Facultad de Medicina

Plasmid-mediated antimicrobial resistance is one of the major threats to public health worldwide. The mechanisms involved in the plasmid/host coadaptation are still poorly characterized, and their understanding is crucial to comprehend the genesis and evolution of multidrug-resistant bacteria. With this purpose, we designed an experimental evolution using Haemophilus influenzae RdKW20 as the model strain carrying the ColE1-like plasmid pB1000. Five H. influenzae populations adapted previously to the culture conditions were transformed with pB1000 and subsequently evolved to compensate for the plasmid-associated fitness cost. Afterward, we performed an integrative multiomic analysis combining genomics, transcriptomics, and metabolomics to explore the molecular mechanisms involved in the compensatory evolution of the plasmid. Our results demonstrate that minimal modifications in the host are responsible for plasmid adaptation. Among all of them, the most enriched process was amino acid metabolism, especially those pathways related to serine, tryptophan, and arginine, eventually related to the genesis and resolution of plasmid dimers. Additional rearrangements occurred during the plasmid adaptation, such as an overexpression of the ribonucleotide reductases and metabolic modifications within specific membrane phospholipids. All these findings demonstrate that the plasmid compensation occurs through the combination of diverse host-mediated mechanisms, of which some are beyond genomic and transcriptomic modifications. IMPORTANCE The ability of bacteria to horizontally transfer genetic material has turned antimicrobial resistance into one of the major sanitary crises of the 21st century. Plasmid conjugation is considered the main mechanism responsible for the mobilization of resistance genes, and its understanding is crucial to tackle this crisis. It is generally accepted that the acquisition and maintenance of mobile genetic elements entail a fitness cost to its host, which is susceptible to be alleviated through a coadaptation process or compensatory evolution. Notwithstanding, despite recent major efforts, the underlying mechanisms involved in this adaptation remain poorly characterized. Analyzing the plasmid/host coadaptation from a multiomic perspective sheds light on the physiological processes involved in the compensation, providing a new understanding on the genesis and evolution of plasmid-mediated antimicrobial-resistant bacteria.

Haemophilus influenzae is a gram-negative bacterium of relevant clinical interest. H. influenzae Rd KW20 was the first organism to be sequenced and for which a genome-scale metabolic model (GEM) was developed. However, current H. influenzae GEMs are unable to capture several aspects of metabolome nature related to metabolite pools. To directly and comprehensively characterize the endometabolome of H. influenzae Rd KW20, we performed a multiplatform MS-based metabolomics approach combining LC-MS, GC-MS and CE-MS. We obtained direct evidence of 15-20% of the endometabolome present in current H. influenzae GEMs and showed that polar metabolite pools are interconnected through correlating metabolite islands. Notably, we obtained high-quality evidence of 18 metabolites not previously included in H. influenzae GEMs, including the antimicrobial metabolite cyclo(Leu-Pro). Additionally, we comprehensively characterized and evaluated the quantitative composition of the phospholipidome of H. influenzae, revealing that the fatty acyl chain composition is largely independent of the lipid class, as well as that the probability distribution of phospholipids is mostly related to the conditional probability distribution of individual acyl chains. This finding enabled us to provide a rationale for the observed phospholipid profiles and estimate the abundance of low-level species, permitting the expansion of the phospholipidome characterization through predictive probabilistic modelling.