PhD Thesis defense by Carlos Serna Bernaldo at the Faculty of Veterinary Medicine of the Complutense University of Madrid

May 14^th, 2025

Aminoglycosides are broad-spectrum antibiotics that are essential for the treatment of infections caused by Gram-negative bacteria, both in human and veterinary medicine. However, in recent decades, their efficacy has been threatened by the increase in antibiotic resistance, mainly due to the action of 16S ribosomal RNA methyltransferases (16S-RMTases). These enzymes, identified in the early 2000s, confer high levels of resistance to multiple aminoglycosides by modifying their target, the ribosome. The emergence of these genes is concerning due to their frequent association with mobile genetic elements, which facilitates their transfer between different bacterial species. This accelerates their spread in clinical settings, both human and veterinary, especially when these elements also carry resistance genes to last-resort antibiotics, such as carbapenemases.

In this context, it is essential to better understand the mechanisms underlying the dissemination of these resistance genes. Therefore, the main objective of this thesis is to investigate how mobile genetic elements, such as plasmids, transposons, and integrative and conjugative elements (ICEs), contribute to the spread of 16S-RMTases. To this end, we conducted four studies based on the genomic and epidemiological analysis of these enzymes, primarily using large publicly available bacterial genomic datasets.

In the first study of this thesis, we performed a general analysis of the distribution and diversity of 16S-RMTases in publicly available bacterial genomes. We used the Blackwell genome collection, comprising 661,405 genomes from the European Nucleotide Archive (ENA) up to 2018. After screening these genomes, we identified 3,214 (0.48%) that contained at least one 16S-RMTase gene. We classified these enzymes into three groups based on their frequency of occurrence: high (armA, rmtB1, rmtC), moderate (rmtF1, rmtI2, rmtE1), and sporadic. The most frequent enzymes showed a global distribution with significant regional variations; for example, rmtB1 was concentrated in East Asia, particularly in China, while rmtC was more common in Europe. Additionally, we observed that these 16S-RMTases were predominantly found in bacteria of the Enterobacteriaceae family, but also in other bacterial genera, and notably in Gram-positive bacteria such as Clostridioides difficile.

In the second study, we conducted an in silico analysis of the global dissemination of 16S-RMTases mediated by plasmids in Klebsiella pneumoniae. For this, we compiled a dataset of 3,838 K. pneumoniae genomes carrying 16S-RMTases, obtained from public databases following a de-duplication and quality control process. We identified rmtB1 and armA as the most frequent 16S-RMTases in this bacterial species, distributed across several sequence types (ST), with ST11 being the most common. Our analyses suggest that the dissemination of these genes is primarily driven by plasmids that can be horizontally transferred rather than by clonal expansions. Specifically, the armA gene was mainly associated with pNDM-Mar, IncM2, and IncC plasmids, while rmtB1 was mostly found on IncFII(pHN7A8)/IncR and IncFII plasmids. We observed that the IncFII(pHN7A8)/IncR plasmids carrying rmtB1 were predominantly related to ST11 in East Asia, indicating successful clonal expansions in this region. Additionally, we detected that the IS26 insertion sequence frequently flanked both armA and rmtB1, suggesting its role in the mobilization of these genes.

In the third study, we investigated the multi-species dissemination of IncR plasmids carrying the armA gene in clinical and environmental bacterial populations from a veterinary hospital in Spain. Through whole-genome sequencing of isolates obtained between 2011 and 2020, we identified four strains of Enterobacter hormaechei subsp. xiangfangensis ST171 from equine clinical samples, all carrying the armA gene on an IncR plasmid. These plasmids showed high similarity to plasmids from a previous K. pneumoniae ST11 outbreak in the same hospital, suggesting possible horizontal plasmid transfer. Environmental sampling in 2022 revealed the presence of these IncR plasmids in several bacterial species, including E. hormaechei, K. pneumoniae, Citrobacter freundii, and Mixta calida, indicating multi-species and polyclonal dissemination in the hospital environment. Phylogenetic analysis confirmed that the environmental isolates were unrelated to the clinical ones, supporting the hypothesis of horizontal plasmid spread rather than clonal expansion. Additionally, conjugation experiments demonstrated that although IncR plasmids lack complete conjugation machinery, they can be mobilized through the conjugation systems of co-resident plasmids.

In the fourth study, we investigated the global dissemination of pan-aminoglycoside resistance mediated by one of the sporadic 16S-RMTases, npmA. We analyzed over 1.9 million bacterial genomes and identified 71 genomes carrying the npmA gene, mostly C. difficile (n = 69) and two clonal isolates of Enterococcus faecium. Most of these isolates carried the npmA2 variant and originated from humans, pigs, and the environment in six different countries. Phylogenetic analysis showed that npmA2 was primarily associated with the ST11 lineage of C. difficile, a lineage widely distributed in human and animal populations. We found that npmA2 was integrated into a novel composite transposon, named Tn7734, flanked by copies of a new IS30 element, which we named ISCld1. This transposon was located within an ICE of approximately 33 kilobase pairs (kbp), designated ICE Tn7740, which was conserved across multiple isolates and lineages of C. difficile. Additionally, the two E. faecium isolates carrying npmA2 also contained the same elements (Tn7734 and ICE Tn7740), suggesting horizontal transfer between bacterial species.

In summary, this thesis has demonstrated that although 16S-RMTases are globally infrequent, their ability to disseminate among diverse bacterial species and environments poses a significant threat. Plasmids such as IncR and IncF, along with other mobile genetic elements like IS26, Tn7734, and ICE Tn7740, play a key role in the mobilization and persistence of these aminoglycoside resistance genes. The identification of new hosts, including Gram-positive bacteria such as C. difficile and E. faecium, expands the understanding of the epidemiology of these resistance determinants.