Progress 07/01/21 to 06/26/25

Outputs
Target Audience:This project primarily served veterinarians, veterinary microbiologists, and animal health researchers focused on polymicrobial diseases caused by Gram negative anaerobic bacteria. These infections-including metritis, diphtheria, liver abscesses, and footrot-are prevalent and clinically significant in livestock production systems. The project also supported stakeholders in the dairy industry, including dairy producers and herd health managers, by generating foundational knowledge to inform the development of alternatives to antibiotics. By identifying bacterial targets involved in growth and virulence, the project contributed to the scientific basis for designing effective non-antibiotic therapeutic strategies. These outcomes address growing concerns about antimicrobial resistance and align with the goals of sustainable livestock production. Veterinary students enrolled in the Bacteriology and Mycology course (DVM-613) at Long Island University College of Veterinary Medicine also represented a key audience. Project findings were incorporated into the metritis module, allowing students to engage with current research and deepen their understanding of disease mechanisms and alternative treatment approaches. In addition, the general public and consumers concerned about antibiotic use in food-producing animals may benefit indirectly through improved animal health, reduced reliance on antibiotics, and enhanced food safety outcomes resulting from this research. Changes/Problems:All planned experimental activities have been successfully completed, although the project experienced an initial delay due to challenges in hiring qualified personnel. This delay affected the early stages of the project timeline but did not alter the overall objectives. No major technical issues were encountered during the reporting period. Current efforts are focused on post-experimental analyses of GC-MS and dual RNA-Seq datasets, manuscript preparation, and dissemination of findings through presentations at CRWAD 2026 and ASM Microbe 2026. The PI will acknowledge USDA NIFA support in all resulting publications and presentations. What opportunities for training and professional development has the project provided?Training Activities: Anaerobic Culture System Training: The project supported the installation of an AS-150 anaerobic chamber (Anaerobe Systems) in Pell 116 and provided hands-on training in the isolation and cultivation of obligate anaerobic bacteria. 2-D and 3-D BEND Cell Culture Training: Veterinary and master's students received practical training in 2-D BEND cell culture techniques. Postdoctoral researchers led the development of 3-D BEND cell culture models, gaining experience in model construction, polymicrobial infection protocols, and transcriptomic sample preparation. GC-MS Training: Postdoctoral researchers were trained in the use of gas chromatography-mass spectrometry (GC-MS) in Pell 116. Dual RNA-Seq Training: Postdoctoral researchers received advanced training in dual RNA sequencing to study host-pathogen interactions, including experimental design, RNA extraction, and bioinformatic analysis of gene expression in both host and microbial transcripts. Microscopy Training: Students and postdoctoral researchers were trained in the use of confocal laser scanning microscopy (CLSM) combined with fluorescence in situ hybridization (FISH) to visualize biofilm architecture, microbial spatial distribution, and host-microbe interactions. Professional Development Activities: Conference Participation: Students and researchers participated in national and international conferences, including ASM Microbe 2024 (Atlanta), CRWAD 2025 (Chicago), and MedVet Pathogens 2025 (Italy). They delivered oral and poster presentations, shared their findings with the scientific community, and received valuable feedback to support their academic development. Mentorship Experience: Postdoctoral researchers served as mentors for students participating in the College of Veterinary Medicine's summer research program, providing guidance in laboratory techniques and data analysis. Manuscript Writing: Postdoctoral researchers gained experience in scientific writing through participation in manuscript drafting and the journal submission process. Online Workshop: Researchers completed a GC-MS data analysis workshop offered by the UC Davis West Coast Metabolomics Center, enhancing their skills in experimental design and metabolomic data interpretation. How have the results been disseminated to communities of interest?Project results have been actively disseminated through conference presentations, manuscript submissions, and data sharing platforms. Findings were presented at ASM Microbe 2024 (June 13-17, Atlanta, GA) in a poster session titled "The polymicrobial infection of anaerobic bacteria in uterine inflammation and disease." At CRWAD 2025 (January 18-21, Chicago, IL), two oral presentations were delivered: (1) "3D bovine endometrium model for studying host-pathogen interactions