PhD Thesis defense by Vicente López Chavarrías at the VISAVET Centre of the Complutense University of Madrid

September 10^th, 2025

Antimicrobial resistance is a major problem for the treatment of diseases caused by zoonotic bacteria such as thermotolerant Campylobacter (C. coli and C. jejuni). The mechanisms by which antimicrobial resistance spreads over bacterial populations, especially via horizontal gene transfer, can have broad consequences depending on the speed at which resistant phenotypes disseminate through food animal hosts, humans and the environment. Particularly relevant are genetic determinants capable of conferring resistance to three or more antimicrobial classes, making treatments more complicated when multi-drug resistant bacteria are involved.

In the case of Campylobacter infections, aminoglycosides and macrolides are the two antimicrobial classes most commonly used to treat clinical cases, when this is necessary. This thesis is focused on the characterization of the distribution and genetic basis of co-resistance mechanisms involving aminoglycosides and macrolides in Campylobacter that could explain possible ways of acquisition, maintenance and dissemination of these resistance phenotypes.

The national programme for antimicrobial resistance monitoring in livestock in Spain served as the framework to conduct this research. Starting from all available data on antimicrobial resistance in thermotolerant Campylobacter from livestock, a ‘top to bottom’ approach of analysis was carried out. Isolates were grouped by phenotype, to assess the importance of those over time and per hosts, and then the genetic elements associated with a particular phenotype were identified using molecular methods. The use of phenotyping methods for microbial characterization is widespread and well developed, but the extent to which these techniques allow making inferences on the specific genetic mechanisms behind the antimicrobial resistance phenotypes observed is limited.

The material and methodology used were suited to achieve three Specific Objectives. The first objective was to evaluate the baseline level of phenotypic resistance to several antimicrobials, mainly aminoglycosides (represented by gentamicin and streptomycin) and macrolides (represented by erythromycin) in Campylobacter isolates retrieved from broilers, turkeys, pigs and cattle between 2002 and 2018, considering also their spatio-temporal distribution. The second objective was to assess the association between the occurrence of phenotypic resistance to both antimicrobial classes. A third objective was to elucidate the genetic mechanisms implicated in the predominant resistance phenotypes observed, with the aim of identifying resistance determinants and mobile genetic elements that could favour their dispersion.

The database analyzed included information on the year, host, sample analyzed, results of the culture procedures and antimicrobial sensitivity testing for all samples analyzed in the context of the antimicrobial resistance surveillance programmes for Campylobacter in food animals during 2002-2018.

In Specific Objective 1, information on approximately 51,000 samples and approximately 11,000 Campylobacter isolates retrieved from those samples was analyzed across host and bacterial species. As expected, higher resistance levels were observed for C. coli compared with C. jejuni, and while very high levels of resistance to tetracycline, ciprofloxacin and nalidixic acid were observed regardless of host and bacterial species, a wider variability was observed between host species with regards to gentamicin, streptomycin and erythromycin.

When considering Specific Objective 2, a strong association between the occurrence of simultaneous resistance to aminoglycosides and erythromycin was observed for all hosts in both C. coli and C. jejuni. The analysis of relative synonymous codon usage in the sequence of the flaA gene, in a subset of 176 isolates selected based on their antimicrobial resistance profile, revealed the existence of clusters of isolates presenting co-resistant phenotypes, particularly for C. coli, what could be indicative of the presence of resistant lineages harboring specific resistance mechanisms.

In Specific Objective 3, 194 isolates selected to include representatives from both bacterial species, the four hosts, and a balance between co-resistant, single resistant or susceptible isolates with regards to their phenotype to aminoglycosides and macrolides was subject to whole genome sequencing to characterize their resistance determinants and the presence of mobile genetic elements potentially associated with these. Overall, resistance to aminoglycosides was attributed to the presence of between one and six out of a total of 10 resistance genes identified. In contrast, resistance to macrolides was mostly due to the presence of A2075G point mutations in the 23S rRNA gene of Campylobacter, with only four isolates carrying the erm(B) gene. Antimicrobial resistance genes were often linked to transposons, plasmids and insertion sequences such as IS1595(ISCo2). A higher number of resistance determinants were found among C. coli resistant isolates compared to C. jejuni, especially the ones retrieved from pigs. This animal species was also the hosts in which more isolates harboring antimicrobial resistance genes on plasmids were found. A multiple correspondence analysis revealed certain host-specific and resistance-specific genetic patterns.

According to the results of this thesis, the occurrence of co-resistance phenotypes in thermotolerant Campylobacter is most commonly due to the simultaneous carriage of antimicrobial resistance genes conferring resistance to aminoglycosides and point mutations for macrolides, which would be transmitted following different dynamics. The phenotypic analyses of antimicrobial resistance data from thermotolerant Campylobacter alone would be insufficient to identify differences in the nature and distribution of the resistance mechanisms involved, often linked with specific bacteria and animal hosts.