The emergence of fungal infections caused by Candida albicans in immunocompromised people represents a major challenge for healthcare systems. This phenomenon is amplified by increasing resistance to antifungal treatments, particularly fluconazole, the first-line antifungal drug. These infections lead to systemic complications, with a significant increase in mortality rates up to 40% in the case of systemic infection. In addition, current methods of resistance detection in clinical microbiology laboratories are costly, time-consuming and require expertise. The aim of our study is to present a novel approach using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry in positive ion mode. This method could enable rapid and cost-effective detection of fluconazole resistance by analyzing lipid variations, focusing in particular on ergosterol, a biomarker exclusive to fungi and the biosynthesis of which is targeted by fluconazole.

The fluconazole treatment protocol was optimized to assess its impact on ergosterol synthesis, followed by a lipid extraction protocol to recover lipids from the samples. The lipid profiles were then analyzed by MALDI-TOF MS, paying particular attention to variations between fluconazole-sensitive and fluconazole-resistant strains. Finally, the mass spectrometry data was subjected to various quality controls before being statistically analyzed to interpret the nuances observed in the lipid profiles.

The first results show that the use of MALDI-TOF in positive ion mode with a fluconazole concentration of 4 µg/mL and a 2-hour incubation on sensitive and resistant clinical strains (DSY294 and DSY296 respectively) reveals significant differences in peak masses between fluconazole-treated and untreated strains. These results correlate with the fluconazole sensitivity and resistance of strain DSY294 and DSY296 respectively.

Preliminary research into the application of MALDI-TOF in positive ion mode shows promising potential for rapid and cost-effective detection of fluconazole resistance in Candida albicans, a crucial element in patient management.

Aims. To determine the geographical and evolutionary phylogeny of Staphylococcus aureus strains of clonal complex (CC) 398 as well as to identify mobile genetic elements (MGEs) carrying antimicrobial resistance and virulence genes.
Methods. The genomic data of an international collection of S. aureus CC 398 from diverse host origins spanning all continents and a period of 30 years were included. A time-calibrated phylogeny was reconstructed using BEAST2 based on a recombination-free core genome single nucleotide polymorphisms (cgSNPs) called using Snippy and Gubbins. Acquired mobile genetic elements were identified using a novel manual approach that we developed and termed “nested screening”.
Results. The time-calibrated phylogeny revealed the presence of distinct phylogroups present in Asia, North and South America and Europe. European methicillin-resistant S. aureus (MRSA) diverged from methicillin-susceptible S. aureus (MSSA) at the beginning of the 1950s. Two major European phylogroups (EP4 and EP5), which diverged approximately 1974, were identified as the main drivers of MRSA CC398 spread in Europe; specifically, an emergent lineage spreading among the European horse population (EP5-Leq), which also contains human-related strains. The major MRSA lineages EP5-Leq, characterized by staphylococcal cassette chromosome mec (SCCmec) IVa and spa type t011 (CC398-IVa-t011), and EP5-Lpg (prevalent among pigs) by CC398-SCCmecVc-t011 diverged approximately in 1996. The lineage-specific antibiotic resistance and virulence gene patterns were mostly mediated by the acquisition of MGEs such as the SCCmec, S. aureus Genomic Islands (SaGIs), prophages and transposons. Different combinations of virulence factors were identified on S. aureus pathogenicity islands (SaPIs), and novel antimicrobial resistance gene (ARG)-containing elements were detected in certain lineages expanding in Europe.
Conclusion. This WGS-based analysis revealed the actual evolutionary trajectory and epidemiological trend of the international MRSA CC398 population considering temporal, geographical and molecular factors and provided a baseline for future global WGS-based One-Health monitoring studies of the adaptive evolution of MRSA CC398 as well as for local outbreak investigations.

Aim
The dissemination of antimicrobial resistance, particularly carbapenem resistance, is predominantly attributed to plasmid transmission among bacterial cells, within the same or different species. In hospital settings, this phenomenon may contribute to outbreaks of multidrug-resistant pathogens. Unfortunately, epidemiological surveillance often focuses solely on bacteria and neglects plasmids. Long-read sequencing has made plasmid sequencing cheap, feasible, and reliable. However, the absence of a standardized plasmid typing methodology and varying accuracy levels based on study objectives remain challenges. This study aims to establish a methodology for delineating epidemiological links and plasmid transmission between strains, demonstrated through two carbapenem-resistant strain outbreaks in our hospital.

Method
The first dataset comprised 7 blaNDM-1 Klebsiella pneumoniae isolates from a single transmission cluster, while the second dataset involved 8 blaKPC-2 Serratia marcescens isolates from another transmission cluster. For both datasets, isolates originated from patients and from the environment (sinks traps). Additionally, 4 blaNDM-1 and 8 blaKPC-2 isolates (K. pneumoniae, S. marcescens and others) unrelated to these clusters and recovered during the same period were sequenced. All strains underwent Nanopore MinION sequencing with R10.4.1 chemistry, followed by assembly using Flye and resistance gene detection using ResFinder. Plasmid sequences were initially analyzed using mob-suite, and plasmid clusters were defined using MGE-cluster. These plasmids were further characterized using core SNPs analysis and accessory genome genes distance.

Results
Two plasmids were found circulating among blaNDM-1 K. pneumoniae strains and two plasmids harboring blaKPC-2 were also found among the S. marcescens outbreak isolates. At the plasmid level, both outbreaks could be extended, as corresponding plasmids were identified in isolates beyond the outbreak in genetically distant strains or in different species. Sink traps not only acted as reservoirs for pathogenic species but also housed a pool of different plasmids carrying the same antimicrobial resistances.

Conclusion
This study revealed the involvement of numerous plasmids associated with transmission clusters. This study underscores the applicability of this methodology in an epidemiological context, allowing the extension of transmission cluster definition to the plasmidic level.

Microtubules are composed of α and β tubulin heterodimers and are only found in eukaryotes. However, bacterial tubulins have been reported in the Prosthecobacter species (1,2) and odin tubulin in Odinarchaeota (3). Uniquely, transmission electron microscopy images have revealed bundles of eukaryote-like microtubule structures in epixenosomes, members of Verrucomicrobiota (4). Yet, it remains unknown whether epixenosomes encode tubulins that form these microtubule-like structures.
Here we report the presence of two tubulin-like genes (etubA and etubB) in the epixenosome genome which cluster with eukaryotic tubulins during phylogenetic analysis. Preliminary genome analysis suggests the absence of known tubulin chaperones necessary for the folding of eukaryotic tubulins. Nevertheless, attempts to heterologously express epixenosome tubulins (Etubs) in a bacterial system were unsuccessful. However, using a eukaryotic system, we have successfully expressed, purified and polymerised the Etubs into short curved protofilaments. Despite a wide screen of polymerisation conditions, we have not been able to reconstitute eukaryote-like microtubule structures in vitro but the Etub polymers do respond to taxol, a microtubule binding drug.
Currently, using single particle cryo electron microscopy, we are resolving a high-resolution structure of the Etub heterodimer to analyse its structure at molecular detail. Additionally, by combining cryo-focused ion beam milling and cryo electron tomography, we are developing a workflow to visualise the in situ architecture of Etubs in a near-native state.
Overall, our study shows the presence of the only reported eukaryote-like tubulins within bacteria and then aims to characterise them and any higher order structures they form both in vitro and in situ. Their level of similarity to eukaryotic tubulins suggests they are likely products of horizontal gene transfer but due to their eukaryote-like nature, it is puzzling how Etubs can be natively produced in bacteria.

Aims
Power-to-Methane (PtM) technology stands at the forefront as a promising solution for the storage of surplus renewable energy aligned with CO2 reduction. In the biological PtM process, methanogens utilize H2 generated from water electrolysis powered by renewable energy and CO2 from industrial waste gas to produce CH4, namely methanogenesis. Our work aims to enhance the robustness of PtM processes by optimizing this methanogenesis step, addressing challenges related to intermittent gas feeding.
Methods
The model strain Methanococcus maripaludis MM901 was cultured in batch cultures to study its starvation-revival dynamics under H2- vs. CO2- starvation by monitoring OD600nm and specific CH4 production rate. To obtain mechanistic understanding of the observed difference, we also (1) measured NAD+ and NADH in the cells during starvation using bioluminescent assays, as well as (2) performed oxygen exposure experiments, where cultures were exposed to air for 1h during starvation followed by monitoring their revival dynamics after O2 removal.
Results
M. maripaludis MM901 exhibited a shorter lag time and higher metabolic activity after H2 starvation compared to CO2 starvation, and an increase in starvation time amplified the difference. We hypothesize that cells experience a reductive stress under CO2 starvation. We measured NAD+ and NADH in cells during starvation and observed significantly higher NADH/NAD+ ratio in CO2 starved cells indicating accumulation of reducing equivalence. O2 exposure also exerts greater inhibition on the revival of CO2-starved cultures over H2-starved cultures shown by a significantly longer lag phase.
Conclusions
In CO2-starved cells where there is H2 leftover, it is possible that O2 reacts with accumulated reducing equivalence, resulting in the production of highly oxidative reactive oxygen species, and subsequently damaged enzymes and DNA in cells. To ensure the process stability and gas product quality, where H2 leftover is preferred over CO2 leftover, further efforts are needed to adapt methanogens to starvation under CO2 limitation.

