PhD Thesis defense by Alberto Hipólito Carrillo de Albornoz at the Faculty of Veterinary Medicine of the Complutense University of Madrid

April 4^th, 2024

Antimicrobial resistance is one of the major global health concerns of the 21st century. The indiscriminate use of antibiotics, among other factors, has led to an exponential increase in antimicrobial- resistant bacteria in recent decades. It is estimated that every year between 1.5 and 5 million people die due to infections caused by multidrug-resistant bacteria. These data highlight the need to take urgent measures to tackle this threat, with a priority on understanding the forces that govern the success of multidrug-resistant bacteria.

The exceptional ability of bacteria to evolve and adapt to antimicrobial compounds is mainly promoted by horizontal gene transfer. A large number of resistance genes are transferred from one bacterium to another through mobile genetic elements and platforms such as plasmids, transposons, and integrons.

Integrons are genetic platforms capable of capturing and storing new genes embedded in small mobile genetic elements called cassettes. Integrons have a conserved structure, composed of a stable platform encoding the integrase gene, the Pc and Pint promoters, and the attI integration site; and a variable array of cassettes. The cassettes’ array is expressed from the Pc promoter following an expression gradient that maintains the cassettes furthest from the promoter silenced. Under stress conditions, the integrase is expressed, allowing to capture exogenous cassettes, as well as the excision and rearrangement of the cassettes present in the array. This rearrangement allows the expression of distal cassettes by relocating them in the first position of the array, via recombination between the attI site placed in the platform and the attC site of the cassette. Therefore, integrons act as low-cost bacterial memories capable of providing adaptation on-demand to their hosts.

Mobile integrons play a crucial role in the increase and spread of antimicrobial resistance, capable of capturing and expressing over 170 antimicrobial resistance genes against most clinically relevant antibiotics. Integrons are highly successful platforms, often found in clinical isolates of multidrug-resistant Gram-negative bacteria. Conversely, the prevalence of antimicrobial resistance cassettes varies widely, even among genes conferring the same antimicrobial resistance. This phenomenon suggests that there are other specific features governing the differential success of resistance cassettes other than the phenotype they confer.

This PhD thesis aims to characterize the forces governing the evolutionary success of resistance cassettes in integrons, as well as to delve into the current model of integron and its regulation. To achieve this, we have quantitate the resistance levels conferred by these cassettes, as well as the cost associated to their expression. Furthermore, we have explored the potential impact of a cassette on the expression of the subsequent collection and assessed their mobility, given their presence in this specific platform.

Despite the significant clinical relevance of this platform, our knowledge about their resistance genes is scattered in the literature, hindering a comparative analysis. To study the forces governing the success of these cassettes in a comparable manner, we have generated the pMBA collection, composed

of 136 resistance cassettes cloned in a genetic environment that mimics their natural context. This collection encompasses 76% of the resistance cassettes described in mobile integrons, being largest collection of resistance cassettes in integrons to date.

The characterisation of the resistance profile of each cassette has confirmed the already known specificity by gene families, while revealing significant differences in the resistance levels conferred by closely related genes. Additionally, we have identified genes that do not confer any resistance, challenging the role of qac and smr gene families as disinfectant resistance genes.

The quantification of the cost entailed by each cassette has revealed that certain families, such as beta-lactam resistance genes, pose a significant cost in E. coli. Conversely, certain genes, such as aacA7, have proven to be beneficial for bacterial fitness in the absence of selective pressure, both in vitro and in vivo. This suggests potential secondary metabolic roles of antimicrobial resistance cassettes in integrons. Furthermore, bacterial competitions conducted both in aerobic and anaerobic conditions have shown that resistance cassettes can drastically modify their effect on bacterial fitness depending on the oxygen availability in the environment, as observed in the case of ereA2. This fact is highly relevant for understanding the ecology of cassettes and their prevalence in clinical and environmental settings.

In addition to the characterisation of the resistance conferred by the cassettes and the cost they impose on their host, we have investigated the influence of a cassette on the subsequent collection and its recombination capacity in the platform. On one hand, we observe that the identity of a cassette can modulate the expression of the collection transcriptionally, adding a new level of complexity to the integron model. This has important implications for potential co-selection phenomena occurring in infections treated with various antibiotics. On the other hand, recombination frequencies obtained for each cassette are highly diverse, with differences of up to a million times between the recombination rates of aacA52 and fosN. Although we have not been able to correlate in silico parameters such as ΔG and pfold of the attC sites of the cassettes with their recombination rate in our experiments, we have found a correlation between cassettes conferring resistance to aminoglycosides and high recombination rates.

Additionally, during the course of this study, we have demonstrated that the aminoglycoside resistance genes present in integrons are not regulated by riboswitches, a topic of controversy in the field until now.

Although we are still far from a complete understanding of the differential success of each resistance cassette, this PhD thesis provides a comprehensive and quantified view of the forces governing the evolutionary success of resistance cassettes in integrons.