to verify these results and better understand the mechanism behind this potent anti-biofilm activity. This deviation has not negatively impacted the project. While we encountered challenges that affected timelines and expenditures, adjustments have been made to keep the project on track toward achieving its objectives. The significant outcomes from the biofilm assaysposition us well for successful completion, though with an extended timeline to accommodate the necessary changes. We anticipate no further delays and expect to meet our end-of-project goals within the revised schedule. What opportunities for training and professional development has the project provided?The project has provided several opportunities for training and professional development: Team members have gained hands-on experience in advanced microbiological and molecular biology techniques, such as isolating and characterizing bioactive molecules, and performing in vitro assays. This technical expertise enhances their lab skills and prepares them for future research. The project involves analyzing large datasets to identify bioactive peptides and small molecules. Lab members have been trained in bioinformatics tools and statistical software, equipping them with the skills to analyze proteomic, and metabolic data effectively. Team members have had the chance to engage in cross-disciplinary discussions, fostering teamwork and the exchange of ideas. Team members have had opportunities to present their findings at internal lab meetings, workshops, and conferences, enhancing their scientific communication skills. Writing reports, andmanuscripts also contributes to professional development. Team members have been trained in ethical guidelines, biosafety protocols, and regulatory compliance, which are essential in conducting experiments. How have the results been disseminated to communities of interest?Team members have presented preliminary findings at national microbiology, food safety, and antimicrobial resistance conferences. These presentations provide opportunities to engage with experts in the field, share insights, and receive feedback. What do you plan to do during the next reporting period to accomplish the goals?1. Continue accomplishingthe goals of our project. 2. Data analysis and reporting. 3. Continue collaborative research efforts. 4. Continue providing team members with training in the required laboratory and analytical techniques. 5. Encourage team members to present their findings in internal and external scientific forums. 6. Apply for a standard grant from USDA/NIFA in the 2025 cycle.

Impacts
What was accomplished under these goals? To confirm that the antimicrobial activity from probiotic NissleE. coli1917 (EcN) is due to thesecretion of abioactive molecule(s) in the cell-free supernatant (CFS), we investigated Effect of the EcN supernatant on the growth ofC. jejuni: We used anagar-well diffusion assay to evaluate the effect of EcN CFS on the growth ofC. jejuni.Briefly, the overnight culture ofC. jejuniwas adjusted to an OD600:0.05 (1×107CFU/mL) in Bacto™ Mueller Hinton (MH) broth. One hundred microliters of the dilutedC. jejuniwere spread evenly on a CHROMagar. The CFS was prepared by centrifugation of EcN whole cultures incubated for 12h, at10,000 × g for 5 minutes. The supernatants were collected and filtered using a0.22 µm filter. One hundred microliters of the CFS were added in wells made on the CHROMagar. We found that after 12hof incubation, the CFS of EcN exhibited a growth inhibition zone of 20 ± 0.7 mm againstC. jejuni. We also used a co-culture assay to test the effect of the CFS onC. jejunigrowth in liquid media. Briefly, 100 µL of overnight-grownC. jejuni(OD600: 0.05; 1×10? CFU/mL in MH broth) was co-cultured with 100 µL of CFS from 12-hour incubated EcN cultures. The mixture was incubated at 42°C under microaerobic conditions for 48 hours. The viable count ofC. jejuni(CFU/mL) was determined at 12, 24, and 48 hours. We found that the EcN CFS significantly inhibited (p<0.05) the growth ofC. jejuniafter 24 h of incubation (~2.5 log reduction) and completely cleared the bacteria after 48 hours. Effect of the EcN supernatant on the biofilm formation and the preformed biofilm ofC. jejuni: To evaluate the effect of EcN on biofilm formation ofC. jejuni, overnight-grownC. jejuni(OD600: 0.05; 1×10? CFU/mL in MH broth) was incubated in the presence of variable concentrations of the EcN CFSs (1:1) making the final volume of 200µL in a96 well plate at 42°C for 48 h under microaerophilic conditions. After incubation, the planktonicC. jejuniwas removed, and the biofilms were gently washed twice with distilled water. The dry biofilms were then stained with 0.1% crystal violet, incubated at room temperature for 20 minutes, washed twice distilled water and dried at room temperature for 20 minutes. The stained biofilms were resuspended in 30% acetic acid, and the biofilm was quantified at 550nm. For effect on pre-formed biofilms ofC. jejuni, 100 µL of the overnight grownC. jejuniwas grown in 96-well plates at 42°C for 48 hours under microaerophilic conditions. After 48h, the biofilms were washed treated with variable concentrations of the EcN CFSs. The plate was further incubated at 42°C for 48h. The biofilms were quantified using crystal violet. We found that the EcN supernatant inhibited the biofilm formation up to 80% and the inhibition of pre-formed biofilm by more than 85% when used at the ratio of 1:1. Effect of the EcN supernatant on the colonization ofC. jejuni: To evaluate the effect of EcN CFS on theadhesion ofC. jejunion the cells, 100 µL of the EcN CFS was added into the wells containing the