associated with metritis" and (2) "Interaction of Fusobacterium necrophorum, Bacteroides pyogenes, and Porphyromonas levii in bovine metritis." In addition, a talk titled "Polymicrobial interactions in the development of bovine metritis" was presented at MedVet Pathogens 2025 (May 26-29, Monash Prato Centre, Italy). A manuscript entitled "Synergistic interactions of Fusobacterium necrophorum, Bacteroides pyogenes, and Porphyromonas levii in driving dysbiosis-associated uterine disease" is currently under review in the journal Microbiome. Two additional manuscripts are in preparation: one focused on bacterial metabolite profiling using GC-MS, and another on gene expression changes in 3-D BEND cells during infection with uterine pathogens. Upon publication, manuscripts will be deposited in PubAg, and associated raw sequencing datasets will be submitted to the NCBI Sequence Read Archive (SRA) to ensure compliance with USDA public access policies. In terms of data sharing, biofilm metagenomic data have been deposited in the NCBI database (accession number: PRJNA1253680), and RNA-Seq datasets will be submitted to the NCBI SRA upon publication of the associated manuscripts. To further promote transparency and reproducibility, key resources developed through the project such as protocols for 3-D culture and representative SEM images have been made publicly accessible on the Jeon Lab website: https://sites.google.com/d/1M9Th2l7NWdjQ8X8AFD_wBAS_ZKjvThaI/p/1DqDtHy-7ctwfrgdATY3ijls04_inST5W/edit?pli=1 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The primary goal of Aim 1 was to determine whether these three opportunistic uterine pathogens interact synergistically to promote bacterial growth and pathogenicity in bovine endometrial epithelial (BEND) cells. To address this, we characterized the planktonic growth and biofilm formation of each species individually and in co-culture. We demonstrated coaggregation among the three species, with F. necrophorum playing a key role in facilitating both bacterial coaggregation and host cell adhesion. P. levii was identified as a major contributor to multispecies biofilm development, producing abundant extracellular matrix and proteases that likely enhance structural integrity and virulence. In contrast, B. pyogenes appeared to modulate overall virulence by attenuating the cytotoxic effects of F. necrophorum and P. levii. Co-culture of all three species significantly increased bacterial attachment and host cell death in BEND cells, indicating a synergistic effect on host-pathogen interactions. To further investigate these interactions, we quantified the relative abundance of each species within the multispecies biofilm using 16S rRNA metagenomic sequencing and visualized the biofilm structure via confocal laser scanning microscopy. Collectively, our findings demonstrate that interactions among these three uterine pathogens are critical for their survival, colonization, and inflammatory potential. This study provides foundational knowledge for developing targeted strategies to disrupt polymicrobial biofilms and control uterine infections. For example, blocking F. necrophorum adhesion to host cells may prevent initial colonization; inhibiting P. levii biofilm matrix production could reduce bacterial persistence and resistance; and leveraging the modulatory effects of B. pyogenes may help mitigate excessive inflammation triggered by more virulent species. The primary goal of Aim 2 was to determine how the metabolic traits and secreted products of three uterine pathogens contribute to their interactions and virulence during endometrial infection. We identified distinct carbon source preferences among the species. F. necrophorum utilized a broad range of carbon substrates, including sugars and organic acids, supporting its rapid growth in the nutrient-limited postpartum uterus. P. levii preferentially metabolized amino acids such as glutamine, while B. pyogenes displayed no strong substrate preference, suggesting minimal nutrient competition among the three pathogens. We also assessed pH tolerance, which revealed species-specific preferences. F. necrophorum maintained high metabolic activity across a wide pH range, with optimal growth between pH 6 and 7. P. levii favored a slightly alkaline environment (pH 7-8) and appeared to regulate environmental pH through the secretion of acidic or basic metabolites. In contrast, B. pyogenes showed optimal growth under acidic conditions, with diminished growth at higher pH levels. Endotoxin levels were elevated in monocultures of F. necrophorum and P. levii, as well as in their co-culture. Notably, the presence of B. pyogenes significantly reduced endotoxin production, indicating a possible modulatory effect. Protease activity was highest in P. levii monocultures and in co-culture conditions where P. levii was present, supporting its role as a key contributor to protease-mediated virulence and tissue degradation. To further investigate the metabolic profiles associated with bacterial interactions and host responses, we collected