Escherichia coli infectionmechanisms and rapid detection of resistance to the association of ß-lactams with ß-lactamase inhibitors, and impact of the Sars-Cov-2 pandemic on the morbidity and mortality of intra-abdominal infection

Resumen

Infections by Escherichia coli in human beings produce a significant disease burden, with important morbidity and mortality, and the increasing resistance to antimicrobials is a challenge for choosing the more effective therapy. In this context, the present PhD Thesis is aimed to produce new knowledges applicable to the treatment of one of the more frequent and severe infections by E. coli¸ the intra-abdominal infections. Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among E. coli isolates. The first objective was to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analysed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). A higher copy number of blaTEM gene in P/T-R isolates was found, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. Currently, the detection of P/T-R relying on conventional methods is time-consuming. To overcome this issue, a cost-effective test based on MALDI-MS technology has been developed, which aims to detect P/T-R and extended-spectrum resistance to ß-lactam/ß-lactamase inhibitors (ESRI) in E. coli. Automated Clover MS Data Analysis software to analyse the protein profile spectra obtained by MALDI-MS from a collection of 248 E. coli isolates (91 P/T-resistant, 81 ESRI developers and 76 P/T-susceptible) has been used. This software allowed to preprocess all the spectra to build different peak matrices that were analysed by machine learning algorithms. The test can efficiently and rapidly (15 min) discriminate between P/T-resistant, ESRI developer and P/T-susceptible isolates and allowed the correct classification between ESRI developers from their isogenic resistance to P/T. Finally, the COVID-19 pandemic by the new SARS-CoV-2 arose new questions derived from the great challenge to the Health Systems and, specifically, in the case of the intra-abdominal infections, about of the impact of the pandemic on the management of them, and looking for data to also improve the clinical approach in these cases. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms. Overall, the data provided by this PhD thesis point to the importance of understanding the mechanisms of resistance acquisition and its early detection in order to have an impact on mortality and morbidity in severe infections.