Aim: B. bacteriovorus is a predatory bacterium capable of attacking and lysing E. coli regardless of its antibiotic resistance (AMR) profile. This predator is particularly interesting for its natural ability to detect and eliminate other Gram-negative bacteria. The aim of this study is to get a holistic overview of the metabolites present and released during the predatory life cycle of B. bacteriovorus on E. coli.

Methods: B. bacteriovorus and E. coli were cultured independently, at 29°C and 37°C with continuous shaking. Subsequently a predation experiment to follow the whole predatory life cycle was initiated. The samples were taken from B. bacteriovorus or E. coli separately, before mixing, and from the interaction of the two (lysis) at regular intervals throughout a six hours timeframe. Each collected sample (pellet or supernatant) was snap-frozen in liquid nitrogen and lyophilized, pellets were additionally resuspended in 80% methanol and dried down. Finally all samples were suspended in acetonitrile for analysis by liquid chromatography–mass spectrometry (LC/MS) to identify the metabolites. An untargeted metabolomics approach was chosen to identify the broadest range of compounds.

Results: A complete overview of the metabolites in the different stages of the predation process was obtained, which allowed to reveal the major metabolic pathways involved in the predation process. A total of 953 compounds were detectable from pellet samples by LC/MS analysis, 460 of which were identifiable based on the reference databases mzVault, myCloud and ChemSpider. The results from the predation experiments showed as most abundant categories, compounds belonging to: central metabolic pathways, secondary metabolites biosynthesis, cofactors, ABC transporters, nucleotide degradation, purine and pyrimidines metabolism.

Conclusion: This study is the first investigation on the overall metabolites present during the predation cycle of B. bacteriovorus. Our investigation elucidates the landscape of essential metabolic pathways that play a crucial role in the destruction of the prey and leading to the production of novel predatory cells. Further, it is paving the way to a deeper understanding of how these specific metabolites could aid in the fight against AMR bacteria.

Phage play a central role in influencing the structure, functional dynamics and evolutionary trajectory of ecosystems, with significant implications in diverse areas including human health, biotechnological applications, biogeochemical cycles and environmental remediation efforts. In the field of phage therapy, phage are typically used as biological agents to target and mitigate bacterial populations that have developed resistance to conventional antibiotics. It is widely believed that predatory phage to control microbial proliferation is generally assumed to not contribute to the spread of antibiotic resistance.
However, our research challenges this assumption by providing evidence that phage predation can create conditions conducive to the spread of antibiotic resistance, particularly in scenarios involving surface-associated microbial growth. We conducted experiments utilizing two strains of Escherichia coli– one serving as a donor of plasmid-encoded antibiotic resistance genes, and the other as a potential recipient. Our findings reveal that the presence of phage predation impedes the spatial segregation of these bacterial strains. This reduced segregation effectively increases the frequency of direct cell-to-cell interactions, thereby amplifying the rate and extent of plasmid-mediated transfer of antibiotic resistance. The primary mechanism driving this phenomenon is a shift induced by phage predation in the locus of maximum bacterial growth. Under predatory pressure, the peak growth area moves from the outer edges of the bacterial biomass towards its interior. This relocation to a more confined space restricts bacterial movement, resulting in the formation of straighter interfaces between the distinct bacterial strains. These straighter interfaces are less prone to merging with adjacent interfaces, which in turn slows down the process of spatial segregation and augments the likelihood of plasmid transfer.
In conclusion, our study reveals an important consequence of phage predation in the microbial environment: it can promote the spread of antibiotic resistance. This finding is particularly important in the context of phage therapy, suggesting a potential unintended consequence that must be carefully considered in its application.

In natural habitats, nutrient availability limits bacterial growth. We discovered that bacteria can acquire nutrients by lysing neighboring cells through contact-dependent antagonism. Specifically, the type VI secretion system increases the growth of antagonistic bacteria during starvation through the uptake of nutrients from lysed cells. Slow lysis, where cells leak nutrients over time before bursting, enhances the nutrient uptake by surrounding cells. The genomic adaptations in antagonists, which show a reduced metabolic gene repertoire, and the prevalence of the type VI secretion system in environmental bacteria from nutrient-limited environments like the global oceans, suggest that bacterial antagonism plays a significant role in nutrient transfer within microbial communities across various ecosystems.

In many natural environments, microorganisms decompose microscale resource patches made of complex organic matter. The growth and collapse of populations on these resource patches unfold within spatial ranges of a few hundred micrometers or less, making such microscale ecosystems hotspots of heterotrophic metabolism. Despite the potential importance of patch-level dynamics for the large-scale functioning of heterotrophic microbial communities, we have not yet been able to delineate the ecological processes that control natural populations at the microscale. Here, we address this challenge by characterizing the natural marine communities that assembled on over 1,000 individual microscale particles of chitin, the most abundant marine polysaccharide. Using low-template shotgun metagenomics and imaging, we find significant variation in microscale community composition despite the similarity in initial species pools across replicates. Chitin-degrading taxa that were rare in seawater established large populations on a subset of particles, resulting in a wide range of predicted chitinolytic abilities and biomass at the level of individual particles. We show, through a mathematical model, that this variability can be attributed to stochastic colonization and historical contingencies affecting the tempo of growth on particles. We find evidence that one biological process leading to such noisy growth across particles is differential predation by temperate bacteriophages of chitin-degrading strains, the keystone members of the community. Thus, initial stochasticity in assembly states on individual particles, amplified through ecological interactions, may have significant consequences for the diversity and functionality of systems of microscale patches.

The ability of bacteria to interact and exchange metabolites with one another is indispensable for their survival in changing environments. Previous studies suggest that such metabolic interactions only occur among cells within a very limited spatial range. These observations motivated us to explore mechanisms that can increase the spatial proximity of interacting cells, and, in this presentation, we will highlight two such critical mechanisms. First, spatial proximity can be maintained by direct cell-cell connection. We found that cells of dimorphic prosthecate bacteria (DPBs) use their stalks to establish direct connections to the cells of diverse ‘host’ organisms, including bacteria, archaea, eukaryotic algae, and fungi. Quantitative analyses through microfluidic experiments and nanoscale secondary ion mass spectrometry (nanoSIMS) indicated that direct cell-cell connections between DPB and ‘host’ cells facilitate metabolic exchanges, consequently improving the growth of the DPB cells. Second, cell motility could improve the spatial proximity of different bacterial populations. To test this hypothesis, we combined individual-based modeling and microfluidic experiments using synthetic consortia composed of motile or non-motile Pseudomonas aeruginosa strains that exchange metabolites for growth. We found that cell motility reduced the clustering of cells from the same lineage, fostering improved spatial proximity between interacting strains and thus facilitating the growth of the consortium. In summary, our findings shed light on the crucial roles of direct cell-cell connection and active cell motility in improving spatial proximity and therefore the metabolic exchange of interacting cells. These mechanisms offer novel perspectives into the survival strategies of microorganisms in ever-changing environments.

Microbes often live in surface-attached formations known as biofilms, where they form complex spatial patterns. Once established, these patterns entirely govern a cell’s local ecology, impacting its growth and evolution. A large body of literature addresses how metabolic interactions affect spatial patterns in microbial biofilms. However, most of these studies used strains of the same species that are identical in every aspect except for single mutations in a metabolic pathway. In natural, multispecies communities, patterns could also be affected by non-metabolic properties of species, such as their shape, friction with the surface, or motility, that could play a relevant role in the pattern formation process. Our aim is to understand to what extent we can understand spatial patterns in dual species biofilms from the knowledge of their metabolic interactions. We grew two-species colonies of Ochrobactrum anthropi (Oa) together with one of a collection of 28 bacterial interaction partners. Oa is auxotrophic for an intermediate of the Thiamine synthesis pathway. Therefore, if Thiamine is absent from the media, Oa can grow only if it is fed Thiamine (or a precursor) by the partner species. We typically observed that the partner species facilitates Oa if Thiamine is absent from the media and competes with it when Thiamine is added. We also observed that Thiamine cross feeding generally increases mixing of the two species, however, the absolute degree of mixing strongly differs between species pairs. We are currently investigating which features of the patterns can be predicted by knowing the metabolic interactions (cross-feeding or competition) as well as which other effects are important to explain them. We hope that our results will contribute to finding general principles for the process of spatial pattern formation.