culture supernatants from mono- and co-cultures, both with and without BEND cells, for untargeted metabolomic analysis using GC-MS. Data processing is currently underway. Together, these results demonstrate that the three uterine pathogens exhibit distinct but complementary metabolic and virulence traits that allow them to coexist and contribute collectively to infection. Notably, several metabolites produced during bacterial interaction or in response to host cells may play key roles in promoting pathogenicity. Targeting these metabolites by neutralizing or removing those that enhance bacterial synergy or host cell damage could represent a novel approach to preventing the progression of metritis. These findings offer a promising foundation for the development of metabolite-based, non-antibiotic therapies for the treatment of polymicrobial uterine infections. The primary goal of Aim 3 was to develop a 3-D endometrial culture model to study host-pathogen interactions during polymicrobial infection and to evaluate host responses associated with metritis development. To achieve this, BEND cells were successfully embedded in a collagen matrix and maintained under both aerobic and anaerobic conditions, resulting in the establishment of a stable 3-D culture system. The cells retained appropriate morphology and viability throughout the culture period. The model was subsequently infected with F. necrophorum, B. pyogenes, and P. levii, individually and in combination, at a multiplicity of infection (MOI) of 10 for 24 hours. Among the pathogens, F. necrophorum exhibited the highest infection potential, forming visible aggregates with BEND cells, while B. pyogenes and P. levii showed weaker adherence. Notably, co-culture of all three species resulted in the greatest level of host cell disruption and bacterial colonization, suggesting a synergistic effect during polymicrobial infection. Confocal microscopy following Hoechst staining was used to visualize the structural organization of BEND cells and the three uterine bacterial species within the 3-D matrix. The imaging confirmed bacterial localization and direct interactions with host cells in the 3-D environment. To assess host and microbial gene expression, total RNA was extracted from 3-D BEND cultures infected with each pathogen alone and in combination. Dual RNA-Seq was performed to capture both host and bacterial transcriptomic profiles. RNA sequencing has been completed, and data analysis is currently underway. Together, these findings demonstrate the successful development of a 3-D bovine endometrial model. This model enables in-depth investigation of host responses to polymicrobial infections and serves as a valuable platform for advancing our understanding of the pathogenesis of uterine disease.

Publications

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The study aims to benefit several target audiences, including veterinarians and scientists concerned with animal diseases caused by polymicrobial infections of Gram-negative anaerobic bacteria. Additionally, it will benefit the dairy industry, dairy producers, and the general public who are concerned about the use of antibiotics in agriculture. The data obtained from this study will also serve as course material for the metritis section in the Bacteriology and Mycology course, making veterinary students an important target audience as well. Changes/Problems:Due to delays in hiring the postdoc, this project has been extended by one year. What opportunities for training and professional development has the project provided? The project has provided research opportunities to 2 veterinary students, 1 MS student, 1 lab technician, and 2 postdocs. This project has enabled us to equip our lab with an anaerobic culture system, facilitating our research on anaerobes. This project has successfully established the 3-D culture of BEND cells. Research findings are being integrated into the teaching materials for the Veterinary Bacteriology and Mycology course. How have the results been disseminated to communities of interest?The results were presented as a poster at the ASM 2024 conference in Atlanta from June 13-17, 2024. The title of the presentation is "The Polymicrobial Infection of Anaerobic Bacteria in Uterine Inflammation and Disease." The first manuscript for this project is currently being prepared and will be submitted to the ISME journal in July. What do you plan to do during the next reporting period to accomplish the goals?We will identify bacterial compounds involved in the interactions between bacteria and the infection of BEND cells. Samples are being prepared for metabolomic analysis. Furthermore, we will examine transcriptomes from both bacteria and 3-D BEND cells during single and multiple infections of the 3 bacteria using dual RNA-Seq, and assess cytokine/chemokine production associated with the innate immune response using the Bio-Plex® 200 system. Through these experiments, we aim to identify genes or proteins that modulate homeostasis and inflammation in the uterus of dairy cows.

