PROBIOTIC NISSLE E. COLI 1917 AS ANTIBIOTIC-ALTERNATIVE TO COMBAT ANTIBIOTIC-RESISTANT CAMPYLOBACTER IN THE FOOD CHAIN

<b>Project Methods</b><br> Our proposed study aimed to elucidate how EcN mediates its action against C. jejuni through:Objective 1: Identification and characterization of the bioactive molecule(s) that contribute to the antimicrobial effect of EcN against C. jejuni. This will be achieved through:1.1. Identification of the bioactive molecule. To determine if the bioactive molecule is SM metabolite and/or AMPs and confirm their anti-C. jejuni activity, the filtered CFSs of EcN will fractionate by partitioning between organic and aqueous phases. The bioactivity of both fractions will be tested using the agar well diffusion assay and the trans-well migration assay. The bioactive fraction will be subjected to dereplication using liquid chromatography-mass spectrometry (LC-MS/MS) to detect the novel bioactive SM metabolite(s).1.2. Purification and characterization ofthe novel bioactive molecule(s). To purify the dereplicated bioactive SM metabolites, we will use preparative High-performance liquid chromatography (prep. HPLC). Additionally, to purify the bioactive peptides, the EcN-filtered CFSs aqueous phase will be precipitated by ammonium sulfate and the precipitate will be collected by centrifugation. The pellets (AMPs) will be aliquoted in potassium phosphate buffer and dialyzed using a 3 kDa molecular weight cutoff (Sigma Aldrich). The obtained AMPs will be freeze-dried and used for further characterization. To characterize the bioactive molecules, the purified molecules will be subjected to LC-MS/MSand the molecular weights of the obtained peptides will be determined by Tricine-SDS-PAGE. To confirm the antimicrobial activity caused by the peptides, we will check the thermal stability of the extracted AMPs (10 mg/mL) at 50 to 121°C. The pH stability of the AMPs will be also determined by dissolving the lyophilized peptides in distilled water at 10 mg/mL and adjusting the pH using 1N NaOH or 1N HCl. The AMPs will be also subjected to proteinase K, trypsin, and pepsin treatments to determine sensitivity to proteolytic enzymes.The 3D structure of the obtained AMPs will be identified using X-ray crystallography. The CFSs of LA, BB-12, and LGG will be used as controls and the inhibitory activity of treated AMPs (by heat, enzymes, pH change) will be investigated using an agar well diffusion assay. Non-treated AMPs will be used as a control.Objective 2: Evaluate the efficacy of the purified molecule(s) in vitro and in chicken enteroids. 2.1. Evaluate the efficacy of the identified molecule(s) in vitro. The purified bioactive molecule(s) will be evaluated for their effect on 1) multidrug-resistant Campylobacterand beneficial microbesas it is important to identify the spectrum of the identified compounds and their effect against the drug-resistant Campylobacter. These Campylobacter strains were collected from poultry as well as humans and they possessed resistance to ciprofloxacin. We will also determine the minimal inhibitory concentration and the minimal bactericidal concentration using a dose-response assay, 2) biofilm formation of C. jejuni using the crystal violet (CV) assay, as biofilms play a crucial role in Campylobacter virulence and enhance the bacterial resistance to antimicrobials and immune clearance, leading to the failure of antimicrobial therapy, 3) Campylobacter motility using semisolid agar motility assay, as motility and chemotaxis allow bacteria to migrate towards or away from favorable environments in response to stimulus and thus contribute to bacterial fitness and virulence, 4) toxicity to chicken macrophage (HD-11) and human intestinal epithelial cells (Caco-2) using lactate dehydrogenase (LDH) assay, and 5) the ability to clear intracellular Campylobacter infections in chicken macrophage (HD-11). We will also evaluate the ability of C. jejuni to develop resistance against the molecule(s) using antimicrobial resistance studies. Since C. jejuni's pathogenicity depends on virulence factors including biofilm formation, motility, adhesion, invasion, chemotaxis, and colonization, it's important to investigate the effect of the identified bioactive molecule(s) on the virulence-associated genes. After growing the sublethal dose of the selected molecule(s) with C. jejuni in liquid media. Total RNA extraction, cDNA synthesis, and qRT-PCR will be performed as described in our previous study. The tested genes will include genes responsible for Campylobacter motility and biofilm formation (faA, faB, fhA, and fhB), adhesion (cadF), invasion (ciaB, and iamA), cytotoxin production (cdtA) and autoinducer production (luxS and pfs). Non-treated bacteria will be used as a control.2.2. Evaluate the efficacy of the identified molecule(s) in the ex vivo intestinal epithelium model (chicken enteroids). A chicken enteroids model was recently developed to conduct standardized high-throughput studies of intestinal epithelial cell biology and innate immune function and to look into strategies to enhance and support intestinal health and host-pathogen interactions. Chicken enteroids will be isolated from the intestine (duodenum, jejunum, ileum, and caeca) of embryonic day (ED18) Hy-Line Brown chickens and the collected tissues will be seeded, digested, and maintained as described before. We will select the molecule(s) (Obj. 2.1) that; 1) inhibit multidrug-resistant C. jejuni, 2) have no resistance, cytotoxicity, and hemolytic activity, 3) didn't affect the beneficial microbes, and 4) inhibit biofilm and motility of C. jejuni. The effect of the selected molecule(s) on adhesion, invasion, and intracellular survival of C. jejuni on chicken enteroids will be testedand the treated enteroids will be collected for transcriptomic analysis. Total RNA extraction will be performed as described in our previous study. Libraries will be generated and sequenced as recently described by Messaoudi's Labusing the NEBNEXT™ ultralow input RNA library kit. Multiplexed libraries will then be sequenced to yield 20 million 100 bp sequences per sample. Reads will be quality trimmed and aligned to the chicken genome [Gallus_gallus-5.0]. We will compare between the transcriptome of non-treated enteroids, enteroids treated with each AMP and infected with C. jejuni, enteroids treated with EcN, enteroids treated with EcN CFSs then infected with C. jejuni, and enteroids infected with C. jejuni alone.Data analysis of expected results. Data generated from this study will be analyzed in GraphPad Prism software using one-way ANOVA, followed by Tukey's posthoc test. The RT-qPCR data will be analyzed using the Mann-Whitney U test, and the fold change of ±1.5 ≥ or ≤ 1.5. Data generated from LC-MS/MS will be analyzed using Proteome Discoverer 2.2 software. Differentially Expressed Genes (DEGs) by RNA sequencing will be analyzed using the R package edgeR and differentially expressed genes (DEG) will be defined as those with fold change >2 and an FDR (False Discovery Rate) <5%. P< 0.05.