Tripartite Motif Protein 28 (TRIM28) is a key host transcriptional regulatory protein and repressor of transposable elements (TEs). Previous research implicated influenza A virus (IAV) infection-induced loss of SUMO-modified TRIM28 in de-repression of immunostimulatory TEs and the subsequent augmentation of host antiviral immunity. We therefore hypothesised that other viruses may have evolved antagonistic mechanisms to control and counteract the TE-derived Antiviral Response (TEAR) if it is indeed a broadly important component of host defences. In this regard, a high-throughput proteomics screen suggested that the Measles virus innate immune antagonist protein, V, interacts with TRIM28, but the significance of this interaction and its function have yet to be explored.
Here, we show that the V proteins from 11 different clinically important paramyxoviruses, including Parainfluenza, Measles, and Nipah viruses, all interact with human TRIM28 in co-immunoprecipitation assays. Remarkably, TRIM28 co-precipitation efficiency with V differs between viruses, and using a chimeric protein approach we could map this property to the unique C-terminal domain of V, and ultimately to a single residue. At a functional level, using Parainfluenza viruses 2 and 5 (PIV2, PIV5) as model systems, we could also show that paramyxovirus infection induces the stress-response activated phosphorylation of TRIM28, similar to IAV infection. However, subsequent phospho-regulated loss of SUMO-modified TRIM28 only occurs with IAV infection, while SUMO-modified TRIM28 is maintained during paramyxovirus infection. The precise mechanism is unknown, but we could show that V proteins do not block deSUMOylation of TRIM28 caused by host SUMO-proteases (SENPs) or by XAF1, a factor recently linked to TRIM28-associated immune responses. Nevertheless, while IAV infection induces TE expression during infection, TE induction during paramyxovirus infection is minimal.
Given that viral co-infections can lead to more severe disease, we are currently assessing the interplay between parainfluenza viruses and IAVs with respect to the TEAR response and viral replication. The aim is to uncover whether potential suppression of TE-induction by co-infecting parainfluenza viruses might modulate the TEAR response to IAV and thereby impact pathogenicity.
Taken together, our results substantiate the emerging concept of the TEAR pathway, and highlight the first description of a potential viral antagonistic strategy.

Stress granules (SGs) are membraneless accumulations of ribonucleoproteins which form in the cytosol of eukaryotic cells upon exposure to a variety of stress stimuli. These granules are proposed to store untranslated mRNA during stress and support the dynamic reprogramming of translation towards stress resolving pathways. Growing evidence of many viruses specifically interfering with SG formation, and the identification of antiviral sensor and effector proteins in SGs suggests an involvement of these condensates in the antiviral host response. However, the characteristics of virus-induced SGs are poorly understood.
Our lab previously showed that SGs induced by the model coronavirus mouse hepatitis virus (MHV) exhibit a changed composition compared to canonical SG. To assess whether these differences are common for virus-induced SGs or rather virus-specific, we extended the characterization of virus-induced SGs to the Semliki Forest virus (SFV), serving as a reference. Microscopy-based investigations, including live-cell imaging, indirect immunofluorescence, and single molecule RNA fluorescence in-situ hybridization, revealed differences in SG dynamics during infection and the abundance of specific SG components between MHV and SFV. To comprehensively compare the SG protein composition, we further applied proximity labelling and quantitative proteomics at different time points of infection. These techniques provided insight into the SG proteome and the temporally resolved microenvironment of our SG bait protein revealing profound differences in the SG protein composition between MHV and SFV. Our results showed a reduced connection of MHV-induced SGs to canonical SG themes when compared to SFV-induced ones. As this observation is similar to our previous comparison between MHV-induced and canonical stress granules, the question arises if the firmer are SGs at all. Taken together, our findings revealed different aspects of these virus-induced granules to be strongly virus-specific, which might reflect different roles of the condensates during infection.
Our SG proteome screen provides a base for further investigations into the role of SGs during virus infection and the interplay between these condensates and different viruses.

To control pathogens, modern agriculture relies on pesticide treatments and breeding of resistant crop varieties. However, pesticides can be harmful to the environment and to human health, and pathogen populations can quickly develop resistance to pesticides and gain virulence on previously resistant varieties. Large scale real-time population genomics and molecular epidemiology studies can improve our understanding of pathogens epidemics and contribute to the development of novel control strategies. While these approaches are commonly used for research, diagnostic and monitoring of human infectious diseases, they are rarely and only partially applied to agricultural pathogens.

In this project we sampled the wheat pathogen Blumeria graminis f.sp. tritici for two consecutive years in Europe and in the Mediterranean region. We used whole genome sequencing to study its population genetics and molecular epidemiology. Overall we assembled a data-set of 416 samples collected from 20 different countries. We found that wheat powdery mildew does not constitute a single panmictic population over the studied region, with the major differences between Northern Europe, Southern Europe and the Middle East. The population in Northern Europe stood out because of it homogeneity over a lager geographic region, which was caused by higher rates of gene flow. We found that both geographic distance and climate shaped the genetic diversity of the pathogen and that the adaptation to tetraploid or hexaploid wheat might also play a role in the differentiation of different populations. In addition, genome scans for selection revealed that different fungicide targets and avirulence loci have been selected in different regions, reflecting heterogeneous agricultural practices and breeding programs. Finally, we investigated the spatio-temporal dynamics of the epidemic, including the direction and magnitude of dispersal, and the evolution and spread of mutations conferring resistance to fungicides or virulence onto wheat varieties containing resistance genes.

Aims
High throughput sequencing (HTS) has revolutionized biodiversity monitoring, enabling extensive data collection on various fungal genera without targeted sampling. Our aim was to test how monitoring data can be used to track soil-borne plant pathogenic taxa, gain information about their spatial and temporal trends, and identify the abiotic and biotic factors that may influence their distributions.

Methods
We compiled data on eight fungal genera (i.e., Armillaria, Fusarium, Gaeumannomyces, Heterbasidion, Paraphoma, Rhizoctonia, Sclerotinia, and Verticillium) that were selected because they include soil-borne plant pathogenic fungi with broad host ranges found in Switzerland. We investigated their occurrence at 30 sites included in the Swiss Soil Monitoring Network using amplicon sequence variants (ASVs) obtained from ITS-based metabarcoding. Samples were collected yearly (2012 to 2016) from ten sites of the land use types (LUTs) forest, permanent grassland, and arable land. The ASVs were analyzed in relation to their geographic location and LUT-preference. Category discrimination analyses were used to explore the associations among the presence of these taxa and the abiotic and biotic factors at each site.

Results
The eight selected pathogenic taxa included 2 to 110 ASVs, of which 44% displayed a widespread distribution and appeared in more than one LUT. Most of the ASVs shared among LUTs (65%) were found in both arable and grassland sites. Fusarium, Rhizoctonia, and Paraphoma were relatively more abundant at arable and grassland sites, and Verticillium was only found at arable and grassland sites. These genera showed negative associations with soil organic carbon, total carbon, and C:N ratio, but a positive association with bulk density. Armillaria was found exclusively in forest sites and showed the opposite trends in its associations with soil nutrients and density.

Conclusion
These analyses highlight how biodiversity monitoring can contribute information on plant pathogen ecology, including the habitat breadth and specificity of the selected genera. The spatial and temporal patterns of potentially pathogenic taxa derived from HTS analyses could aid in more targeted studies, and ultimately, contribute to the development and implementation of sustainable control strategies.

Many beneficial microorganisms contribute to plant resistance towards biotic and abiotic stresses. Recently, there has been growing evidence that plants are protected from diseases by their microbiome and the volatile organic compounds (VOCs) they emit could have a major role in this process.
Emission of VOCs is an important means of communication among microorganisms. These volatiles have various effects, they contribute to the stabilization of microbial communities, they can attract or repel different species, promote growth or display antimicrobial properties. Recently, several pieces of evidence have shown that the volatilome emitted by a microorganism depends on the volatiles of its surrounding. Thus, the volatilome of two different microbes grown together is different from the sum of the two individuals grown separately. These differences may include inhibition and promotion of various compounds, and the production of new compounds. However, the set up used so far to highlight these kinds of interactions has several drawbacks, including artificial volatile overaccumulation, potential oxygen limitation and the impossibility to assign a producer to the compounds newly emitted during the interaction.
To solve this problem, we have developed a solution enabling us to trap the whole volatilome of an organism and to use it to expose another one unilaterally. By combining this method with comparative Gas Chromatographic–Mass Spectrometry (GC-MS), it is thus possible to study the volatile-mediated interactions more precisely by identifying more easily the compounds responsible for the changes in the volatilome and the emitter of any newly produced compound.
We have used this new procedure to study volatile-mediated interactions between the biocontrol fungus Trichoderma simmonsii and the two plant pathogens Botrytis cinerea and Fusarium oxysporum. Our results show that the perception of each pathogen's volatilome triggered a specific response, resulting in significant changes in the VOCs emitted by Trichoderma. Trichoderma's volatilome modulation was overall stronger when exposed to the VOCs from Fusarium than to the VOCs from Botrytis, which correlated with increased siderophore production when co-incubated with this fungus. Our newly developed method will not only help to better understand volatile-mediated interactions in microbes but also to identify new molecules of interest that are induced by VOCs, as well as the putative inducing signals themselves.