monolayer cells and incubated for 4h followed by the infection of the cells with 107CFU/mLC. jejunifor 2 h. Afterincubation, the cells were washed three times with DPBS, and the adheredC. jejuniwere enumerated on CHROMagar plates after lysis of the cells with 0.1% Triton X-100. Forinvasion, the cells were treated with gentamycin (150 mg/ml) and incubated for 2 hours to kill the extracellularC. jejuni.After the incubation, the invadedC. jejuniwas enumerated as described above. To assess theability of EcN CFS to kill the survivedC. jejuniin the HT-29 cells, the cells were then treated with gentamycin (10 mg/mL) for 24 hours, and bacterial counts were determined as described above. We found that the pre-treatment of the HT-29 cells with EcN CFS significantly reduced (p<0.05) the adhesion (~1.5 log reduction) and invasion (~2.5 log reduction). The EcN CFS completely cleared bacteria survived inside the cells after 24h. Effect of the EcN supernatant on the expression ofC. jejunivirulence-associated genes: The virulence-associated genes ofC. jejuniinclude biofilm formation, invasion, motility, and cytotoxin production. Briefly, 100 µL of overnight-grownC. jejuni(1×10? CFU/mL) was co-cultured with 100 µL EcN CFS for 12h. The bacteria were then pelleted, and the total RNA was extracted with the RNeasy® Mini Kit. The cDNA was synthesized using RT2First Strand Kit. The qPCR was performed using PowerTrack™ SYBR Green Master Mix for qPCR following the instructions of the manufacturer. We found that the supernatant significantly downregulated the expression of genes associated with virulence factors ofC. jejuni. We observed the downregulation of genes associated with motility likeflhA(~1-fold),flhB(~2-folds), cytotoxin production such ascdtA(~1.5-folds),cdtB(~9-folds),cdtC(~1-fold), colonization such ascadF(~1.5-folds),ciaB(~2-folds); quorum sensing such asluxS(~1.5-folds) inC. jejuni, and protein synthesis includingrrS(~1-fold),slyD(~1-fold), andilvC(~1.5-folds). Detection of the bioactive molecules in the EcN supernatant To identify the EcN bioactive molecules, EcN was subcultured for 3, 12, and 24h in LB-broth. The EcN cultures at each time point were centrifuged at 10,000 × g for 5 minutes and the supernatants were filtered using a0.22 µm filter. We used the agar-well diffusion assay described above to test how well the EcN CFSs stopped the growth of C. jejuni at each time point.We found that the CFS that was cultured for 12h demonstrated the highest zone of inhibition (21±0.3 mm), followed by 3h (20±0.3 mm), and 24h (18±0.2 mm).To identify the size of EcN bioactive molecules, EcN-CFS wasgrown in LB broth for 12h and was filtered using MicrosepTMadvance centrifugal device of filter sizes 1K, 3K, 10K, and 30K. An agar well diffusion assay was performed to assess the ability of both the concentrated residue and filtration from all four filters to inhibit the growth ofC. jejuni.We found that the concentrated residue ≥30 KD demonstrated the highest zone of inhibition (20±0.2 mm). This experiment was repeated using minimal media (M63). The supernatant active part was used to perform proteomic and metabolomics. Samples are still under processing. We identified some potential proteins and currently working on their purification to confirm the target. Extraction of Natural product compounds To identify and isolate the bioactive active molecules synthesized by EcN, the overnight grown EcN was sub-cultured in the ratio of 1: 500 in 5 liters of LB broth for 12h. The EcN culture was then centrifuged at 500 rpm for 20 minutes. The pellets were washed with 500 mL of distilled water. For extracting the bioactive compounds from the supernatants, XAD-16 polymeric resin (4%) was added to the supernatant and incubated overnight with shaking. The resin was then filtered and washed with 3L of distilled water and subsequently extracted using 2 liters of methanol. The brown oily organicextracts were then evaporatedin avacuoyielding whitish-brown extracts. Similarly, the cell biomass/ pellets were extracted using methanol (800mL) and evaporated. Both the supernatants and biomass extracts were then analyzed with HPLC to identify and compare the metabolites. Also, the supernatant extracts were put through an HP-20 resin column and eluted with methanol to get 16 extract fractions and one fraction from the cell biomass. On evaluating all the 16 fractions against the growth ofC. jejuni,we identified that 14 fragments (F1-F9, and F11-F15) demonstrated different levels of growth inhibition,with F7 demonstrating the highest level (91.6%). The cellular biomass extract demonstrated an inhibition of 85.87%. Fraction F7 is currently being fractionated for further analysis to identify and purify the bioactive molecules responsible for anti-Campylobacteractivity.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Bibek Lamichhane and Yosra A. Helmy (2024). Evaluate the efficacy of E. coli Nissle 1917 (EcN) supernatants on Campylobacter jejuni infections in vitro. 2024 CCTS Spring Conference, 9th April 2024, University of Kentucky, Lexington, KY, USA. Bibek Lamichhane and Yosra A. Helmy (2024). In vitro evaluation of probiotic E. coli Nissle 1917 supernatants in inhibiting Campylobacter jejuni growth and virulence. ASM KY-TN Spring 2024 Conference, April 19-20, 2024, University of Kentucky, Lexington, KY, USA. The 3rd place poster presentation competition award. Bibek Lamichhane, Heather True, Brianna Doratt, Ilhem Messaoudi, Yosra A. Helmy (2024). Evaluate the efficacy of novel probiotic strains on Campylobacter infection in vitro. The Conference of Research Workers in Animal Diseases (CRWAD), Jan. 20 -23, 2024. Chicago, USA.