PhD Thesis defense by Irene Aldea Ramos at the VISAVET Centre of the Complutense University of Madrid

July 1^st, 2025

Antibiotic resistance is a public health problem facing society, with fewer and fewer therapeutic options available for the treatment of infections caused by resistant bacteria. Production animals can transmit these resistant bacteria to humans through the food chain, so the use of antibiotics in these animals is restricted at both European and national level.

In this thesis, the role of the gut microbiota of animals (from day-old chicks to 84-85 weeks old laying hens) in the spread of antimicrobial resistance of Escherichia coli was studied in a commercial layer farm, providing a closed scenario in which to study the transmission and persistence of resistance genes.

E. coli is able to survive in the food chain and could spread antimicrobial resistance genes between bacteria due to its ability to horizontally transfer gene platforms. The presence of antimicrobial resistant E. coli in the gut microbiota of animals such as pigs and broiler chickens is well documented; however, the population dynamics of resistant E. coli in commercial table egg production have been scarcely studied. This study is the first longitudinal study of the transmission of antibiotic resistance genes in E. coli from laying hens in Spain, and one of the few in the world. The main objective of the thesis is to improve knowledge on the transmission dynamics of resistant E. coli in commercial laying hens (egg production) and to determine to what extent table egg production represents a risk to public health through contamination of food and/or the environment.

The research focused on identifying E. coli resistance genes from animals and eggshells using classical microbiology and masive sequencing techniques. This work analysed 687 animal and egg isolates from commercial egg production farms, with 271 isolates characterised by whole genome sequencing (WGS). Of these, 218 non-repetitive isolates were retained for further analysis, and of these, 113 isolates had at least one antimicrobial resistance gene. Up to 33 resistance genes were detected in these isolates (tetA, blaTEM-1B, aadA1, sul2, strA and strB being the most frequent).

Furthermore, the dynamics of antimicrobial resistance between different stages of egg production were studied by phylogenetic analysis from day-old chicks to pullets and laying hens. Based on phylogenetic distance, 30 different clones composed of two to eight isolates were detected. At least one plasmid replication origin was detected in 195 isolates and, in addition, eleven different patterns for resistance gene propagation were detected on this farm, improving understanding of the different roles played by integrons, multi-resistance regions, plasmids and clones. It was found that the most common models were plasmid-clone assemblies involving individual or linked resistance genes. In addition, events related to the gain or loss of resistance genes by plasmids, or the gain or loss of resistance gene-containing plasmids themselves by isolates, were recorded.

In conclusion, this thesis deepens the knowledge of the dynamics of antibiotic resistance genes and the different transmission mechanisms found in E. coli in an antibiotic-free environment, determining that, despite the non-use of antibiotics on farms, there must be sources of these resistance genes in this type of farms.