Aims
Proteus mirabilis are Gram-negative bacteria that are found in the environment and also forms part of the commensal flora in the gastrointestinal tract of both humans and animals. This species does not harbour any intrinsic β-lactamase genes, explaining the natural full susceptibility to β-lactams. OXA-23 enzymes are carbapenem-hydrolyzing class D β-lactamases originating from Acinetobacter radioresistens and are the main source of acquired resistance to carbapenems in Acinetobacter baumannii but are rarely reported in Enterobacterales. Here, we report the emergence of OXA-23-producing P. mirabilis clinical isolates in Switzerland.
Methods
Between 2018 and 2023, nine non-duplicate P. mirabilis isolates were submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance for the investigation of antimicrobial resistance. Susceptibility testing was performed by disk testing and PCR analyses were used to detect the OXA-23 allele. Whole genome sequencing (WGS) was performed using the Illumina platform.
Results
Isolates were submitted from 6 laboratories, across six Swiss cantons, and all were obtained from urines. All isolates exhibited a similar phenotype with resistance to amoxicillin, amoxicillin-clavulanic acid, piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanic acid, and exhibited intermediate susceptibility to temocillin. WGS analyses showed that the isolates could be divided into two groups; group 1 (n=7) had snp differences ranging from 2-41 snps, and the isolates in group 2 (n=2) differed by just a single snp. Unsurprisingly, 3/4 isolates submitted from a single hospital differed by ≤6 snps, suggesting a dominant clone in this hospital environment. The blaOXA-23 gene was found to be located within a 41.2 kb genomic island, flanked by the IS15DII IS6-family transposon. Similarly that observed isolates from France, the blaOXA-23 gene was located within a transposon, Tn6704, which also contained the ISAba-type insertion sequences, ISAba1, ISAba125 and ISAba14.
Conclusions
This study identified the emergence of two closely related clones in Switzerland which were closely related to that previously identified in France, suggesting cross-border dissemination of OXA-23-producing P. mirabilis. The increasing identification of OXA-23-positive P. mirabilis in Switzerland is concerning since these strains may serve as a reservoir to aid the silent dissemination of the blaOXA-23 gene in other bacterial species.

Aims. The increase of multidrug resistance in Enterobacterales constitutes a major global health threat. Acquired resistance to broad spectrum -lactams is dominated by the emergence of broad-spectrum ß-lactamases (ESBLs) and carbapenemases. The rapid detection of these major antibiotic resistance mechanisms is critical to ensure a successful clinical outcome and to prevent their dissemination. Here, we developed a rapid biochemical test for a concomittant identification of ESBLs and carbapenemases in Enterobacterales.

Method. The rapid Combo ESBL/Carba NP test is a colorimetric test based on citrate-capped gold nanoparticles (AuNPs) aggregation in the presence of acid products resulting from cefotaxime or imipenem hydrolysis. The ESBL and the carbapenemase activity were evidenced by a color change of the AuNPs solution from red to purple, blue, or green in the presence of cefotaxime (reversed by tazobactam) and imipenem, respectively. A total of 76 characterized clinical enterobacterial isolates of worldwide origin were used to evaluate the test performance, among which 22 carbapenemase producers, 7 carbapenemase + ESBL, 29 ESBL, 3 ESBL + cephalosporinase, 8 cephalosporinase, 4 penicillinase and 3 wild type isolates.

Results. The sensitivity and specificity were found to be respectively 98.4% (95% CI 91.4% - 99.7%) and 92.9% (95% CI 68.5% - 98.7%). Of note, none of the cephalosporinase producers was detected as ESBL producers. All results were obtained within 60 minutes, with all ESBL detection within 30 minutes.

Conclusion. The rapid Combo ESBL/Carba NP test is rapid, highly sensitive, specific, easily interpretable, and easy to implement in routine. This is the first rapid phenotypic test providing a combined detection of all types of ESBL and carbapenemases, regardless of the gene types.

Numerous bacteria, including the opportunistic human pathogen Legionella pneumophila, respond to various stresses by entering a dormant state known as "viable but non-culturable" (VBNC). In this state, the cells display viability markers but cannot be grown on standard laboratory media, rendering them undetectable by conventional diagnostic tools reliant on cultivation.

L. pneumophila is a widespread bacterium found in fresh water sources, both natural and artificial, where it can infect and replicate inside amoeba and other protozoa. Utilizing mechanisms similar to those used in amoeba, L. pneumophila can infect and destroy human macrophages. Upon inhalation of pathogen-laden aerosols, L. pneumophila replicates within alveolar macrophages, leading to a severe pneumonia known as Legionnaires' disease.

This project investigates the induction of VBNC L. pneumophila through heat stress and their “resuscitation” upon encountering different amoeba species. Incubation of L. pneumophila at 60°C, 55°C, or 50°C for 30 min, 5 h, or 30 h, respectively, caused a complete loss of culturability. We found that this treatment rapidly inactivated the Icm/Dot type 4 secretion system (T4SS), and the inactivation prevented the resuscitation of heat-induced VBNC L. pneumophila by amoeba. Neither the formation of a Legionella-containing vacuole (LCV) nor intracellular replication was detected in A. castellanii, A. polyphaga or D. discoideum amoeba upon infection with VBNC L. pneumophila. Intriguingly, already the uptake rate of VBNC L. pneumophila was severely diminished in amoeba. Current research aims at characterizing bacterial and host factors involved in the VBNC state, with an emphasis on the pivotal role of ribosomes during dormancy/hibernation.

Aim: Whole-cell screening of a comprehensive chemical library containing 400’000 compounds for anti-mycobacterial activity revealed 9 promising drug lead candidates (1). The compound, abbreviated here as X1, piqued our interest, due to its effectiveness in the low μM range against internalized M. tuberculosis (MTB), without exhibiting any host cell toxicity. WGS analysis of spontaneous compound X1 resistant Mtb showed three independent SNP in gpsI a gene involved in mRNA metabolism. We aim to confirm gpsI as target of X1 and investigate its mode of action and mode of resistance.
Methods: We used biochemical methods to investigate the target interaction of X1. We cloned wild type gpsI and mutated gpsI in expression vectors and purified it. Poly(A) degradation and synthesis in presence and absence of compound was monitored in real time using Thioflavin T as RNA probe. Reaction parameters were assessed by Michaelis-Menten kinetic and bactericidal properties of X1 were determined by dose and time-dependent kill curves. Further, drug target interaction was investigated by Cryo-EM.
Results: Recombinant Mtb GpsI showed PNPase activity (RNA degradation and Synthesis). In presence of 4 μM compound X1, enzymatic activity was completely abolished. Titration of X1 showed dose dependant inhibition, half maximal inhibition was reached at 2 μM. Michaelis Menten Kinetic revealed that Vmax but not Km value was reduced, pointing towards a non-competitive inhibitor. Mutant enzymes showed complete and partial X1 resistance profiles. Bactericidal activity of X1 against MTB was investigated, revealing that X1 kills 99% of MTB bacilli within 48 hours at concentration of 4.4 mg/L. The homotrimeric core structure of GpsI-X1 complex was resolved by Cryo-EM at resolution of 2A. X1 binds to the phosphate binding site, forming two H-bonds and several weak interactions with GpsI.
Conclusion: GpsI is a functional PNPase. Compound X1 targets and blocks GpsI activity in a dose dependant matter. Mutant enzymes show similar in vitro activity as wt GpsI and exhibit different resilience profile (half to full tolerant) to inhibitor X1. X1 possesses potent bactericidal properties, killing 99% of bacteria within 3 days. X1 binds to the phosphate binding site, stabilizes RNA and thereby blocks GpsI activity.

Urinary tract infections (UTI) are one of the most common human acquired and recurring infections. With a recurrence rate of 30% within the first 6 months, UTIs lead to high antibiotic prescription, which in turn exacerbates the current antimicrobial resistant crisis.
Existing research models investigating UTI are murine in vivo models or in vitro models based on cancer cell lines. These do not accurately reflect many of the human bladder’s features including physiology, immunity and the tissue’s structure, making it difficult to effectively develop new adequate therapies and study the conditions that uropathogens face.
Here, we employ the newly developed 3D-UHU microtissue model (PMID:37939183), recapitulating key human bladder features, including full stratification into 7 cell layers, differentiation into three urothelial cell subtypes and urine tolerance. This model allowed us to test the bladder infection strategies employed by uropathogenic Pseudomonas aeruginosa (PA). We showed that PA preferentially forms biofilms in this human-like microenvironment. Aiming at classifying the heterogeneity underlying PA biofilms, we monitored their ability to form biofilms, while infecting our bladder model with 8 strains isolated from the University Hospital Basel. Additionally, we are investigating the effectiveness of two novel anti-biofilm molecules in combination with standard-of-care antibiotics to clear PA infection. Preliminary tests at high concentrations of the anti-biofilm agents (100µM) have proven a strong effect in biofilm disruption.
Overall, this combinatorial approach will pave the way for a better understanding of P. aeruginosa pathophysiology in UTIs, as well as for the development of a more effective course of treatment.

