College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item Three Clostridium species with Health Imparting Properties: In vitro Screening for Probiotic Potential(2024) Mochama, Victor Moronge; Obanda, Diana; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research aimed to unlock the probiotic potential of the genus Clostridium, which is often overshadowed by the predominant focus on pathogenic species. The study specifically targeted three promising Clostridium species: C. disporicum, C. celatum, and C. vincentii, which have shown potential in mitigating diet-induced obesity. Despite the challenges presented by the anaerobic growth requirements of Clostridium bacteria, the study capitalized on their capacity to sporulate. This characteristic provides an avenue to use them as probiotics, with resilient and dormant spores capable of surviving food processing and harsh stomach conditions. The resilience of these spores was examined by exposing them to oxygen, heat, gastrointestinal juices, and bile salts. The spores survived oxygen exposure, exhibited resilience to both bile salts and gastric acids, and demonstrated a survival temperature of 70°C. When exposed to suitable germination conditions in vitro, the spores successfully germinated. The study assessed the colonization potential of the bacteria by evaluating their adhesion ability, and all bacteria were found to have the adhesion ability. Furthermore, a safety assessment was conducted by examining hemolytic activity and antibiotic susceptibility to selected antibiotics. The bacteria were found to be susceptible to the antibiotics and did not exhibit hemolytic activity. Bile salt hydrolase (BSH) activity and antibacterial activities were also assessed, and none of the bacteria exhibited BSH activity or antibacterial activity. Antioxidant tests revealed that C. vincentii had the highest antioxidant properties. Assessment of anti-inflammatory properties showed that C. celatum downregulated the gene expression of cytokine inflammation markers IL-6, IL-1, and iNOS while upregulating TGF-β expression. In summary all 3 bacterial species showed good probiotic potential from the in vitro tests. Particularly the formation of resistant spores that later germinated to vegetative cells that produced molecular patterns with antioxidant and anti-inflammatory properties. This necessitates further studies on their probiotic properties.Item EVALUATION OF THE COPPER HOMEOSTASIS AND SILVER RESISTANCE ISLAND AND ITS ROLE IN PERSISTENCE OF SALMONELLA ENTERICA(2023) Haendiges, Julie; Tikekar, Rohan; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Salmonella enterica is one of the leading bacterial cause of foodborne illness in the UnitedStates. Although there are many serovars, only a small subset causes human illness. Since Salmonella is ubiquitous in the environment, the Food and Drug Administration has established regulations for food processors to ensure that the products are free of contamination. Low-moisture foods are commonly ready-to-eat, and due to the low water activity do not promote growth of bacteria. However, Salmonella has been shown to persist in these foods. There havebeen two outbreaks and multiple recalls in the United States due to contaminated pistachios. Based on a retrospective study, results show that there is evidence of a contamination in the growing orchard and a significant number of Salmonella isolates from the environments contain the Copper Homeostasis and Silver Resistance Island (CHASRI) cassette. This raises several questions: what is the prevalence of CHASRI among different Salmonella isolates from food and environmental sources? Does presence of CHASRI enable Salmonella to survive better against copper stress? And whether presence of CHASRI provide cross-protection against other stresses such as desiccation and thermal treatment? This dissertation attempts to answer those questions. The prevalence of the CHASRI in Salmonella was determined by the use of publicly availablewhole genome sequencing data. The CHASRI was found in 61 different serovars and types of sources. The presence of the CHASRI in isolates from low-moisture foods that have caused previous outbreaks (peanut butter, nuts, spices) was interesting to note, and leads to future studies on correlations between this island and virulence. Based on results of phylogenetic analysis of CHASRI sequences from closed genomes, we determined there were four types of CHASRI found in Salmonella. Traditionally, the Salmonella Genomic Island-4 (SGI-4) is found but in addition the CHASRI can incorporate by itself, within a variant of SGI-4, or via a rare plasmid. Interestingly, the sequence of the CHASRI from SGI-4 and the variant SGI-4 were highly different. The high SNP differences in sequence along with the difference in the arsenic operon led to the conclusion that these variants arose independently. A