Impacts
What was accomplished under these goals? Aim 1) Identify the interactions of F. necrophorum, B. pyogenes, and P. levii for enhancing growth, colonization, and pathogenicity. The major goal of this aim is to determine if these 3 opportunistic uterine pathogens have synergistic interactions in terms of bacterial growth and pathogenicity to bovine endometrial epithelial (BEND) cells. In Aim 1 there are 3 specific objectives: 1 - investigate the growth rates of F. necrophorum, B. pyogenes, and P. levii in culture media, both separately and together; 2 - determine if these pathogens form a biofilm when grown separately and together; 3 - examine attachment and cytotoxicity to BEND cells separately and together. We have successfully obtained results on planktonic growths, biofilm formation, coaggregation with other species, bacterial adherence to BEND cells, and cytotoxicity of live bacteria in BEND cells. Additionally, we observed the proportion of each bacterium in multi-species biofilm using 16S metagenomic sequencing and confirmed the structure of biofilms using confocal laser scanning microscopy. Our findings demonstrate that F. necrophorum is a key pathogen for coaggregation with other anaerobic bacteria and attachment to host cells. P. levii produces extracellular matrix and plays a major role in multispecies biofilm formation. B. pyogenes produces acidic metabolites, which may help create a favorable environment for the growth of other anaerobic bacteria. Co-culturing these 3 bacteria increases attachment and invasion of host cells, possibly leading to cell death. Taken together, the interaction of these 3 uterine bacteria is essential for their survival, growth, and ability to cause inflammation in the uterus. Aim 2) Characterize the in vitro metabolic profiles of F. necrophorum, B. pyogenes, and P. levii to determine what metabolic pathways are involved in their interactions and what bacterial metabolites are associated with endometrial infection. Our focus for this aim is to identify metabolic pathways involved in the interaction of F. necrophorum, B. pyogenes, and P. levii, as well as bacterial metabolites associated with endometrial infection. In Aim 2 we will: 1 - screen carbon sources that are used by F. necrophorum, B. pyogenes, and P. levii; 2 - identify bacterial products that are toxic to BEND; 3 - characterize extracellular and intracellular metabolites produced by F. necrophorum, B. pyogenes, and P. levii during infection of BEND using Gas Chromatography Mass Spectrometry (GC-MS). Objective 1 and 2 have been accomplished, and Objective 3 is currently in progress. We investigated carbon sources metabolized by uterine pathogens using a 96-well plate containing water (control) and 95 different chemical compounds. Notably, F. necrophorum showed a high utilization of β-hydroxyburytic acid, whereas P. levii preferred to use amino acids such as glutamine. There was no preference for B. pyogenes. This indicates that F. necrophorum, B. pyogenes, and P. levii have different preferences for carbon sources; therefore they do not seem to compete for nutrient in the uterus. Additionally, we measured metabolic activity of the 3 uterine pathogens at different pH levels ranging from pH3.5 to pH10. F. necrophorum showed metabolic resilience across a broad pH spectrum, with optimal conditions at pH 6 and pH 7. B. pyogenes exhibited metabolic activity within the pH range of 6 to 10, while P. levii showed activity within the pH range of 7 to 10, without displaying a distinct preference for a specific pH. Taken together, our findings demonstrate that F. necrophorum has a greater capability for adapting to environmental changes in nutrients and pH, which may explain its rapid growth in the uterus after calving. The 3 bacteria were cultured in CM broth individually and in combination for 24 hours, and culture medium containing bacterial metabolites was collected to measure endotoxin quantitation and protease activity. Endotoxin levels from the mono-culture of F. necrophorum and P. levii were significantly higher than those from the co-culture of the 3 bacteria, while protease activity levels were higher in the co-cultures than in the mono-cultures. These findings suggest that bacterial interaction increases protease activity, which may be cytotoxic to host cells. Furthermore, we have been collecting samples for metabolomic analysis to identify extracellular and intracellular metabolites released during bacterial interaction as well as during infection of BEND. The collected samples will be submitted to the University of California West Coast Metabolomics Center for untargeted Gas Chromatography-Time of Flight Mass Spectrometry analysis. Aim 3) Investigate the host responses to F. necrophorum, B. pyogenes, and P. levii using a 3-D culture model of the endometrium. The goal of this work is to generate a 3-D culture model of endometrium to study multiple infections by