Periodic Reporting for period 2 - CARTNET (Combatting Antimicrobial Resistance Training Network)
Período documentado: 2020-01-01 hasta 2021-12-31
CARTNET has addressed the challenge of antimicrobial resistance in bacterial pathogens that can be transmitted between humans and animals including methicillin resistant Staphylococcus aureus (MRSA) and pathogenic E. coli. For these pathogens, resistance is becoming critical as less and less treatment options are available while at the same time multidrug resistance is becoming more frequent. In consequence society is increasingly needing options for antimicrobial therapy not just for treating human infections but also for reducing the pool of resistant bacterial pathogens in animals, such as livestock, from which antibiotic resistance may be transmitted. The project aimed to develop alternative solutions approaches and compounds to target antibiotic resistant pathogens and did so by investigating phage therapy to target pathogenic E. coli in poultry; by limiting the spread of phages needed by livestock MRSA for human infections; by developing new antimicrobials that target essential signal transduction systems and by exploring novel antimicrobials expressed from the environmental organisms. These objectives were addressed by structure guided improvement of small molecule inhibitors, by eliciting expression of silent antimicrobial gene clusters in genera of actinomycetes and based, by metagenomic data as well as screenings identify novel compounds in various habitats, by isolating phages to be used in phage therapy and by identifying intrinsic and transferable antibiotic resistance mechanisms that could guide identification of risk markers pointing to strains prone to resistance development.
CARTNET has taken several different routes to search for novel ways to treat infections with antibiotic resistant pathogens that can be transmitted between humans and animals. For development of compounds with specific targets, we have focused on the bacterial histidine kinases that are widespread among bacteria and are central in major signal transduction cascades of which several are essential under normal growth conditions. Using a combination of structure-guided and ligand-based techniques to design novel molecules against this group of targets more than a hundred compounds belonging to 4 different series have been synthesized and examined. Ultimately, we identified 3 novel series of histidine kinases binders and binding was validated as was the ability to inhibit catalytic activity. We also searched more broadly for antibacterial activities by eliciting the greatest possible number of antimicrobial compounds from actinomycetes. This work resulted in a tridecapeptide and the corresponding biosynthetic gene cluster was identified. As an alternative strategy to control resistance development, we have also predicted "risk" variants that in Staphylococcus aureus make a strain more prone to develop resistance to vancomycin. This has been accomplished by novel bioinformatic methods combining mutations and gene expression in strains evolved to vancomycin resistance. Also, the spread of resistance is being limited by studying the transfer of plasmids and bacteriophages between strains. This has resulted in the characterization of new transfer mechanisms such as the integration of phages into non-conserved bacterial integration sites and the development of new phages. Phages are also being exploited as antimicrobials and to this end we have demonstrated that lytic phages can be identified that targets pathogenic E. coli strains infecting poultry. These investigations highlight the diversity by which resistance can develop and they point to potential solutions. Collectively the research conducted in CARTNET shows that there are multiple paths to be taken to address bacteria resistance to antibiotics and that many of the approaches taken show promising results that may lead to new therapeutic options.
The overall objectives of CARNET have been to address antimicrobial resistance in human and animal pathogens by identifying new antimicrobials using structure guided improvement of small molecule inhibitors, by finding effective elicitors able to trigger expression of silent antimicrobial gene clusters in genera of actinomycetes , by metagenomic data analysis and activity based screenings of actinomycetes and other soil related organisms as well as by studying intrinsic and transferable antibiotic resistance mechanisms that can be targeted by novel compounds or bacteriophages. As part of CARTNET we have developed an extensive synthesis and screening platform for the development of two component sensor histidine kinase inhibitors that will be applicable not only when searching for antimicrobial activity but in general for manipulations of bacterial signal transduction pathways involved in controlling for example biosynthesis of important metabolites exploited within biotechnology. When studying intrinsic resistance and the development of decreased susceptibility to vancomycin our results show that it may be possible to determine the resistance development pattern based on the initial mutations present in a strain. This may be the case for development of resistance to other antimicrobials in other bacteria and could form the foundation for development of new diagnostic tools that could aid clinicians when choosing antimicrobial therapeutic strategies when knowing the exact genome sequence of a bacterium. The studies of interactions between bacteria and the bacterial viruses, phages have revealed that integration of phages in bacteria genomes promote evolution of phages subsequently leading to increased establishment in new bacterial hosts. Since phages often encode virulence factors such as toxins, phage integration enhances the risks associated with a pathogen. The results demonstrate a novel mode of interaction between phage and bacterium and will help to understand how other phages are able to establish in bacteria and potentially will also provide insight into how eukaryotic viruses interact with human cells.