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Antimicrobial resistance (AMR) is of paramount importance in the context of One Health, an integrated and unifying approach that aims to achieve a sustainable balance in the well-being of people, domestic and wild animals, plants, and their shared environments. Whenever bacteria become resistant to the therapeutic effects of antibiotics, they can cause infections that are difficult to treat effectively, increasing the risk of severe disease progression and death. Although AMR can develop naturally over time and is per se “ancient”, the excessive use of antibiotics in human and veterinary medicine over the past century has significantly accelerated its emergence and spread. Opportunistic Gram-negative enterobacteria, particularly Escherichia coli (E. coli ) and Klebsiella pneumoniae (K. pneumoniae) strains, increasingly exhibit resistance to multiple classes of clinically used antibiotics, thus presenting multidrug-resistant (MDR) phenotypes. To make matters worse, some of these strains combine multidrug resistance with high-level virulence, posing a threat to both immunocompromised and healthy individuals. Consequently, MDR E. coli and K. pneumoniae have been designated as high-risk pathogens by the World Health Organization, underscoring the urgent need for new antibiotic development.
This thesis is motivated by the fact that only a limited number of international high-risk clonal E. coli and K. pneumoniae lineages stand out across all One Health dimensions and dominate the broad pool of MDR enterobacteria. While we only know little about the underlying drivers and contributing factors impacting their occurrence, emergence, and adaptation across different ecologies, this thesis employs a diverse range of bioinformatics and phenotypic approaches to identify the key factors important for the success of these lineages, also in rather under-explored settings. It includes three main components: (i) the analysis of genomic survey data of MDR E. coli isolates from ecologies in sub-Saharan Africa, (ii) the application of functional genomics and phenotyping techniques to characterize bacterial virulence and assess its clinical relevance in a food-borne E. coli strain, and (iii) the investigation of evolutionary pathways that promote the development of resistance to a novel drug combination and exploring compensatory mechanisms in a K. pneumoniae strain. To achieve these objectives, this research integrates genomics and transcriptomics with molecular biology and functional studies encompassing a comprehensive set of in vitro and in vivo virulence and resilience assays to explore MDR bacteria in-depth.
We provide compelling evidence for the broad occurrence of successful high-risk clonal lineages in the One Health context and their circulation among clinics, wildlife, and food in international locations. In the first study, we isolated extended-spectrum β-lactamase (ESBL)-producing E. coli strains from houseflies collected from various wards at the University Teaching Hospital of Butare (Rwanda). In a follow-up study, we then examined in-depth the genomes of additional ESBL-producing E. coli from the same clinic and obtained from hospitalized patients, their caregivers, associated community members, and pets. The analyses revealed that the sample sets from this sub-Saharan African context consisted predominantly of globally recognized E. coli lineages, including sequence types (ST)131, ST167, ST410, and ST617. They play a pivotal role in the further dissemination and stabilization of AMR across diverse habitats within the One Health context. Moreover, our genomic results emphasize that these One Health-related high-risk clonal lineages exhibit the ability to successfully combine multidrug resistance with high-level bacterial virulence.
To gain a more detailed understanding of the sophisticated interplay of virulence and AMR, we developed and refined a set of in vitro and in vivo methods for virulence phenotyping. These methodologies enabled us to characterize pathogens based on crucial clinical aspects such as biofilm formation, siderophore secretion, resistance to complement-mediated killing, and their capacity to cause mortality in Galleria mellonella larvae. By using a food-borne E. coli strain from an internationally recognized high-risk clonal lineage, we verified the remarkable combination of a MDR phenotype with clinically significant virulence properties, including synthesis of curli fibers and cellulose as part of biofilm formation, extensive secretion of siderophores, resilience against complement-containing human serum and pronounced mortality in the infection model.
Nevertheless, the success of One Health-related high-risk clonal lineages does not rely solely on an “ideal” synergistic interplay between bacterial virulence and AMR. It also depends on their ability to rapidly mitigate the fitness costs associated with AMR acquisition, as these costs manifest in the form of reduced competitiveness and virulence in the absence of antibiotics. However, this is at odds with the observation of the global distribution of One Health-related high-risk clonal lineages across various One Health dimensions, even in environments with expectedly low selection pressures. To comprehensively address this, we conducted experimental evolution studies selecting for ceftazidime-avibactam-resistant mutants, which illuminated the rapid adaptations to changing environments. The adaptations and compensatory mechanisms were seemingly driven by major bacterial regulators, including the envelope stress response regulator RpoE on genomic and transcriptomic levels.