Salmonella Senftenberg strain (CFSAN047523), isolated from pistachios, was used to createthree knockouts (∆cus, ∆pco, and ∆CHASRI). Previous studies have looked at the minimum inhibitory concentration (MIC) of strains with and without the CHASRI but have omitted the minimum bactericidal concentration (MBC). In this study, we used the knockouts to test for both MIC and MBC. While the MIC was similar for the strains, the MBC was greater in the wild type and partial CHASRI knockouts. Growth and inactivation kinetics were measured in different concentrations of copper sulfate. At higher levels of copper sulfate, the presence of the CHASRI made cells more resilient to inactivation by copper sulfate. Evidence shows that the stress response in Salmonella has the ability to crosstalk and provideprotection against multiple stresses. To investigate this phenomenon further, our isolates were tested against a multitude of stresses to evaluate for cross-protection that may be due to theCHASRI. Cells undergoing copper stress were better equipped to survive lethal copper concentrations and desiccation if the CHASRI was present. The presence of Salmonella in final pistachio products that have been fully processed identifies that some adaptation and stress response is occurring in the processing facility. Inoculated pistachios with the wild type and ∆CHASRI strain were thermally processed to test for survivors. This study showed that the presence of the CHASRI gave the isolate an advantage to survive thermal processing after desiccation. Overall, this study presents the prevalence of the CHASRI in Salmonella enterica as well as theimportant role it plays in copper tolerance. The evidence of cross-protection and tolerance to copper leads to future research regarding gene expression and virulence assessment.Item CAUSATIVE AGENTS FOR FOWL TYPHOID AND PULLORUM DISEASE IN POULTRY AND APPROACH TO CONTROL(2023) Julianingsih, Dita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In response to escalating consumer demand, a significant number of conventional US poultry farms have transitioned to antibiotic-free and chemical-free practices, particularly those adopting pasture/organic methods. However, recent reports highlight a resurgence of bacterial diseases in both conventional and pasture poultry farms, resulting in elevated bird mortality rates and reduced profitability. This comprehensive study investigates the prevalence of S. Gallinarum and S. Pullorum, causative agents of fowl typhoid and pullorum disease, in integrated crop-livestock/pasture farm environments and meat products. A total of 1,286 samples from 7 farms and 13 retail markets were examined, revealing that S. Pullorum and S. Gallinarum are common in both farm poultry environments and market products. Antibiotic resistance patterns, determined through an antibiogram assay, indicated high resistance to multiple antibiotics. S. Pullorum and S. Gallinarum were discovered in 2.7% and 1.5% of samples, respectively, at the pre-harvest stage. Only 1.6% of the meat samples recovered from retail markets had S. Gallinarum detected in them at the post-harvest level. Concurrently, a different study investigates the possibility of Orange Cold-press Valencia Terpeneless, a citrus oil variety, acting as a natural antimicrobial in poultry farming. This study tackles the problems caused by a decline in the usage of antibiotics, which has resulted in an increase in bacterial infections. Citrus oil exhibits potential as an antimicrobial agent, inhibiting the growth of S. Pullorum and S. Gallinarum, with consistent MIC and MBC values. Time-dependent experiments with 0.4% citrus oil show total suppression of bacterial growth, which is confirmed by environmental simulations. Furthermore, the study reveals that both Salmonella strains have downregulated their virulence genes, which may indicate a change in the pathogenicity of the bacteria. Overall, the findings highlight the crucial importance of surveillance programs and preventive measures. Citrus oil is presented as a promising natural alternative for antibiotics in the treatment of Salmonella-related infections in the poultry farming industry.Item MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.Item BACTERIAL GROWTH AND INACTIVATION IN HETEROGENEOUS ENVIRONMENT(2023) Tsai, Shawn; Tikekar, Rohan V.; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation explores the growth and inactivation of Salmonella enterica subsp. enterica Serovar Typhimurium in oil-in-water emulsion systems, with a particular focus on the effects of emulsifier type, oil fraction, temperature, molecular weight, and surface charge. First, investigation was conducted on the effects of emulsifier type (Tween 20, Tween 80, Triton X-100) and oil content (20%, 40%, 60%) on growth and thermal inactivation of S. Typhimurium in emulsions. The results indicated that while emulsifiers