Cost and benefit of beta-lactam resistance in Streptococcus pneumoniae: interplay between the resistance determinants and the cell elongation/division components

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)

Okres sprawozdawczy: 2019-11-01 do 2021-10-31

Streptococcus pneumoniae (the pneumococcus) is one of the most important human pathogens that can cause a broad spectrum of diseases such as otitis media, pneumonia, bacteraemia and meningitis. Pneumococcal diseases lead to over a million deaths per year and especially the individuals with a compromised immune system are affected. Beta-lactam antibiotics, such as penicillin, have been successfully used for many decades to treat pneumococcal infections. However, penicillin-resistant S. pneumoniae strains, which are often resistant also to other classes of antibiotics, have increased dramatically since the 1980s and pose serious problems in the treatment of infections. This led the World Health Organization in 2017 to include S. pneumoniae on a list of priority pathogens for which research, discovery and development of new antibiotics is urgently needed. Despite the availability of antibiotic therapy and highly effective pneumococcal conjugate vaccines, which cover a minority of the 100 known pneumococcal serotypes, S. pneumoniae remains a critical clinical problem.
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.

During the StreptoMANIAC project, we performed detailed comparative genomic analyses using different approaches and thereby focusing on clinical isolates which belong to one specific clonal complex or are closely related to this lineage. The members of this complex are often multiple antibiotic-resistant strains and display different levels of resistance to beta-lactam antibiotics. Remarkably, one strain belonging to this clone is beta-lactam sensitive due to the absence of altered mosaic PBPs. We used this unique situation to study how development of beta-lactam resistance occurs and also searched for other non-PBP beta-lactam resistance determinants. Through genome comparison, we identified six large regions in the beta-lactam resistant strains where the mutations accumulated mostly and defined 36 genes in which the presence of mutations in the DNA also resulted in amino acid changes at the protein level. We further analysed their sequences and grouped these proteins either according to their function or according to the localization of their corresponding gene in the genome. This allowed us to identify other putative genes/proteins that are associated and may contribute to beta-lactam resistance.
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.

Historically, studies on antibiotic resistance in human pathogenic bacteria have always focused narrowly on the few determinants that are directly involved in the mechanisms of resistance and their clinical consequences. However, it was suspected even previously that in many cases there was a trade-off between a selective advantage in the presence of antibiotics versus a selective disadvantage in the absence of them. This somewhat contrasted with the emergence and spread of antibiotic resistant clones in the community.
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.

Genomic comparison between a β-lactam sensitive (top) and resistant (bottom) S. pneumoniae strains

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)

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