Understanding complex systems dynamics entails the discerning of the underlying mechanisms governing interactions between species and with their environment. Here, we aim to infer the mechanisms that drive community dynamics using high-frequency time series data and an established equation-free modelling framework that addresses the complexity of high-dimensional interconnected systems. To reach this goal we are using a natural planktonic community as our study system and focus on understanding how changing interactions are linked to the emergence of cyanobacterial blooms. We conduct in situ investigations of cyanobacteria and their growth rate within the plankton community of primary producers and consumers, utilizing daily observational data spanning 5 years. Our focus lies on identifying the key drivers governing cyanobacteria growth rates, comparing responses across four different blooming taxa and study how interactions affect bloom initiation. To accomplish that we analyze daily cyanobacterial abundance data from Lake Greifensee, a eutrophic Swiss lake, calculate growth rates (cell divisions minus losses), and reconstruct the attractor manifolds of the system to estimate the magnitude and sign of interactions between species and biotic / abiotic factors in state space using locally weighted multivariate linear regressions. These factors include nutrient supply, physical parameters of the water column, meteorological conditions, grazer abundance, and competing phytoplankton densities, all recorded in situ. Our analysis reveals the complex interactions of biotic and abiotic environmental factors that facilitate cyanobacterial blooming. This study highlights a knowledge gap: the importance of biotic interactions (e.g. grazing, competition, facilitation) in cyanobacterial bloom initiation.

Dispersal processes shape aquatic microbial communities, but their respective role is difficult to disentangle in field studies. While dispersal limitation enhances colonization by random taxa, the dissimilarity among newly formed communities is reduced by high dispersal rates (mass effect). By contrast, the subsequent development of such communities will mainly be determined by biotic interactions. To disentangle the importance of these factors, we studied compositional and functional changes of freshwater bacteria in 20 parallel experimental microcosm communities during contrasting dispersal regimes. The Elo-rating, an index used in competitive chess was applied to assess the success of bacterial taxa during the incubations. Dispersal limitation generated high beta diversity and the emergence of replicate community ‘types’ dominated by Acidovorax, Pseudomonas, or Aeromonas genera. Compositional stability and evenness of these types varied over subsequent growth cycles, reflecting differences in functional properties. Overall, carbon use efficiency increased during cultivation, with some communities of unique composition outperforming the dominant ‘types’. Homogenizing dispersal led to high compositional similarity and reduced gamma diversity. While neutral and competitive models (Elo-rating) together largely explained community assembly, Pseudomonas outperformed predictions, possibly due to exclusive genomic traits. Thus, high beta-diversity caused by initial dispersal limitation can subsequently be ‘purified’ into community types by deterministic processes. However, dispersal limitation may represent an insurance for highly productive microbial taxa that are competitively excluded during community coalescence.

Farming of phytoplankton (both eukaryotic microalgae and cyanobacteria) has the potential to revolutionize agriculture. Algae fix carbon into proteins, lipids, and other biopolymers at faster rates than any other autotrophs on the planet. The ancient association between algae and heterotrophic bacteria is strong yet nuanced, and its implications are only beginning to be understood. Despite the ubiquity of algae-bacteria interactions, commercial scale cultivation of algae and bacteria has tended to focus on each group in isolation. My work at Eawag makes links between algal taxonomy, algal exometabolites, and bacterial communities, with the goal of making fundamental insights that can inform industrial cultivation of microbes for sustainable bioproducts. I will outline ongoing work to characterize algal exometabolomes using a broad high-throughput screening, as well as our exploratory work to characterize the microbiomes of these 84 algae strains under investigation and a targeted survey for genes related to bioplastic production by microbes. A more holistic overview of how mineralized carbon flows into algal and bacterial biomass will help us more effectively and economically grow such microbes at the massive scales required to address pressing issues at the climate-energy-water nexus.

The Arctic has been warming at an unprecedented rate. In response, Arctic plants are experiencing prolonged growth seasons and higher productivity. On the global scale, the plant response to these changes is commonly referred to as “Arctic Greening” but above ground changes are not homogeneous. While soil microorganisms are known to play an important role in many ecosystems, their connections to the above ground vegetation in the greening Arctic is largely unknown. To evaluate the role that microorganisms play in Arctic Greening, we sampled and analysed different sites on Svalbard. These sample sites consist of (i) native tundra soils that are characterized by low nutrient input, (ii) soils around bird cliffs that are amended with nutrients such as nitrogen by bird droppings, and (iii) soils with influence and disturbance from humans and agriculture. 31 topsoil samples from the three site types were sequenced for 16S rRNA and a quantitative PCR of 16S rRNA and ITS was performed. This study shows that these three high Arctic tundra ecosystems have specific impacts on the soil microbial communities. Anthropogenic disturbance leads to a microbial community with more cells, that is lower in diversity and that has a very steep distance decay of community similarity. This is due to a few dominant groups such as Clostridia and Bacteroidia and the phylum of Desulfobacterota profiting from high nutrient input and outcompeting other community members. Soils surrounding bird cliffs have a higher abundance of fungi over procaryotes, a significantly more diverse soil community and very slow distance decay. This is likely due to less selective pressures and more dispersing effects caused by the spread bird droppings, the large water flow in the beginning of the growing season and potentially by fungi. Important biological above ground associations with community composition are the graminoids that are strong drivers of greening observed in sites (ii) and (iii) and the dwarf shrubs that were dominant in (i). Below ground parameters affected by climate change such as the soil moisture and deep soil temperature also associated with the communities, while signs of exogenous input such as total nitrogen, total organic carbon and manganese correlated as well. A more comprehensive understanding of the biogeographical processes that influence these microbes is crucial for clarifying their role in Arctic greening and predicting how this will evolve with climate change.

1. Introduction
Coronaviruses are a family of enveloped spherical viruses, with a monopartite linear ssRNA+ genome. They have been described to cause from mild to lethal infections in mammals and birds, and more recently have been put at the forefront of global attention after the COVID-19 pandemic. Mouse Hepatitis Virus (MHV), similar to SARS-CoV and MERS-CoV, is classified within the Betacoronavirus genus, thus it provides a model system to study coronavirus tropism, pathogenicity and lifecycle.

2. Aims
This project is focused on studying coronavirus recombination, a process thought to be a driver of coronaviruses spillover, emergence and evolution. Despite its important role, it is poorly understood. Additionally, we want to investigate the Replicase-Transcriptase Complex (RTC) organization and environment.

3. Materials & methods
We are studying coronavirus recombination via two different strategies. First, we produced fluorescent-labelled viruses by TAR cloning technology to visualize their localization within the infected cells. We plan to combine this method with High Resolution Microscopy to enhance the visualization.
And second, by Cryo Electron Microscopy (CryoEM) we plan to investigate what happens in the cells upon coinfection with two different viruses.

4. Results
Preliminary data has shown overlap of fluorescence signal from two differently labelled viruses in the same areas of the cell, thus suggesting viral replication of two different viruses in close proximity. Currently, we are at the stage of optimizing the methods to combine these results with High Resolution Microscopy. Additionally, to improve our resolution, with CryoEM we are working on obtaining tomograms of the Double Membrane Vesicles (DMVs) in single infection and coinfection settings during a MHV infection.

5. Conclusion
The results of this project will shed light on coronavirus co-infection and recombination. Understanding this complex mechanism is crucial for deepening our knowledge and understanding on virus infections, as well as developing diagnostic methods and therapeutics for both humans and animals for future pandemics prevention.

Enteroviruses are a group of single-stranded RNA virus that are associated with multiple diseases in humans and animals. Their transmission relies on their ability to persist in the environment. In aquatic environments, multiple abiotic and biotic factors contribute to viral inactivation. While abiotic factors such as temperature, pH and UV-light exposure have been studied, biotic factors remain uncharacterized. Our lab has previously shown that lake water microbial communities reduce the infectivity of Echovirus-11 (E11) and Coxsackievirus-A9 (CVA9), and that this reduction is associated with the production of bacterial proteases. Here, we aim to further characterize these bacterial communities and their role in viral inactivation. We have developed a pipeline to isolate and grow bacterial communities from Lake Geneva using chemostats. Chemostats are open culture systems with a continuous inflow of nutrients and removal of microbial metabolic waste. This culture system allows bacterial cultures to be maintained in exponential growth phase for an extended period of time. Once the bacterial communities are established, we will characterize their viral inactivation capacity, and their community structure and metabolism. To characterize bacterial capacity to inactivate enteroviruses, E11 and CVA9 will be inoculated into the chemostat communities and viral infectivity will be monitored over time. In addition, to characterize our lake communities and their metabolism, a combination of “-omics” and biochemical approaches will be used. Metagenomics will be used to infer the taxonomic profiles of the communities, metatranscriptomics their gene expression profiles, and metabolomics their metabolite production. Data analysis will be focused on identifying bacterial taxa and metabolic processes responsible for viral inactivation in the chemostat communities. All in all, our research provides a foundation to better understand the fate of enteroviruses in the environment and their interaction with environmental microbial communities.