F. necrophorum, B. pyogenes, and P. levii and to elucidate a host-pathogen interaction related to homeostasis and metritis development. In Aim 3 we will: 1 - construct a 3-D culture model of the endometrium; 2 - identify transcriptomes for both bacteria and BEND using dual RNA-Seq; 3 - determine cytokine/chemokine production associated with innate immune response. Objective 1 has been accomplished, and Objective 2 and 3 are currently in progress. BEND cells were cultivated in a 1:1 mixture of Ham's F12 and Eagle's Minimal Essential medium with Earle's BSS (D-valine modification) with 1.5 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 0.034 g/L D-valine, 10% heat-inactivated fetal bovine serum, and 10% heat-inactivated horse serum. The culture media were supplemented with 1% of a combination of penicillin and streptomycin (100 units/ml) for the initial cultivation and maintenance of cells. For the 3-D culture, collagen (5 mg/ml) was used as a matrix for assembling BEBD cells. On day 1, BEND cells (25, 000 cells) in DMEM with 10% horse serum were mixed with collagen (21.8 µl of 5 mg/ml collagen), and a total of 125 µl was added to each well in a 24-well tissue culture plate. Then, encapsulated cells were incubated at 37°C and 5% CO2 for 15 min for the gelation of collagen before adding the cell-specific medium (complete medium). On day 2, the 3-D cultures were moved to the anaerobic chamber at 37°C. On day 3, 3-D culture was infected with the 3 bacteria individually and in combination at MOI 10 and 100. The infected cultures were incubated in the anaerobic chamber at 37°C for 24 and 48 h. For immunostaining, the cultures were fixed with a 2% (w/v) glutaraldehyde in PBS (1 X) solution. Both cellular and bacterial nuclei were visualized by Hoechst staining. Briefly, 100 µl of Hoechst (15 µg/ml in PBS) was added to each co-culture well and incubated for 1 h at 37°C. The wells were washed with PBS thrice for 1 min each. 0.5 ml of glycerol was added to the wells, and the culture plates were stored at 4°C until imaging. Imaging was conducted on a Zeiss LSM 900 confocal microscope. With increased bacterial cell number and infection time, the number of BEND cells decreased in the 3-D culture. With MOI 100 and 48 h infection time, the number of BEND cells decreased drastically compared to that of MOI 10 and 24 h infection time. F. necrophorum was highly infectious and formed aggregates in the 3-D culture compared to B. pyogenes and P.levii. Adhesion of bacteria to BEND cells was clearly observed especially with B. pyogenes and P.levii. Bacterial cells were clearly visible and distinguishable with Hoechst staining. As the 3-D cell culture of BEND cells is established, we plan to use it to study the interactions between the host and the 3 uterine pathogens.

Publications

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:The study aims to benefit several target audiences, including veterinarians and scientists concerned with animal diseases caused by polymicrobial infections of Gram-negative anaerobic bacteria. Additionally, it will benefit the dairy industry, dairy producers, and the general public who are concerned about the use of antibiotics in agriculture. The data obtained from this study will also serve as course material for the study of Gram-negative anaerobic bacteria in Bacteriology, making veterinary students an important target audience as well. Changes/Problems:Dr. Jeon and Dr. Cao have conducted experiments and put significant effort into this project due to the challenges of hiring a postdoctoral fellow. Starting in January 2024, a postdoctoral researcher with expertise in 3-D culture techniques will join the project. Additionally, we will provide research opportunities to undergraduate, graduate, and veterinary students who are interested in contributing to this project, whether on a short-term or long-term basis. What opportunities for training and professional development has the project provided?The project has provided training in laboratory research to undergraduate and veterinary students. How have the results been disseminated to communities of interest?At this time, the results have not been disseminated. The first manuscript for this project is currently being prepared and will be presented at the conference of Research Workers in Animal Diseases (CRWAD) in Chicago in 2024. What do you plan to do during the next reporting period to accomplish the goals?We plan to observe bacterial interactions in multi-species biofilms using Zeiss Scanning Electron Microscopy at Columbia University, as well as the confocal laser scanning microscopy at Long Island University. We will identify bacterial compounds involved in interactions between bacteria or infection of BEND cells using GC-MS. Currently, the GC-MS instrument is being operated at LIU. Furthermore, we will develop a 3-D culture of BEND cells to characterize the host immune response to F. necrophorum, B. pyogenes, and P. levii.

