Christian G. Giske

Multidrug-resistant bacterial infections constitute a major challenge, resulting in significant morbidity and mortality. One of the cornerstones of the management of AMR infections is development of antimicrobials. In recent years several novel antimicrobials have been developed, but the emergence of resistance has been described, and novel approaches are needed to improve the efficacy of current treatment regimens, while preventing development of resistance. Bacteriophages are viruses that selectively target bacteria, causing lysis of bacterial cells. Phage therapy has emerged as a potential tool to mitigate the effects of AMR. Using phages in combination with antimicrobials is a largely unexplored although our evidence, and that of others suggests that phages can improve the efficacy of antibiotics.  However, there is limited knowledge about how to combine them, how this works in vivo and the impact of joint treatments on the immune system. Herein, we describe a project aimed at generating important information on how to optimally combine phages and antimicrobials by using several infection models – in silico, in vitro, ex vivo cell culture, patient-derived organoid models, and animal models. We will also investigate the emergence of phage resistance and development of antibodies in animals receiving long-term treatment with phages. This project explores the utility of phage-antimicrobial combinations in various infection models and will direct roadmaps for clinical use.

Patients with cystic fibrosis are at risk of developing infections caused by Pseudomonas aeruginosa. Normally, these infections are challenging to treat and require intravenous treatment for two weeks. Moreover, the patients frequently remain colonized with P. aeruginosa and with time the strains accumulate antimicrobial resistance mechanisms, thus compromising future treatment. Despite recent emergence of novel antimicrobials, development of resistance is observed already before the new antimicrobials are introduced on the market. Novel strategies are urgently needed for the management of these infections, and it has been proposed to combine traditional antimicrobials with bacteriophages, viruses that selectively infect and kill bacteria with very high specificity. Herein, we describe a multidisciplinary approach with isolation and characterization of bacteriophages and evaluation of their properties in lung organoid models, murine pneumonia models, and finally in a clinical trial where combination of bacteriophages and antimicrobials is compared to standard of care treatment. The infection models described herein simulate acute-on-chronic infections, and we will use multi-omics strategies to assess the immune responses. Phages that will be used in the project have largely been isolated and their detailed bioinformatical characterization is ongoing. Discussions are also ongoing with regulatory agencies on clinical trial design to allow for rapid clinical translation.

Antimicrobial resistance (AMR) is a major threat to global health. The situation is particularly alarming in Low- and Middle-Income Countries (LMICs), since easy-to-use tools for identifying AMR bacteria are lacking. The goal of this project is to develop and use two fluorescence microscope-based assays to detect carriage and infections of AMR bacteria. The project is a collaboration with the neonatal ward at Muhimbili National Hospital (MNH) in Dar es-Salaam, Tanzania. Tanzania is one of ca 85 LMICs where simple fluorescence microscopes are used to diagnose tuberculosis. We will repurpose these microscopes for AMR detection assays, which means that there is no cost for infrastructure. We will focus on neonates because they are a highly vulnerable population prone to acquire hospital infections. We believe that interventions based on this project will have immediate clinical impact on neonatal care. However, the methods developed are applicable in any hospital ward. The first assay is based on PNA-FISH and can be used to identify AMR bacteria in complex samples. We will focus on detecting K. pneumoniae and E. coli in fecal samples. The second is a genetic analysis that will identify resistance genes in the detected bacteria. The project includes assay development in Sweden, a screen for AMR bacteria at the neonatal ward at MNH, teaching of the assays to MNH staff, and implementation of the assays for infection control interventions in the neonatal ward at MNH.

Work with bacterial whole genome data and data analysis<br/><br/>NOResearch stay abroadThe work in 2024 has focused on the molecular epidemiological study of bloodstream infections with bacteria in the Bacteroides fragilis group. We have compiled clinical information and performed bioinformatic analyses on whole genome data from the bacteria. As previously mentioned, we have now sequenced the entire genome of 381 strains of bacteria in the Bacteroides fragilis group from bloodstream infections in the period 2008-2022 using IonTorrent technology. Additional sequencing with Oxford Nanopore technology of Bacteroides fragilis division 2, which has the highest incidence of resistance to meropenem and piperacillin-tazobactam, was completed in 2024. The work in 2024 has mainly consisted of bioinformatic analyses to process these data. The analyses that have been performed are:<br/>- Assembling the sequences into the most coherent genome possible (assembly)<br/>- Mapping of resistance genes<br/>- Mapping of virulence genes<br/>- Mapping of mobile genetic elements<br/>- Mapping of the degree of relatedness between the different isolates (phylogeny). We have tried several methods, and found that the one that gives the best results is core genome phylogeny using the programs Panaroo and RAxML. This means that the programs identify genes that are present in at least 99% of the bacteria (core genome), and use variation in these to map the degree of relatedness between the different strains. In this way, we have produced phylogenetic trees (family trees) of all the bacterial isolates, and can see whether there are special groups (clones) of bacterial strains that accumulate as the cause of infections within a given period of time.<br/><br/>Furthermore, clinical data has been extracted according to the project protocol, in order to be able to compare these with the microbiological findings. This allows us to gain an overview of whether certain patient characteristics (e.g. type of infection, or previous hospitalizations) give a higher risk of infection with resistant strains. We can also perform exploratory analyses to discover any previously undescribed associations between genes and resistance or virulence. Furthermore, we will map the occurrence of mobile genetic elements, in order to gain knowledge about how resistance genes can spread.<br/>