Aims: Lignocellulosic biomass and its associated wastes are available sources of carbon and energy for microorganisms. These materials are however complex to be degraded into useful molecules, requiring physical or chemical pre-treatments before an enzymatic treatment. After a final detoxification step, the generated building blocks (glucose, xylose, etc.) may become suitable substrates for microorganisms to produce added-value products. Here a microorganism, Trichoderma reesei was coupled with at least another microorganism strain to produce an intracellular biopolymer called poly(3-hydroxyalkanoate) (PHA) using solely cardboard as substrate.

Methods: A minimal medium was designed for supporting the growth of T. reesei and the following strains: Pseudomonas putida, Wickeramomyces anomalus, and an isolate from a composting unit. Cardboard was used as only carbon and energy source. Shake flasks tests were conducted for each microorganism alone and for combinations of microorganisms (synthetic consortia). A benchtop bioreactor culture was conducted with T. reesei and P. putida for evaluating the feasibility of such a process at the larger scale.

Results: Shake flask tests showed growth for all cultures in presence of T. reesei and no growth in its absence, showing that its presence was essential for usage of carbon from the cardboard. P. putida accumulated low amounts of PHA (4.18 wt% of the total dry cell weight). Enzymatic tests evidenced the presence of several enzymes, endo- and exocellulases, beta-glucosidase, and endoxylanase, in the medium. In the bioreactor, T. reesei grew well (µ = 0.1 h-1), but P. putida was not able to grow or to significantly accumulate PHA, probably due to an unsuited pH. The yield Y(X/C) for T. reesei was determined to be 0.33 g/g.

Conclusion: A synthetic consortium based on two microorganisms, a medium, and growth conditions were designed and evaluated for use of lignocellulosic materials such as cardboard to produce PHA. The product was indeed obtained in a reproducible manner, indicating the feasibility of the proposed concept. Based on these preliminary results, further efforts will be conducted on the optimization of the PHA productivity. This involves a careful balance bewteen the growth of T. reesei and the PHA production.

The Burgundy truffle is an abundant ectomycorrhizal symbiont that occurs in a wide range of temperate climates. Despite its prized underground ascocarps, its complex lifecycle and possible response to anthropogenic climate change are largely known.We ran a citizen science monitoring of Burgundy truffle ascocarp production with a 3-week resolution in 23 natural populations at the center of its European distribution over up to 11 years. We genotyped more than 3000 truffles using microsatellite markers to assess the genetic structure and diversity of these populations in space and time. Preliminary results support well-differentiated genetic groups on a small spatial scale and throughout the study area, with often low levels of individual admixture. Within populations, genotypes from different genetic groups co-occurred, while only a few perennial genotypes dominated, producing most of the ascocarps. Further analysis indicate that genetic diversity was generally low in most populations and fluctuated over time with unpredictable trend. Our data suggest that among-population gene flow in Burgundy truffles is limited, even if they grow only a few kilometers apart. This restricted dispersal ability could prevent the northward migration of advantageous alleles from more heat- and drought-tolerant southern populations.
Conclusion – Given that ascocarp production in truffle populations is highly sensitive to dry conditions, our data call for genomic studies on local adaptation of this valuable ectomycorrhizal fungus, which could guide the use of well-adapted provenances for truffle cultivation and restoration.

Aims
Soil fungal communiUes associated with trees play an essenUal role in forest ecosystems by
providing water, nutrients, and protecUon against bioUc and abioUc stress. However, it
remains unclear how these communiUes respond to drought stress when associated with their
host forest tree species.

Methods
At six sites across Switzerland severely affected by drought condiUons, following a water stress
gradient, and well characterized environmentally over the past 10 years, we tracked the fungal
communiUes associated with healthy and decaying beech root systems using DNA
metabarcoding.

Results
Fungal richness and diversity correlated posiUvely with an increasing water stress and slightly
between beech health status. Fungal communiUes were dominated by ectomycorrhizal (EcM)
and saprotrophic fungi, and their relaUve abundance was altered by beech health status but
not by drought. The EcM fungi Lactarius was significantly associated with drier sites and
healthy beech root systems, while saprotrophic fungi were mainly associated with decaying
beech. Fungal community assemblies were more determinisUc in decaying beech than in
healthy beech root systems. Specifically, the stochasUcity of fungal communiUes’ assembly
increased with a rising water stress only for healthy beech, and was mainly driven by
saprotrophs.

Conclusions
Drought-related condiUons rather than other environmental parameters and health status of
the beech trees were the major drivers of fungal communiUes, even though their assembly
processes differed between fungal funcUonal traits. This study highlights the importance of
drought condiUons for the assembly of fungal communiUes. It also points to an interplay
between fungal funcUonal traits and host health status when facing environmental
constraints.

Key words
Water stress, fungal community, stochasUcity, EcM, Lactarius

Aims
Only five genomes of E. devriesei strains from different human, animal, and environmental sources are available at the contig or scaffold levels on the NCBI database. Here, we describe the first complete genome of an E. devriesei from larvae.

Methods
E. devriesei strain Ed-CK-24 was isolated from homogenized tissues of Z. morio larvae, which we have been using to establish a new in vivo model of gut colonization (https://data.snf.ch/grants/grant/206400, https://doi.org/10.3389/fmicb.2024.1381051). Species identification (ID) was achieved using the MALDI-TOF MS. The strain was sequenced and assembled using a combined short-read (Illumina) and long-read (Nanopore) sequencing approach to obtain the complete de novo genome. Species ID was confirmed using JSpeciesWS and OrthoANIu software. Antimicrobial susceptibility tests (ASTs) were performed using a microdilution MIC system and antimicrobial resistance genes (ARGs) were identified with AMRFinder and CARD-RGI. Phylogenetic analyses based on the 16S rRNA gene sequence and a core-genome alignment were also performed.
Results
The genome of Ed-CK-24 consisted of one circular chromosome (2,777,134-bp), one circular megaplasmid (633,497-bp) and one circular replicon-type plasmid (57,656-bp). The average coverage was 761.6x and the GC content 40.2%. Ed-CK-24 showed 97.4% (JSpeciesWS) and 97.9% (OrthoANIu) similarity with the E. devriesei type strain DSM22802 (Bovine, Belgium, 2016; GenBank: GCA_001885905.1). 16S rRNA phylogenetic analyses revealed that Ed-CK-24 shared 100% homology with other deposited E. devriesei strains. In addition, core-genome analysis revealed that Ed-CK-24 was most closely-related to strain ERR9969252 (Human, Australia, 2010; GCA_963528665.1) and to strain CTOTU50461 (Environment, Germany, 2013; GCA_032102565.1). ASTs indicated resistance to cephalosporins (intrinsic resistance), clindamycin, fusidate, tiamulin and sulfamethoxazole. Tiamulin and clindamycin were associated with the presence of the lincosamide-pleuromutilin resistance gene lsa(A) on the megaplasmid. The virulence gene bpsC was identified on the chromosome.

Conclusion
This study provided the first complete genome of an E. devriesei isolated from Z. morio larvae and identified antimicrobial resistance and virulence genes. These findings contribute to expanding our knowledge regarding this rarely isolated Enterococcus species and suggest that the Ed-CK-24 genome may serve as a reference for future studies with this specie

Aims
Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms).
Methods
Campylobacter jejuni (n=127) and Campylobacter coli (n=28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB).
Results
A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems.
Conclusion
The high rate of genetic diversity observed amongst broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown

The life cycle of Chlamydiae includes a biphasic developmental sequence. Additionally, in response to stressors such as IFN-γ, temperature changes or iron deprivation, the bacteria can survive in an aberrant state. This persistent, non-proliferative state is reversible, however, the precise mechanisms leading to the transition from replicative, reticulate bodies (RBs) to aberrant bodies (ABs) have not been characterised. By comparing the transcriptomes of RBs and ABs, we have identified differentially expressed genes that were predicted to be part of a two-component regulatory system (TCS). TCSs are predominant approaches used by bacteria to sense and respond to their environment.

AB formation was induced in Chlamydia trachomatis and Waddlia chondrophila infected cells by depleting iron using the chelator 2,2’-bipyridyl (BPDL). To study reversion to RBs, the stressor was removed. RT-qPCR was performed to quantify RNA levels. Expression of the TCS candidate genes were compared between RBs and ABs. In parallel, immunofluorescence microscopy was carried out to compare gene expression to morphological changes associated with ABs. Similar experiments are currently being performed on other stress stimuli.

Results indicate that the two examined TCS predicted genes, are strongly down-regulated in ABs compared to RBs. After 8 hours following BPDL removal, expression levels returned to those found in control infections. Surprisingly, upregulation in gene expression was observed 16 hours prior to morphological changes by microscopy. Downregulation in gene expression was not observed in ABs induced by other stress stimuli, indicating a relationship between iron and the TCS in Chlamydiae.