Impacts
What was accomplished under these goals? The primary impact of this work will be to modulate microbial interactions to control and treat metritis caused by polymicrobial infections of opportunistic uterine pathogens. Microbial interactions play an important role in disease development by increasing bacterial populations, releasing toxic components, and facilitating evasion of immune responses. Therefore, understanding microbial interactions will lead to the identification of potential drug targets for metritis that can either prevent direct contact between bacteria or eliminate key metabolites contributing to bacterial virulence. This research has potential benefits for the development of therapeutics targeting liver abscesses, foot rot, and calf diphtheria caused by Gram-negative anaerobic bacteria. Furthermore, this work has a positive impact on dairy farm sustainability and public health by reducing the use of antibiotics in food animals. Aim 1) Identify the interactions of F. necrophorum, B. pyogenes, and P. levii for enhancing growth, colonization, and pathogenicity. The major goal of this aim is to determine if these 3 opportunistic uterine pathogens have synergistic interactions in terms of bacterial growth and pathogenicity to bovine endometrial epithelial (BEND) cells. In Aim 1 there are 3 specific objectives: 1 - investigate the growth rates of F. necrophorum, B. pyogenes, and P. levii in culture media, separately and together; 2 - determine if these pathogens form a biofilm when grown separately and together; 3 - examine attachment and cytotoxicity to BEND cells separately and together. We have successfully obtained results regarding planktonic growths, biofilm formation, coaggregation with other species, and adherence to BEND cells. Cytotoxicity of live bacteria in BEND cells is currently in progress. Additionally, we plan to observe the structure of biofilms using confocal laser scanning microscopy and scanning electron microscope. Metritis is caused by polymicrobial infection of Gram-negative anaerobic uterine pathogens: F. necrophorum, B. pyogenes, and P. levii. These bacteria isolated from the uterus of diary cows with metritis exhibit distinct morphologies and characteristics. F. necrophorum is characterized as pleomorphic, filamentous bacteria, while B. pyogenes appears as pleomorphic, short rods, and P. levii presents as uniform short rods. In terms of planktonic growth, F. necrophorum showed relatively faster growth compared to other anaerobic bacteria, despite its deficiency in biofilm formation. On the other hand, both B. pyogenes and P. levii displayed slow planktonic growth but possessed the capability to form biofilms either individually or in conjunction with other strains. To identify physical interactions between bacterial cells, F. necrophorum, B. pyogenes and P. levii were cultured individually or together in chopped meat broth for up to 48 hours. F. necrophorum formed aggregates, resulting in a clear supernatant. B. pyogenes and P. levii did not form clumps individually but exhibited the formation of aggregates when cultured with F. necrophorum. These aggregates may involve adhesins on the surface of F. necrophorum binding to receptors or specific molecules on the surfaces of B. pyogenes and P. levii. Next, we examined whether uterine pathogens can interact with each other through released compounds. One strain was placed at the bottom of the well, while another strain was incubated together in a separate insert. There was no significant difference in the growth of F. necrophorum and P. levii. However, when B. pyogenes was cultured with P. levii, their populations significantly increased compared to B. pyogenes alone or with F. necrophorum. This shows that products released from P. levii may facilitate the growth of B. pyogenes. Furthermore, we evaluated the adherence ability of bacteria to BEND cells. In single culture, we observed that 25% of F. necrophorum attached to BEND cells, while B. pyogenes and P. levii exhibited lower attachment, with only 4% and 8%, respectively. However, in the co-culture of these 3 strains, both B. pyogenes (10%) and P. levii (16%) showed a significant increase in adherence to BEND cells, reaching levels comparable to that of F. necrophorum (16%). Taken together, our findings demonstrate synergistic interaction between F. necrophorum, B. pyogenes and P. levii, where F. necrophorum plays a significant role as a bridging organism, promoting the association with B. pyogenes and P. levii. This association has implications for bacterial growth, biofilm formation, and adherence to host cells, leading to the development of polymicrobial infections in the uterus. Aim 2) Characterize the in vitro metabolic profiles of F. necrophorum, B. pyogenes, and P. levii to determine what metabolic pathways are involved in their interactions and what bacterial metabolites are associated with endometrial infection. Our focus for this aim is to identify metabolic pathways involved in the interaction of F. necrophorum, B. pyogenes, and P. levii, as well as bacterial metabolites associated with endometrial infection. In Aim 2 we will: 1 - screen carbon sources that are used by F. necrophorum, B. pyogenes, and P. levii; 2 - identify bacterial products that are toxic to BEND; 3 - characterize extracellular and intracellular metabolites produced by F. necrophorum, B. pyogenes, and P. levii during infection of BEND usingGas Chromatography Mass Spectrometry (GC-MS). Objective 1 and 2 have been accomplished, and Objective 3 is currently in progress. We investigated carbon sources metabolized by uterine pathogens using a 96-well plate containing water (control) and 95 different chemical compounds. Notably, F. necrophorum showed a high utilization of β-hydroxyburytic acid, whereas B. pyogenes and P. levii preferred to use amino acids such as glutamine. The uterus of dairy cows experiences low nutrient availability and elevated levels of ketone bodies such as β-hydroxyburytic acid. Therefore, the availability of carbon sources in the uterus may explain the rapid growth of F. necrophorum. Furthermore, as postpartum cows increase their demand for amino acids for wound hearing, tissue repair, and immune function, B. pyogenes and P. levii are likely to increase their populations accordingly. Next, we investigated whether metabolites produced by F. necrophorum, B. pyogenes, and P. levii are toxic to host cells. BEND cells were seeded into each well of a 96-well plate and then incubated with water (control) and 95 bacterial metabolites individually. After 24 hours of incubation, live and dead cells were counted using a fluorescence microplate reader. We observed that bacterial metabolites generated after the utilization of D-Melezitose, α-Ketovaleric acid, and stachyose exhibited the highest cytotoxicity to BEND cells, which was greater than 30%, while the control showed cytotoxicity at about 15%. These bacterial products will be identified using GC-MS.

