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

Outputs
Target Audience:Target audience include poultry scientific community interested in antimicrobial resistance, organic poultry, educators in sustainable agriculture, largescale organic poultry production, and small-scale organic poultry production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI and graduate student (Rithu Chandran) attended the Poultry Science Association Annual Conference at Louisville, KY, during July 15-18, 2024 How have the results been disseminated to communities of interest?The graduate student presented the in vitro experiment research findings using the select phytochemicals in the Student Oral Research Presentation at the Poultry Science Association Conference in Louisville, KY. Abstract #72 - Effect of Select Phytochemicals on Mitigation of Horizontal Gene Transfer of Plasmids Harboring Antibiotic Resistance Genes between Foodborne Extended Spectrum Beta-lactamase producing Salmonella and commensal E. coli. [Page 40 of the following link - 2024_PSA_Annual_Meeting_Abstract_Book.pdf] What do you plan to do during the next reporting period to accomplish the goals?The microbiological and sequencing data analysis will be updated in the next reporting period.

Impacts
What was accomplished under these goals? Objective 2: Bird trials are currently undergoing at the University of Florida during Fall 2024. Sixty day-old chicks were randomly assigned to six groups of 10 birds each. Cuminaldehyde feed supplementation was initiated from the day of arrival of chicks. In addition, the chicks were also provided oxytetracycline through drinking water from days 0 to 5 days. After the 24-hour washout period for the antibiotics on Day 6 with regular drinking water, the respective treatment groups receiving the bacterial inoculation was done on Day 7. Prior to inoculating birds with the respective bacterial cultures, two birds fromeach group were euthanized and necropsied to check for the presence of SH in the flock. The details of the treatment groups used in the experiment is as follows: Groups 1, 2 and 3 were the uninoculated chicks provided with 0%, 0.1% and 0.2% of Cuminaldehyde treatment in feed. Group 4 involved chicks orally co-inoculated with S. Heidelberg strain 18 (10^7 CFU) and E. coli J53R (with rifampicin resistance), respectively, with no cuminaldehyde treatment Groups 5 and 6 were chicks orally co-inoculated with with S. Heidelberg strain 26or 18 (10^7 CFU) and E. coli J53R (with rifampicin resistance), respectively, and given 0.1% and 0.2% of the phytochemical treatment, respectively. Cloacal swabs were collected and streak cultured on selective agar plates on day 10 to ensure the colonization of the donor and recipient in the birds. On day 17 (which is day 10 post infection), birds from all treatment groups were euthanized (CO2 inhalation) and necropsied for sampling cecal contents to enumerate E. coli transconjugant frequency. A subset of the cecal content were snap frozen and saved in -80? for sending out samples to Phase Genomics, Inc., for metagenomics and Hi-C sequencing.

Publications

Progress 08/01/22 to 07/31/23

Outputs
Target Audience:Target audience include poultry scientific community interested in antimicrobial resistance, organic poultry, educators in sustainable agriculture, largescale organic poultry production, and small-scale organic poultry production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project involved training an graduate student to conduct basic bacteriological procedures, perform in vitro HGT experiments, bioinformatic applications such as NCBI BLAST, Primer BLAST, sequencing aligning and annotation, plasmid isolation, purification and whole plasmid sequencing analysis. Dr. Abraham Pellissery and graduate student (Rithu Chandran) attended the NIAMRRE Workshop: Antimicrobial Resistance Conference at University of Florida, during May 16-18, 2023. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The bird trials relevant to the study will be conducted during late Spring 2024 semester and during Summer 2024. The Salmonella Heidelberg isolate, with plasmid derived CTX-M-1 gene, will serve as the donor bacteria, and E. coli J-53-R (rifampin resistance marker- chromosomally integrated) will serve as the recipient bacteria for the in vivo chicken trial experiments. The chicken trials will be performed using the aforesaid donor-recipient combination as an oral challenge to assess for the efficacy of in-feed supplemented phytochemicals to reduce horizontal gene transfer in the bird's cecal microbiota. Preliminary bird experiments (at the University of Connecticut) will focus on standardizing the in-feed supplementation of phytochemicals that effectively reduce HGT in the gut environment without affecting the production parameters of the broiler chickens. Upon standardization of the phytochemical dosage, 4 bird trials will be conducted and the last trial from the set of broiler chicken studies will focus on collecting snap-frozen cecal samples for subjecting them to deep metagenomic sequencing and Hi-C sequencing.

Impacts
What was accomplished under these goals? Objective 1: The project officially started during January 2023. The graduate student was hired for the Spring semester and continues to be trained and working to accomplish the goals under this objective. The project has completed genome sequencing and annotation of the whole genome as well as the plasmid derived from Salmonella Heidelberg isolates 26-4057, 18-9079 and 19-9089. The plasmids have been annotated for an intact tra gene machinery as well as functional beta lactamase gene elements (antibiotic resistant genes) such as as blaTEM and CTX-M-1 encoding resistance for 3rd and 4th generation cephalosporins (ceftriaxone and cefipime). Based on the sequencing information, preliminary conjugation experiments were conducted to confirm the anti-HGT efficacy of phytochemicals in vitro to validate its use for the upcoming chicken trials. From these studies, it was decided to proceed with either the 18-9079 (harboring IncI plasmid) or 26-4057 (harboring IncN plasmid) for the upcoming bird trials. Objective 2: This objective will be conducted and reported as part of next year's progress report.

Publications

Progress 08/01/22 to 10/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:The PI has moved to University of Florida as a research faculty in the Department of Comparative Diagnositc and Population Medicine. The grant and funding are in the process of being transferred to University of Florida from University of Connecticut. The relevant paperwork regarding the grant transfer has been processed by the University of Connecticut. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Expected to start project during Spring 2023 or by Fall 2023. The PI was recently hired (w.e.f. 10.19.2022)as a research faculty at the Department of Comparative Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida.

Publications