Chlamydiae encode a limited number of TCSs, compared to other pathogenic bacteria. As TCSs normally detect environmental stress stimuli, our results suggest that these genes may regulate persistence during iron starvation. Further knowledge of the biological mechanisms triggering the development of persistent bacteria can give insights into mechanisms at play during chronic chlamydial infections.

Aim:
To identify a previously unknown interspecies communication peptide from Klebsiella pneumoniae and analyze its effect on Streptococcus pneumoniae (pneumococcus) in vitro and in vivo with a view to investigating its potential as a therapeutic for pneumococcal diseases.
Methods:
The peptide was identified from the K. pneumoniae secretome by mass spectrometry and the effects on S. pneumoniae analyzed by optical density measurement, time-kill assays, transformation assays and microscopy. The effects on the transcriptome were determined by RNA-Seq and on colonization using primary human airway epithelial cells and an infant rat model. Peptide toxicity was assessed using primary human airway epithelial cells and zebrafish larvae.
Results:
The K. pneumoniae secretome contained a peptide that suppressed growth of genetically diverse clinical pneumococcal isolates, including antibiotic-resistant strains, in defined medium and human cerebrospinal fluid. Bacteriostatic growth inhibition was dependent on uptake via a functional Ami permease and caused downregulation of genes involved in amino acid and protein metabolism. Furthermore, the peptide caused irregular bacterial shapes, decreased chain length and decreased genetic transformation. Pneumococcal adherence to primary human airway epithelial cells and colonization of rat nasopharynx were also decreased. We did not detect toxicity of the peptide in vitro or in vivo.
Conclusion:
We identified a K. pneumoniae peptide which targets the pneumococcal Ami permease to inhibit pneumococcal growth and colonization. The peptide has potential as a therapeutic for pneumococcal diseases, including treatment of antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

The coprophile ink cap mushroom Coprinopsis cinerea is a well-established model organism for agaricomycetes. Fungivorous nematodes are ecologically significant predators of fungal mycelium. The chemical defense of C. cinerea against these predators includes both a constitutive (autonomous) and an inducible part. Latter part is manifested by the production of a series of nematotoxic intracellular proteins in the vegetative mycelium of this fungus upon attack by the stylet-harbouring fungivorous nematode Aphelenchus avenae1,2,3. Interestingly, this inducible chemical defense mechanism is not restricted to the sites of the predation but propagates along specific hyphae both acropetally and basipetally3. The signalling pathways that are responsible for triggering the inducible anti-nematode defense response in C. cinerea and the nature of signals mediating its propagation along hyphae remain unclear. Moreover, the physiological relevance of the constitutive and inducible chemical defense of C. cinerea against fungivorous nematodes has not been assessed so far.

Recently, we tested the vegetative mycelia of a series of monokaryotic and dikaryotic C. cinerea strains for their susceptibility to grazing by A. avenae. We found that on some strains, nematode populations thrive rapidly, while on others, nematodes struggle to propagate. We set out to unravel the genetic basis of the difference between permissive and prohibitive strains using forward genetics. To achieve this goal, we have crossed a permissive and a prohibitive strain and isolated 123 F1 progenies. Phenotyping revealed that 84 strains of the progenies were prohibitive while the rest was permissive. We will apply bulk segregant analysis (BSA) with and without backcrossing of single progenies to the parents to identify the gene loci associate with the permissive phenotype. To further validate the candidate genes and investigate their roles in the expression of nematotoxic effectors, we will apply reverse genetics i.e. create respective knout-out strains using CRISPR-Cas9. The results of this study will hopefully reveal the signalling pathways involved in the defense response of C. cinerea against A. avenae and demonstrate that this response is physiologically relevant for the fungus.

Aims
Legionella pneumophila, the causative agent of Legionnaires’ disease, is often found in the plumbing systems of buildings from where it can be transmitted to humans via inhalation or aspiration of contaminated water drops. Routine water sampling from the potable water system of an occupational building in Basel over 25 years allowed analysis including whole genome sequencing (WGS) of the colonizing L. pneumophila isolates. This is the most detailed, long term building survey of L. pneumophila recorded.

Methods
Annual routine testing of the drinking water system at the building from 1994 to 2018, was performed in accordance with national guidelines. Isolates (n=113) were grown on Buffered Charcoal Yeast Extract Agar for 48-72 h. DNA was extracted using the Qiagen EZ1 with the DNA tissue kit. WGS used the NextSeq500 PE150 after Illumina DNA prep, resulting in >40x mean read depth per sample. Assembly with Unicycler v0.3.0b was used in core genome MLST (cgMLST) analysis in Ridom Seqsphere+ 9.0.10 according to the species scheme on https://www.cgmlst.org. Single nucleotide polymorphism (SNP) analysis was performed in CLC Genomics Workbench v22.0.2 and the resulting alignment analysed with Gubbins v3.3.0.

Results
Overall, 309 water samples were collected at 38 time points over the period of 25 years. L. pneumophila was recovered from 120 water samples (38.8%) and 113 L. pneumophila isolates from 26 time points were included in this study. After initial decontamination measures that were successful for approximately 12 years, an increase in the total number of Legionella as well as of L. pneumophila-positive site was noticed in 2008. WGS showed all L. pneumophila to be ST45 (SBT scheme). The isolates are very closely related, all clustering within cluster limits (4 cgMLST alleles). Recombination analysis showed that of >6000 identified SNPs between isolates, 94% fall within recombinations. SNP analysis of the remaining SNPs does not show temporal signal or clustering by location.

Conclusion
Over 25 years, a single ST lineage colonised the water system of this building. This ST is known from other publications to be able to cause disease; however, no infections with Legionella spp. have been reported as being associated with this building. The phylogeny of isolates can be interpreted as inferring good water circulation, possible recolonisation from a common source after cleaning, and possible evolutionary bottlenecks within the water system.

Wastewater-based infectious disease surveillance has helped to inform dynamics of SARS-CoV-2 in Switzerland since July 2020. Wastewater analysis complements traditional epidemiological indicators, such as reported cases, offering a unique perspective on disease dynamics. Since November 2022, wastewater sampling has been expanded to include monitoring of other priority respiratory diseases. Routine sampling is conducted with high temporal (5 days a week) and spatial (14 treatment plants across Switzerland, representing 26% of the population) resolution. Wastewater samples are received at a central laboratory weekly, total nucleic acids are extracted, and RNA concentrations of SARS-CoV-2, Influenza A and B, and Respiratory Syncytial Virus are quantified with a multiplex digital PCR assay offering absolute quantification. Digital PCR’s accuracy is achieved by partitioning samples into thousands of individual reactions, enabling absolute quantification without the need for standard curves. Additionally, amplicon-based sequencing and associated bioinformatics pipelines facilitate the detection of variant signature mutations and estimation of SARS-CoV-2 variant proportions in wastewater. The wastewater data collected within the scope of the work has also been used to estimate the effective reproductive number of SARS-CoV-2 and Influenza over time, complementing clinical-based measures of disease dynamics. The work highlights the potential for low cost, environmental surveillance of populations to inform Swiss infectious diseases. Intended expansions include work on surveillance of other priority pathogens, including gastrointestinal diseases, seasonal coronaviruses, and antimicrobial resistance.

The presence of enteric viruses in water is an ongoing global concern with negative human and ecosystem health impacts. Sunlight is an important inactivation mechanism for viruses, but inactivation rates can be impacted by interactions with abiotic and biotic factors. Filter-feeding organisms, like zooplankton, have been shown to remove viruses from water, but how this uptake impacts the efficacy of sunlight inactivation rates is not understood. Hence the aim of this research is to quantify the changes in sunlight inactivation in the presences of zooplankton. To achieve this aim, two model zooplankton species were evaluated Tetrahymena pyriformis (ciliated protozoa) and Brachionus calyciflorus (a rotifer) with two viruses MS2 phage and Echovirus-11 (EV-11). Batch experiments were conducted using a solar simulator (Sun 2000, Abet Technologies) in a temperature-controlled water bath (24°C), In addition batch experiments were conducted in the dark. Proper controls were employed to compare inactivation rates. Water samples were collected for up to 3-days and infective viral concentrations were quantified. Data will be presented showing comparison of dark and light removal rates for each zooplankton species. Data show that removal of MS2 and EV-11 exposed to sunlight is enhanced in the presence of ciliates (T. pyriformis), and protection effects are not observed. In contrast, sunlight inactivation of EV-11 is reduced in the presence of rotifers (B. calyciflorus), but the same protection is not observed for MS2 in the presence of rotifers. The results obtained to date highlight the complexity of zooplankton-viral interaction, with the potential for zooplankton to enhance as well as reduce viral inactivation. Further work is needed to elucidate the possible mechanisms that cause the differences in sunlight inactivation of viruses in the presence of zooplankton.

