Periodic Reporting for period 1 - StreptoMANIAC (Cost and benefit of beta-lactam resistance in Streptococcus pneumoniae: interplay between the resistance determinants and the cell elongation/division components)
Période du rapport: 2019-11-01 au 2021-10-31
Beta-lactam resistance in S. pneumoniae involves the modification of target enzymes for this class of antibiotics, the penicillin binding proteins (PBPs) as well as non-PBP components. In clinical isolates, three PBPs, namely PBP2x, PBP2b and PBP1a, are the main players in the development of beta-lactam resistance and display a so called "mosaic" structure, which is the result of interspecies gene transfer followed by recombination events. These altered PBPs have reduced affinity for beta-lactams while the enzyme function remains unaffected, giving a selective advantage for the resistant strains to grow in the presence of beta-lactams. In some penicillin-resistant S. pneumoniae clones, non-PBP determinants are also involved and contribute to the resistance phenotype. The main objectives of the StreptoMANIAC project were to study the interplay between the different beta-lactam resistance determinants, thereby focusing on the molecular mechanisms and on the physiological and biochemical consequences of acquired resistance in S. pneumoniae clinical isolates.
Furthermore, we elucidated the physiological and biochemical consequences of the acquisition of the main beta-lactam resistance determinants on cell growth and division, and evaluated the overall fitness of S. pneumoniae mutants carrying the altered mosaic PBPs. We examined if and how these PBPs affected growth, morphology and cell viability and performed the analysis of the peptidoglycan, the major component of the bacterial cell wall, in strains carrying different resistance determinants with and without antibiotics. This last aspect was never addressed before and the results provide new insights in the understanding how bacteria respond to antibiotic stress. We then focused on the interplay between the PBPs and the non-PBP resistance determinants, which also plays a role in pneumococcal resistance to beta-lactams, and determined the interaction profiles of different proteins using a bacterial two-hybrid system. In addition, using fluorescent microscopy, we performed localization studies of these determinant in live S. pneumoniae cells.
The results of the StreptoMANIAC project were discussed among the participants and the collaboration partners involved in the study and one part of the project was presented at the 48th virtual SIM2020 Congress “Antimicrobial resistance: the sustainable challenge” as selected short talk and poster. Participation and presentation of the results at other national or international meetings/conferences was not possible due to the ongoing COVID-19 pandemic and its global implications, as all scheduled meetings were cancelled or postponed, first to 2021 and then to 2022, without being converted online.
In the StreptoMANIAC project, we used the human pathogen S. pneumoniae and resistance to beta-lactams, which is paradigmatic in this respect. Using a genomic comparison, we studied, in a closely genetically related group of clinical isolates belonging to the same clonal complex, which changes occurred along with resistance and identified a set of genes/proteins that are candidates to be responsible for the selective trade-off. We then analysed the cell wall of some of these strains in the absence or in the presence of a beta-lactam antibiotic. Although the analysis is still ongoing and the final results will be available in the near future, the preliminary results are already very promising and represent a major step towards understanding the bacterial response to antibiotic stress. Another part of the project was dedicated to study the role of the non-PBP components and their impact in the physiology of S. pneumoniae. Taken together, the results provide a clearer picture underlining that successful and stable beta-lactams resistance to in S. pneumoniae involves more players than those already known, which can help to explain the different levels of resistance observed in different clinical isolates sharing a similar genetic background and resistance determinants.
The social and economic importance comes from the larger context in which the project is framed. Antibiotic-resistance is a global public health emergency, for which there are not easy short-term solutions. This project has been crucial to understand that the stages for stable development and spread of beta-lactam resistance in S. pneumoniae are much more complex than originally thought and requires changes other than the target enzymes. This provides new information regarding additional players in the process and would allow to extend this type of study also to other pathogenic bacteria.