Publications

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The target audiences that this study will serve to benefit include veterinarians and scientists concerned with animal disease caused by polymicrobial infection of Gram-negative anaerobic bacteria in addition to the dairy industry, dairy producers, and general public concerned with use of antibiotics in agriculture. The data obtained from this study will be used as course material for Gram-negative anaerobic bacteria in Bacteriology, so veterinary students will also be target audiences. Changes/Problems:It has been difficult to hire a postdoctoral fellow to work on this project. Due to COVID-19 and the high cost of living in NY, it has taken more time to hire for this position. What opportunities for training and professional development has the project provided?The project has provided training in laboratory research to veterinary and graduate students. How have the results been disseminated to communities of interest?At this time results have not been disseminated. We are planning to present our results at the conference of Research Workers in Animal Diseases (CRWAD) in Chicago on January 20-24, 2023. What do you plan to do during the next reporting period to accomplish the goals?Multi-species biofilm formation and BEND cell viability in response to live bacteria are being investigated and we will soon complete all experiments as proposed in Aim 1. Our focus during the next reporting period is Aim 2, which identifies metabolic pathways involved in the interaction of F. necrophorum, B. pyogenes, and P. levii as well as bacterial metabolites associated with endometrial infection. We will screen carbon sources that are used by F. necrophorum, B. pyogenes, and P. levii, identify bacterial products that are toxic to BEND, and characterize extracellular and intracellular metabolites produced by F. necrophorum, B. pyogenes, and P. levii during infection of BEND using GC-MS. Metabolic profiles identified from GC-MS will be deposited in the Global Natural Product Social Molecular Networking database. Also, we will construct the bovine 3-D endometrial epithelial cell model that will be used in Aim 3. The data generated in the first and second year of the project will be published.

Impacts
What was accomplished under these goals? Aim 1) Identify the interactions of F. necrophorum, B. pyogenes, and P. levii for enhancing growth, colonization, and pathogenicity. The major goal of this aim is to determine if these 3 opportunistic uterine pathogens have synergistic interactions in terms of bacterial growth and pathogenicity to bovine endometrial epithelial (BEND) cells. In Aim 1 there are 3 specific objectives: 1 - investigate the growth rates of F. necrophorum, B. pyogenes, and P. levii in culture media, separately and together; 2 - determine if these pathogens form a biofilm when grown separately and together; 3 - examine attachment and cytotoxicity to BEND cells separately and together. Obj. 1 has been accomplished. Obj. 2 and 3 has been accomplished in mono-species, but co-culture of the 3 species is still on-going. We have isolated strains of F. necrophorum (SJ11), B. pyogenes (KG36), and P. levii (SJ44) from the uterus of dairy cows with metritis, which have been confirmed with 16S rRNA gene sequencing. They have been cultured at 37°C in the AS-150 anaerobic chamber in chopped meat broth or on Wilkins Chalgren (WC) agar supplemented with vitamin K1-hemin solution and 5% horse blood. To determine growth rates of individual species (Obj. 1), bacteria were inoculated into chopped meat broth and incubated in the anaerobic chamber at 37°C overnight. Then, 100 uL of the overnight culture was inoculated with 3 mL of fresh chopped meat broth to make the OD600 of 0.1 corresponding to approximately 108 CFU/mL. Planktonic growth of uterine bacteria were measured at 3, 6, 9, 12, and 24 hours of incubation at OD600 using a colorimeter. We observed that F. necrophorum grew rapidly, reaching a plateau at 6 hours of incubation. B. pyogenes and P. levii grew relatively slow as they reached the maximum growth value of 2 between 12 and 24 hours of incubation. For co-culture of the 3 bacterial species (Obj. 1), we used permeable polycarbonate membrane inserts with a pore size of 0.4 μm. Briefly, 108 CFU of F. necrophorum in 1 mL of chopped meat broth were added into the wells of a 24-well plate. Then, an insert containing 108 CFU