The anaerobic food chain describes the sequential degradation and utilization of biopolymers by a community of microorganisms in the absence of dioxygen. The anaerobic digestion (AD) process utilizes such microbial degradation networks to reduce organic waste volume while producing biogas as a renewable energy source. More recent interest has developed in applying AD to produce fatty acids as useful precursors for repurposing organic wastes. Previous research has characterized the different trophic layers and microbial metabolic groups carrying out digestion and identified key biological and operational factors affecting AD dynamics. However, it is still challenging to design a stable process that continuously produces targeted fatty acids with high productivity and purity by finely tuning the AD microbiota. As an initial step towards understanding the interactions of different factors and the dynamic feedback between microbial activity and the environment, we carried out enrichment experiments of microbiota taken from three different AD reactors with varying feedstock, operational conditions, and geographical locations. Minimal media containing glucose, cellobiose, or no carbon substrates (as negative controls) at three different pH levels (5.5, 6.5, and 7.5) were used. The production of metabolites (short-chain fatty acids, H2, CO2, and CH4) was monitored. Despite the different inocula, we observed the enrichment community converging functionally depending on substrate and pH conditions. Through multidimensional analysis of metabolites and 16S sequencing results, we will evaluate the relevance of substrate and pH in selecting community function

We are lacking antivirals for different viruses. In addition to targeting viral protein, focusing on viral RNA is a promising strategy to address this gap in antiviral therapeutics.

We previously showed geneticin’s inhibition of various SARS-CoV-2 variants by targeting its -1 programmed ribosomal frameshift. This mechanism involves a pseudoknot, an RNA structure common in coronaviruses but uncommon in humans. To identify more potent inhibitors, we conducted virtual screening with a drug-like library on an RNA ensemble derived from molecular dynamics, using a refined SARS-CoV-2 pseudoknot structure. One of the hits demonstrated reduction of SARS-CoV-2 frameshift and exhibited antiviral activity in the micromolar range.

However, cellular studies revealed a different SARS-CoV-2 RNA folding, driving our focus on identifying molecules binding to alternative structures that could result in a conformational block, preventing the formation of the pseudoknot. Consequently, we are selecting potential inhibitors by virtual screening and using a refined dual luciferase construct in which the RNA can fold as identified in the cells.

This novel drug development strategy is applicable to various viruses. We are currently employing this approach on rhinoviruses by determining the predominant RNA cellular structure with DMS probing, selecting druggable pockets with antisense oligonucleotides, and using in silico screening to identify potential drug candidates. Overall, targeting viral RNA is an unexplored field with promising therapeutic opportunities.

Respiratory aerosol particles have gained attention as a major transmission route for respiratory diseases in the last years. Our previous research suggests that small aerosol particles on a sub-micron scale acidify in seconds to minutes, reaching a pH of approximately 4. Gas phase adjustments, e.g. acidification, can further reduce the predicted aerosol pH. In this context, we set out to investigate the inactivation mechanism of SARS-CoV-2 at a pH below 3 with a focus on its surface glycoprotein Spike (S). S consists of the S1 subunit containing the receptor binding domain (RBD) binding to ACE2 and the S2 subunit responsible for fusion with the host membrane.
Herein, we study SARS-CoV-2 after acidification and subsequent neutralization for infectivity and copy numbers. We use light and cryo-electron tomography (cryo-ET) to determine virion integrity as well as Immunofluorescence (IF) and biolayer interferometry (BLI) approaches to study binding on a molecular level.
The infectivity of SARS-CoV-2 particles decreases within minutes when the pH drops below 3, while genomic copy numbers remain stable. Cryo-ET showed virions were largely intact after acidification with signs of a disrupted inner structure. While we saw no decrease in the in vitro binding of recombinant S subunits (including the RBD, S1, the S1+S2 monomer and trimer) to ACE2 in BLI after acidic treatment, we found that the vast majority of SARS-CoV-2 virions lost the ability to bind to target cells after acidic treatment in an IF based assay. Additionally, we observed a reduction of RBD signal on SARS-CoV-2 virions treated with acidic pH in IF, but an increase of S2 signal of virions.
Overall, our data show a loss in binding capacity of SARS-CoV-2 virions to the target cell after encountering acidic pH below 3. Considering that, our in vitro BLI assays demonstrate that the RBD is still intact and capable of binding to its ACE2 receptor, our data suggest that the decrease in virion binding to cells is attributed to S1 shedding rather than conformational changes of S.

Anisogamous species, which produce gametes with significant morphological differences, are thought to have evolved from isogamous ancestors1. Lack of evolutionary records and experimental evidence has made it difficult to understand how selection could act on isogametes to select for increased asymmetries. I will show our recent data indicating that the morphologically indistinguishable fission yeast P- and M-gametes, which differentially express only a handful of genes, experience distinct selective pressures. We initially discovered that transcription of meiosis-specific genes, including the highly conserved meiotic cohesin rec8, occurs ahead of fertilization. Surprisingly, only P-gametes produce Rec8 protein during mating, whereas Rec8 encoded by the M-gamete genome becomes detectable only at the time of partner fusion. This asymmetry in Rec8 production is driven by distinct pheromone signaling between partners, and P-gamete-produced Rec8 is critical for meiotic chromosome segregation. Strikingly, early expression of Rec8 also places a fitness cost on P-gametes; P-gametes that engage partners but fail to fuse exhibit increased genomic instability when allowed to reproduce asexually, which is prevented by removal of the rec8 gene. Finally, we show that P-gametes are at a competitive disadvantage to M-gametes in evolving populations that undergo cycles of mating and asexual reproduction, and that the observed difference in fitness depends on the rec8 gene. Taken together, we demonstrate that distinct investments of P- and M-gametes in zygotic development impose different fitness costs. Our work provides the first example of how subtle molecular asymmetries can drive distinct selective pressures on isogametes during evolution.

In complex and heterogeneous environments, the hyphae of filamentous fungi and fungal-like Oomycetes are known to provide a support for the dispersal of other microorganisms. The use of these “fungal highways” is regulated by the interplay of both physical and biological constraints. Therefore, the ability of different species to establish fungal highways must be verified experimentally. Several experimental devices exist that can test specific pairings. However, these methods are time consuming and cannot be applied at a large scale and high-throughput format. Today, three-dimensional (3D) printing offer fast translation between digital design and a finished product. This allows testing intricate designs with greater reproducibility, and faster production times.
In this study, we used 3D printing to develop an experimental tool to allow fast and high-throughput evaluation of bacterial dispersal on hyphal networks in 96-well microplates. Different materials and designs were tested to produce a “crossing bridge” that is traversed by the fungus-bacteria partners. The design allows for the simultaneous testing of multiple species and the inclusion of any culturing media. By combining the transport with a redox active dye, the arrival of the partners to the target well can be quickly monitored. The devices were evaluated with several fungal and bacterial species and the performance of designs was compared. The optimal topology was selected to evaluate the effect of multi-trophic conditions on the effectiveness of the transport. This study provides an easy-to-implement approach for evaluating the effective transport of bacteria by fungi and fungi-like hyphal networks.

Aims :
The pathogenic yeast Candida auris has been classified as urgent threat by the Center for Disease Control and Prevention (CDC) because of its potential to cause outbreaks of candidemia. Some characteristics of C. auris include adherence to inert surfaces, biofilm formation and aggregation. C. auris can also form pseudo-filaments under specific conditions. The transcription factor Ume6 was shown to play a role in virulence and morphogenesis (e.g. filamentation) of Candida albicans. However, its role in C. auris remains unclear. The aim of this study was to investigate the role of Ume6 and its main downstream effectors (Als4498, Scf1, Hgc1) in C. auris morphogenesis and virulence.

Methods :
To assess the impact of UME6 hyperactivation and deletion, we constructed the UME6HA strain (in which UME6 was under the control of the ADH1 promoter and tagged at its C-terminal locus by a 3xHA tag) and the ume6Δ strain, respectively, in a non-aggregative C. auris strain from clade IV. Transcriptomic analysis was performed in the UME6HA strain and compared to its background strain. Genes that were overexpressed in the UME6HA strain were subsequently deleted in the UME6HA background. To study biofilm, we used crystal violet assay for quantification, SEM microscopy and confocal microscopy. Flow cytometry was used for adhesion assay and also Imaging flow cytometry for to quantify length of the fungal cells.

Results :
Deletion of UME6 did not result in any significant phenotype regarding morphogenesis. Hyperactivation of UME6 in the UME6HA strain induced aggregation, pseudohyphae formation and biofilm formation. Based on transcriptomic analysis, three genes (ALS4498, SCF1 and HGC1), which were highly overexpressed in UME6HA strain were subsequently deleted in UME6HA background. Deletion of ALS4498 (an adhesin) abolished aggregation and resulted in decreased biofilm formation. Deletion of SCF1 (adhesin known as surface colonization factor) only resulted in loss of aggregation, while deletion of HGC1 (transcription factor controlling morphogenesis) only resulted in loss of pseudohyphae formation.

Conclusion :
The transcription factor Ume6 plays a crucial role in morphogenesis of C. auris by controlling :
- Aggregation via Als4498 and Scf1
- Adhesion via Scf1
- Pseudofilamentation via Hgc1
- Biofilm formation via Als4498 (and Scf1, Hgc1)