In conclusion, the results of this thesis shed light on the fundamental principles that govern the character and interplay between AMR and bacterial virulence and advance our understanding of the contributors and drivers of successful MDR international high-risk clonal lineages in the One Health context. This is also important for effective and alternative intervention strategies to prospectively further address the global threat of AMR.
The discovery of antibiotics around one century ago was a milestone for medicine. However, despite the warning of Alexander Fleming in 1945, antibiotics were used poorly, resulting in many antibiotic-resistant pathogens. Patients infected with resistant pathogens need to get treated with additional antibiotics or, as a last resort, trust completely on their immune system. This causes 700,000 deaths per year. Most clinically used antibiotics have been derived from soil microorganisms, while other niches stayed unexplored. Exploring new niches inhabiting antibiotic-producing microorganisms may result in novel antibiotics. Furthermore, expanding the search from frequently investigated soluble metabolites to volatiles may open up numerous compounds as potential future antibiotics. This thesis is about the search for antimicrobial volatiles produced (among others) by microorganisms from social spider ecosystems, a niche that was little explored until now.
Volatiles are characterized by their high vapor pressure at ambient temperatures, allowing them to distribute fast in both the gas and water phase. They can spread quickly even in complex ecosystems using the air and potentially fulfill functions like communication and antimicrobial defense. Especially, volatiles with antimicrobial activities caught the attention of many scientists because of their potential role in pathogen defense, as we have reviewed (Article I). Volatiles are usually produced in the primary metabolism and belong to diverse chemical classes, like hydrocarbons, aromates, alcohols, aldehydes, acids, esters, amides, and thiols. Their antimicrobial spectrum ranges from antifungal, to antibacterial, anti-oomycete, and even broad-spectrum activity. Volatiles are ubiquitously produced. Especially Bacillus and Streptomyces species are often reported to produce antimicrobial volatiles. Knowledge about antimicrobial volatiles – for example, details about their modes of action – is lacking yet, but these compounds may help to overcome the antimicrobial resistance crisis in the future. Volatiles could be used in medicine and agriculture, either alone or in combination with traditional antibiotics, opening new strategies against antimicrobial resistance.
A promising source of (volatile) antimicrobials is the ecosystem of social arthropods. Due to their lifestyle in dense colonies, they likely spread pathogens between individuals, making antimicrobial defense crucial. Since the presence of antimicrobial volatiles was reported in social insect ecosystems, we investigated the unexplored volatilome of the Namibian social spider Stegodyphus dumicola (Articles II and III). In the first study, we analyzed the in situ volatilomes of the spiders’ nest, web, and bodies using GC/Q-TOF and revealed that more than 40 % of the tentatively identified volatiles were already known for their antimicrobial activities (Article II). We proved the antimicrobial activity of five pure compounds found in the samples, among others against the suggested spider pathogen Bacillus thuringiensis. These results indicate the potential role of antimicrobial volatiles for pathogen defense and could ultimately help explain the spiders’ ecological success.
Volatiles from the spider volatilome can originate from various sources, including microorganisms, surrounding plants, the spiders themselves, the spiders’ prey, so we analyzed the volatilomes of microbial nest members in a second study. The microbial nest members we selected for this were the bacteria Massilia sp. IC2-278, Massilia sp. IC2-477, Sphingomonas sp. IC-11, and Streptomyces sp. IC-207, and the fungus Aureobasidium sp. CE_32 (Article III). Several volatilomes showed antibacterial and/or antifungal activities against two suggested spider pathogens. The subsequent volatilome analyses using GC/Q-TOF revealed the presence of many volatiles that have already been described as antimicrobials. Five pure volatiles were tested against two suggested spider pathogens, revealing all volatiles as antibacterial, antifungal, or both. These results support the potential role of antimicrobial volatiles in social spider pathogen defense and indicate microbial nest members as the origin of (novel) antimicrobial volatiles.
Together, the articles that constitute this thesis highlight the antimicrobial power of volatiles (Article I), indicates the volatilome of the ecosystem of S. dumicola as a potential pathogen defense (Article II), and finally reveal the spider nest microbiome as a source for antimicrobial volatiles (Article III). This knowledge not only adds to the understanding of social spider ecosystems (and likely other social arthropod ecosystems) but also has the potential to open a novel source for antimicrobial compounds that may help to counter the antimicrobial resistance crisis.