did not affect the bacterial growth rate or lag phase, the presence of high oil content (60%) prolonged the lag phase in emulsions. In addition, Tween 80 and Triton X-100 emulsifier solutions exhibited protective effects against thermal inactivation. Next chapter was focused on evaluating the impact of temperature on growth (7, 22, 37°C) and inactivation (55, 58, 60°C) kinetics of S. Typhimurium in emulsion systems with same emulsifier and oil fraction as previous chapter. The results indicated that temperature had a significant impact on bacterial kinetics, with increasing temperatures leading to faster growth and inactivation rates. Next, the effect of emulsifier molecular weight and surface charge on the growth and thermal inactivation of S. Typhimurium in emulsions was examined. To control the molecular weight with similar structure, whey protein was selected for experimentation. By adjusting the pH, it was possible to change the surface charge in whey protein. Results indicated that whey protein hydrolysate (WPH) with a lower molecular weight did not exhibit a lag phase in Salmonella growth. However, whey protein isolate (WPI) with higher molecular weight demonstrated no difference in the lag phase when compared to bacterial growth in TSB. Similar effects were observed with a positively charged emulsifier (WPI+). The findings suggest that the molecular weight of emulsifiers has a more significant impact on bacterial growth than their surface charge. Regarding the evaluation of inactivation, emulsifier solutions exhibited no significant difference compared to TSB, while emulsions stabilized by WPH and WPI+ showed some protective effects on S. Typhimurium. This observation can be attributed to the ability of positively charged emulsifiers to interact with the bacterial membrane, providing protective effects during thermal treatment. Lastly, to gain a comprehensive understanding of the mechanism concerned with the impact of emulsifier and oil inclusion on bacterial growth and inactivation behavior, S. Typhimurium was cultured in different emulsion-related environments and evaluated for nine stress-related genes (rpoE, rpoH, otsB, proV, fadA, fabA, dnaK, ibpA, ompC) after 20 hours of incubation at 37°C and after thermal treatment at 55°C for 45 min. It was found that ibpA was upregulated in all emulsifier environments, regardless of the presence of oil, indicating that IbpA was synthesized in emulsifier environments. Moreover, increased expression of fabA was also observed in Triton X-100 stabilized 60% emulsion, indicating poor heat resistance due to increased membrane fluidity. In the combination of gene expression data, our results showed that emulsifier solutions without oil exhibited a greater number of regulatory mechanisms compared to those containing oil, indicating that the presence of oil did not provide as much protection after thermal treatment. Based on these findings, the stress-related mechanism was constructed by the expression of those selected genes. Overall, this dissertation provides valuable insights into the factors influencing bacterial growth and inactivation in oil-in-water emulsion systems, as well as bacterial stress response in these systems. These findings provide important insights into the growth and inactivation behavior of S. Typhimurium in oil-in-water emulsion systems and the stress response mechanisms involved. Understanding these factors is crucial for developing effective control measures to ensure food safety and prevent foodborne illness outbreaks caused by this pathogen. This information can be used to optimize the formulation and processing of emulsion-based food products to minimize the risk of bacterial contamination and ensure their safety for consumption.Item POSITIONAL CLONING OF BROAD-SPECTRUM LEAF RUST RESISTANCE GENE, LR57, FROM AEGILOPS GENICULATA, A TERTIARY GENE POOL MEMBER OF WHEAT(2023) Schulden, Taylor Francis; Rawat, Nidhi; Erwin, John; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The tertiary gene pool of wheat includes wild relatives like Aegilops geniculata (UUMM, 2n=4x=28) that are valuable genetic reservoirs for novel abiotic and biotic resistance genes. However, modern wheat varieties share limited genomic commonality with these gene pool members presenting barriers to recombination and genetic mapping of desirable genes. We mapped a broad-spectrum leaf rust resistance gene Lr57 located on chromosome 5Mg of Ae. geniculata using a simple but powerful methodology for high-resolution genetic mapping in tertiary gene pool members of wheat. Five gene candidates were revealed all with possible defense related functions. Strategic application of differential expression analysis, Virus-Induced Gene Silencing, and mutagenesis analysis reduced the candidate gene of interest to a singular and novel ID-NLR resistance gene containg a protein kinase, NB-Arc, and LRR