Biology subjects

Calvo-Villamanan, A.

Publications and source records attributed to Calvo-Villamanan, A..

4 recordsLinked to original sources

bioRxiv2026

Coupling metabolic enhancement to plasmid spread enables programmable antimicrobial control

The rise of multidrug-resistant pathogens underscores the need for precise antimicrobial strategies that extend beyond conventional antibiotics. Conjugation-based approaches offer a powerful yet underexploited means of delivering targeted genetic interventions directly within microbial communities. In this work, we combined selective killing modules with rationally optimized conjugative vectors to target antibiotic-resistant pathogens and clinically relevant antimicrobial resistance plasmids. First, we engineered and validated toxin-intein modules, programmable cassettes that restrict toxic activity to highly specific regulatory contexts. Specifically, we developed and validated modules targeting Shigella spp., Salmonella enterica, and bacteria carrying the resistance plasmid pOXA-48, demonstrating a tunable system capable of selective activity at both the species and strain levels. To identify the most effective delivery platform, we compared mobilizable and conjugative systems and found that, in vitro, conjugative plasmids consistently outperformed mobilizable ones by approximately one order of magnitude. To further optimize delivery, we streamlined the broad-host-range plasmid RP4 and enhanced its functionality by incorporating either the metabolic fos locus, which confers a fitness advantage to cells carrying the delivery vehicle; a type IV pilus operon that promotes mating-pair stabilization and enables efficient conjugation in liquid environments; or both features combined. Using these engineered RP4 derivatives, we integrated the toxin-intein module targeting pOXA-48 and evaluated its performance in complex microbial communities. In this setting, the RP4 variant carrying both the fos locus and the type IV pilus operon effectively blocked the spread of pOXA-48. Together, this work advances the use of conjugative plasmids as robust and programmable platforms to combat antibiotic resistance and enable microbiome engineering. Beyond introducing highly specific antimicrobial modules and a new generation of optimized conjugative vectors, our results identify ecological competitiveness and plasmid transfer dynamics as critical determinants of the success of such interventions.

microbiology↗

bioRxiv2025

Plasmids promote antimicrobial resistance through Insertion Sequence-mediated gene inactivation

Antimicrobial Resistance (AMR) is a major threat to public health. Plasmids are mobile genetic elements that can rapidly spread across bacterial populations, promoting the dissemination of AMR genes in clinical bacteria. In addition, plasmids are enriched in insertion sequences (IS), which are small transposable elements able to translocate between genetic locations. Importantly, IS transpositions commonly lead to gene inactivation, which can in turn promote AMR (e.g. through the modification of the antibiotic target). In this study, we combined experimental, bioinformatic and computational approaches to investigate the role of plasmids as catalysts of AMR through IS-mediated gene inactivation. Our results revealed that plasmid pOXA-48, which encodes two IS1 elements, increases the rate of resistance acquisition to multiple antibiotics in clinical strains of Klebsiella pneumoniae through IS1-mediated gene disruption. Moreover, a large screen of genome databases confirmed that the inactivation of genes through plasmid-encoded IS elements is an extended mechanism of AMR evolution. Finally, both our experiments and computational model revealed that conjugative plasmids can promote this route of AMR acquisition while invading complex bacterial communities. Overall, our study reveals that conjugative plasmids fuel AMR not only through the dissemination of resistance genes, but also through IS-mediated gene inactivation, promoting the evolution of multidrug resistance in bacteria.

microbiology↗

bioRxiv2025

Dissecting pOXA-48 fitness effects in clinical enterobacteria using plasmid-wide CRISPRi screens

Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We designed and performed CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical enterobacteria. Our results revealed that pOXA-48 gene-level effects are conserved across clinical strains, and exposed the key role of the carbapenemase-encoding gene, blaOXA-48, as the main responsible for pOXA-48 fitness costs. Moreover, our results highlighted the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncovered new genes implicated in pOXA-48 stability. This study sheds new light on the biology and evolution of carbapenem resistant enterobacteria and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.

microbiology↗

bioRxiv2024

Plasmid-chromosome transcriptional crosstalk in multidrug resistant clinical enterobacteria

Conjugative plasmids promote the dissemination and evolution of antimicrobial resistance in bacterial pathogens. However, plasmid acquisition can produce physiological alterations in the bacterial host, leading to potential fitness costs that determine the clinical success of bacteria-plasmid associations. In this study, we used a transcriptomic approach to characterize the interactions between a globally disseminated carbapenem resistance plasmid, pOXA-48, and a diverse collection of multidrug resistant clinical enterobacteria. Although pOXA-48 produced mostly strain-specific transcriptional alterations, it also led to the common overexpression of a small chromosomal operon present in Klebsiella spp. and Citrobacter freundii strains. This operon included two genes coding for a pirin and an isochorismatase family proteins (pfp and ifp), and showed evidence of horizontal mobilization across Proteobacteria species. Combining genetic engineering, transcriptomics, and CRISPRi gene silencing, we showed that a pOXA-48-encoded LysR regulator is responsible for the plasmid-chromosome crosstalk. Crucially, the operon overexpression produced a fitness benefit in a pOXA-48-carrying K. pneumoniae clinical strain, suggesting that this crosstalk promotes the dissemination of carbapenem resistance in clinical settings.

microbiology↗