of B. pyogenes and/or P. levii in 100 μL of chopped meat broth were placed into the wells. The plate was incubated in the anaerobic chamber at 37°C for 24 hours, inserts were removed, and wells were measured at OD600 on the GloMax® discover microplate reader. F. necrophorum showed no difference in growth between mono-culture and co-culture.B. pyogenes increased in co-culture with P. levii, which was significantly greater than mono-culture, co-culture with F. necrophorum, and co-culture with both F. necrophorum and P. levii. Growth of P. levii was significantly lower in co-cultures with F. necrophorum or B. pyogenes than mono-culture of P. levii. This shows that P. levii have a synergistic effect on growth of B. pyogens, but not vice versa. Secreted products of P. levii when cultured with B. pyogenes will be identified in Aim 2. Next, we determined the biofilm formation of F. necrophorum, B. pyogenes, and P. levii in 96-well flat-bottom polystyrene microplates (Obj. 2). Briefly, 200 µl of bacterial suspensions in log-phase at OD600=0.1 or 0.4 were added to each well of the microplates and incubated at 37°C for 72 hours in the anaerobic chamber. The supernatants were discarded, and the wells were washed once with distilled water. After 15 min of staining with 150 µl of 0.1 % crystal violet, the biofilm was washed twice with distilled water and allowed to dry. To solubilize the stain, 100 µl of 30% acetic acid was added to each well, and the absorbance was measured at 600 nm with the GloMax microplate reader. The results showed that inoculum of ≥OD600 0.1 can efficiently form the biofilm by B. pyogenes and P. levii at 72 hours, and biofilm biomass increased with inoculum increase. The biomass reached 0.838 in B. pyogenes and 0.784 in P. levii. On the other hand, F. necrophorum was defective in biofilm formation. Multispecies biofilm formation by F. necrophorum, B. pyogenes, and P. levii is being examined using confocal laser scanning microscopy (Zeiss). Next, we investigated the ability of these 3 species to adhere to BEND cells (Obj. 3). BEND cells in fresh medium supplemented with 10% serum but containing no antibiotics at a concentration of 2 X 105 cells/mL were incubated with F. necrophorum, B. pyogenes, and P. levii corresponding to 106 CFU (MOI of 5:1). After 3 hours of incubation at 37°C with 5% CO2, the medium of infected cells were collected to count the number of non-adhered bacteria, and the cells with adhered bacteria were scraped from the wells after incubation with 0.05% trypsin-EDTA for 10 min at 37°C. Serial dilutions from the medium and BEND cells were plated onto WC agar and anaerobically incubated for 48 hours at 37°C to count viable bacteria as the number of CFU/mL. The percent of adhered bacteria were calculated by dividing the number of adhered bacteria by the addition of the number of adhered and non-adhered bacteria. As a result, we found that 92% of F. necrophorum, 54% of B. pyogenes, and 13% of P. levii adhered to BEND cells with a significant difference among species. To determine cytotoxicity of these 3 bacteria to BEND cells (Obj. 3), we performed MTT assay. Heat-killed bacteria were prepared in 10-fold increasing doses relative to CFU determined after initial bacterial culture, and were applied to BEND cells for a period of 24 hours. Then a total of 10 μL of 5 mg/mL MTT was added to each well. Cells were incubated for 2 hours in MTT prior to washing in warm DPBS. Following incubation with MTT, cellular formazan was solubilized by the addition of 100 μL of dimethyl sulfoxide per well and incubated for 15 min at room temperature in the dark. Optical density of each well was measured at 540 nm using a microplate reader and data were normalized as fold change from the medium alone treated cells. The data showed that exposure of BEND cells to heat-killed bacteria at doses ranging from 102 to 108 CFU/mL had no effect on cell viability. Next, we examined expression of pro-inflammatory cytokines in BEND cells exposed to heat-killed bacteria using qPCR. We found that F. necrophorum at a dose ≥106 CFU/mL increased expression of IL-1β, IL-6 and IL-8, while B. pyogenes and P. levii had little or no effect. Together, these heat-killed uterine pathogens at low numbers have no cytotoxicity to BEND cells, but high numbers of F. necrophorum are capable of inducing pro-inflammatory response in BEND cells.

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