domain. Using multiple strategies, validation of Lr57 candidate was completed. Gene complementation by transformation of Lr57 candidate is currently being conducted.Item SURVIVAL OF ESCHERICHIA COLI AND CHANGES IN PHYSICOCHEMICAL PARAMETERS IN AQUAPONIC SYSTEMS DURING BASIL AND LETTUCE PRODUCTION(2023) Quach, Emily; Yonkos, Lance; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Aquaponics (APs), a soilless production system, integrates aquaculture and hydroponics to provide local fresh produce while conserving natural resources. The absence of soil in APs eliminates one potential food safety risk present in typical soil-based production systems, but APs may become contaminated from a variety of sources. Escherichia coli TVS 354 long-term survival was evaluated in bench-scale, deep-water APs units. In addition, pathogen presence on basil and lettuce at the time of harvest and changes in the population density of mesophilic aerobic bacteria in APs were measured. Results showed E. coli populations significantly decreased 24 h post-inoculation in water samples and remained undetectable by day 1 post-inoculation. Lettuce harvested on day 60 had detectable E. coli on lettuce leaves and roots at harvest. These results provide new insight on E. coli survival in harvested plants, indicate potential risks for foodborne illnesses, and unreliability of water testing as a monitoring tool.Item AUGMENTING SEQUENCING TECHNOLOGY FOR BETTER INFERENCE IN SOIL MICROBIOME ANALYSIS(2023) Epp Schmidt, Dietrich; Yarwood, Stephanie A; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The advent of DNA sequencing revolutionized the field of microbiome research. Many organisms, by virtue of their codependence and/or growth rate, are either impossible or extremely challenging to get into pure culture. Sequencing allows important taxonomic and phylogenetic information to be obtained independent of culturing. Development of the sequencing technology itself has allowed for high throughput workflow that has allowed low cost and extensive sampling of microbiomes across environments. The co-development of reference datasets for taxonomy and functional assignments, along with open-source bioinformatics pipelines has further empowered scientists to explore microbiomes in many environments. However, there are limitations to sequence data that have constrained the ecological inferences in microbiome research. One such limitation, the compositional nature of sequence data, has impeded our ability to make accurate inferences about the environmental drivers of taxon abundance and covariance across conditions. In this dissertation I explore the use of quantitative PCR in combination with sequencing techniques to generate “Quantitative Sequencing” data (QSeq) that mitigates the limitations of compositionality on inferences relating to taxon abundance and covariance across environmental gradients. In chapter 1, I reviewed key characteristics of the soil environment and sequencing as a mechanism for sampling. In chapter 2, I leveraged modeling, synthesis, and literature review methods to establish the questions and data characteristics that demand QSeq methodology. I show that even small amounts of variation in total abundance make determining the effects of environment (biotic and abiotic factors) on any given taxon unreliable without QSeq. In Chapter 3, I extend the logic of quantitative sequencing to improve metagenome prediction from PICRUSt2. Using data synthesis methods, accounting for 16S gene abundance consistently improved the accuracy of predicted functional genes. This was confirmed by high correlations between predicted and measured gene abundance (QPCR). There was however a large variation in prediction accuracy, likely due in part to database biases and in part to decoupling of bacterial function from taxonomy. In Chapter 4, I applied QSeq in the context of an experimental, long-term farming system that has large gradients in total abundance with depth, and I used QSeq to identify taxa that changed in abundance due to different farming system management and soil depth. Finally in Chapter 5, I used QSeq to identify putative N-fixing taxa that responded to glyphosate in four experimental farming systems. I show that the abundance of these taxa were decoupled from other effects of glyphosate on N-fixation in soybean across farming systems.Item Investigating the hyperdiversity of fungal endophytes in wild Rubiaceae tropical plants and coffee plantations.(2022) Castillo Gonzalez, Humberto; Yarwood, Stephanie A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal endophytes are an essential component of a plant’s microbiome, their effect spreads to fitness, disease dynamics, stress tolerance, water acquisition and nutrient uptake. Plant ecosystems, from natural forest to plantations bear the indelible signature of its presence. The current investigation was designed to understand the diversity of endophytes in the Rubiaceae family, in plants associated to natural and managed ecosystems. The effect of location, leaf developmental stage, tissue type, host genotype, and anthropogenic interference was evaluated through amplicon sequencing. Costa Rica served as base for the sample collection. Leaves and sapwood from a variety of tropical plant species were collected in old-growth natural forests and foliar tissue from domesticated coffee plants were sampled in two plantations under different management. Fungal diversity was assessed by metabarcoding using the ITS2 nrDNA region fITS7 – ITS4, and library sequencing was completed by Ion Torrent. We identified a hyperdiversity of endophytes inhabiting these plants and were able to isolate a total of 659 fungi from coffee leaves. This investigation provides relevant information about overall community composition, the ecological drivers of community assemblage and the characteristics of the fungal endophytic communities, including potential interactions among the identified taxa. Endophytes may harness the potential to transform agriculture and conservation science, however we currently lack the knowledge to engineer microbial communities through breeding or management. It is essential to continue the efforts on understanding community functions and dynamics, and how host, endophyte interactions, and other ecological and human- related mechanisms influence their diversity in both forest species and agronomically important crops.Item ANTAGONISTIC MECHANISM OF METABOLITES FROM LACTOBACILLUS CASEI AGAINST FOODBORNE ENTEROHEMORRHAGIC ESCHERICHIA COLI(2022) Aditya, Arpita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Enterohemorrhagic Escherichia coli O157: H7 (EHEC), a foodborne enteropathogen, remains a significant public health concern since its discovery in 1982. With an incredibly low infectious dose (10-100 bacteria), this pathogen can cause self-limiting diarrhea, vomiting, and abdominal cramps. However, more complicated disease conditions such as bloody diarrhea or hemolytic colitis have been known to develop depending on the serotype involved in the infection, and on immune status and/or age of the patients. Due to its Shiga toxin (Stx) production ability, EHEC infection may lead to a kidney-related problem known as hemolytic uremic syndrome (HUS), which requires advanced medical care. Unlike other bacterial illnesses, therapeutic administration of antibiotics to treat EHEC infections is not recommended due to their controversial association with Stx production. As a result, only preventative/prophylactic and immune-supportive strategies are followed for EHEC infections. Using the antibacterial properties of probiotic bacteria and the metabolites they produce are promising alternative strategies for preventing EHEC infections. We have targeted the probiotic bacteria Lactobacillus casei to determine the mechanism of this alternative strategy. In our study, we have executed microbiological, molecular, chromatographic, and metagenomic approaches to determine the antagonistic mechanisms of action of their metabolites, specifically conjugated linoleic acid (CLA) produced by Lactobacillus casei, against the growth and metabolism of EHEC. The metabolites of wild-type L. casei (LCwt) were augmented by supplementing it with a prebiotic-like dietary component, namely peanut flour (PF) (LCwt+PF), while another LCwt was also genetically engineered (LCCLA) to over convert CLA from linoleic acid (LA). These modifications showed effective results in controlling EHEC both in vitro and in ex vivo conditions. Total metabolites present in cell-free culture supernatant (CFCS) of LCwt, LCwt+PF, and LCCLA were able to control the growth of EHEC without negatively hampering the relative abundance of Firmicutes and Bacteroidetes present in rumen fluid (RF). Among these CFCSs, CFCSCLA exerted the most desirable outcome by eliminating EHEC. In vitro studies demonstrated that, a lower concentration of purified CLA worked synergistically with other metabolites of LCwt and augmented their inhibitory activity against EHEC. The orchestrated effect of metabolites has been observed to downregulate the virulence genes, disrupt the cell membrane, interfere with cell division, and damage their genomic DNA. The probable effect of these metabolites, specifically CLA, on Stx production and neutralization was also investigated by assessing host cell cytotoxicity. Total metabolites of Lactobacillus spp. as well as CLA itself, showed improvement in cell survivability when exposed to Stx. Our findings established a ground to explore the effect of specific metabolites obtained from probiotic bacteria in control and prevention of EHEC. The findings also showed a promising association of purified CLA in neutralizing Stx which can be further explored to use it in therapeutic purposes.