Susanne Häussler

Objectives: This study aimed to investigate pathogen-specific prevalence, 30-day mortality, and host factors associated with infection in a large tertiary-care hospital cohort, and to evaluate whether comorbidity patterns and machine-learning models can predict individual outcomes. Methods: We conducted a 39-month retrospective cohort study of 16,282 hospitalized patients with positive bacterial cultures at a tertiary-care university hospital. Cox proportional hazards models identified mortality risk factors. Comorbidity-pathogen associations were examined using logistic regression. Five machine-learning classifiers were trained to predict 30-day mortality. Results: Enterococcus faecium contributed deaths comparable to Staphylococcus aureus and Escherichia coli despite markedly lower prevalence, reflecting the highest case fatality rate across all infection sites (adjusted HR 1.91, 95% CI 1.65–2.22). Multidrug resistance or sex were not independently associated with mortality. Comorbidity profiles were strikingly uniform across pathogens and infection sites; only hematologic malignancies were overrepresented in E. faecium bloodstream infections. Machine-learning models achieved limited predictive performance overall, with higher sensitivity only in elderly patients and those with E. faecium infections. Conclusions: Nosocomial mortality is shaped by pathogen-specific case fatality rather than prevalence alone. Uniform host vulnerability profiles constrain individual risk prediction and argue for dedicated E. faecium surveillance in tertiary-care settings.

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility ^1,2,3 and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems ⁴ . Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa . Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

Pseudomonas aeruginosa is a versatile, opportunistic pathogen, well-known for its adaptability to the challenging conditions encountered during infection of the human host. In chronic biofilm-associated infections, variations in motility and LPS structure are commonly observed adaptive traits. In this study, we identified differences in the expression of the O-specific antigen (OSA) biosynthetic cluster as a key driver of motility variation among a large number of clinical PA14 clade isolates. Despite retaining flagellar presence and function, OSA-deficient mutants exhibited increased hydrophobicity, which enhanced surface attachment and, in turn, reduced surface motility while promoting biofilm formation. The modified lipopolysaccharide structure with a lack of OSA also resulted in increased serum sensitivity, reduced phagocytic uptake, increased aminoglycoside resistance, and enhanced expression of the type III secretion system. These findings suggest that the commonly observed loss of OSA in P. aeruginosa isolates recovered from chronically infected cystic fibrosis lungs may be selectively advantageous. Importantly, we demonstrate that clinically relevant phenotypes influenced by the presence of OSA, such as motility and antibiotic resistance, become detectable only under test conditions that promote bacterial attachment. Similarly, the role of OSA expression in bacterial pathogenicity is conditional, with neither its presence nor its absence uniformly favoring infection. Together, our findings support a model in which OSA expression may be beneficial during early infection, whereas reduced OSA expression may promote traits associated with persistence at later stages. IMPORTANCE: Loss of the O-specific antigens (OSA) in Pseudomonas aeruginosa isolates recovered from chronically infected cystic fibrosis lungs is a commonly observed phenotype that may confer a selective advantage to this opportunistic pathogen. In this study, we demonstrate that P. aeruginosa strains lacking OSA become more adhesive, limiting their motility but enhancing biofilm formation. Furthermore, OSA-deficient strains exhibit increased serum sensitivity, while they are phagocytosed less, express elevated type III secretion system genes, and show enhanced antibiotic resistance. Our findings suggest that altered OSA expression may contribute to stage-dependent infection phenotypes, where reduced OSA expression during later stages drives biofilm formation and antibiotic resistance, thereby enabling persistence.

Post-transcriptional transfer RNA (tRNA) modifications dynamically regulate translational capacity, yet whether the tRNA epitranscriptome is temporally coordinated with gene expression programs during bacterial growth remains unknown. Here, we combine time-resolved nanopore tRNA sequencing, liquid chromatography–tandem mass spectrometry modification profiling, transcriptomics, and proteomics across the Pseudomonas aeruginosa PA14 growth cycle to show that tRNA-modifying enzymes, the corresponding tRNA modifications, and modification-dependent genes are expressed in a coordinated temporal cascade. GidA-dependent 5-methylaminomethyl-2-thiouridine (mnm ⁵ s ² U) modifications exemplify this program: GidA expression peaks during early growth, followed by rising mnm ⁵ s ² U levels coinciding with maximal expression of genes enriched in rare AGA and UUA codons, with a concomitant enrichment of virulence-associated functions. This sequential pattern is consistent with a feedforward architecture in which the modification machinery acts in advance of translational demands. This mechanism is especially relevant for a set of horizontally acquired genes, suggesting that temporally programmed tRNA epitranscriptomic remodeling may help bridge local codon usage deviations between horizontally acquired genes and the core translational context.

Background. Genomic surveillance of Gram-negative pathogens is generally selective, focusing on suspected outbreaks or resistant isolates. The extent of what such programmes miss remains unknown. It has not been quantified in a large, unselected dataset. Methods. We prospectively whole-genome sequenced isolates of Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa from two tertiary-care hospitals in Copenhagen and Hannover, regardless of resistance phenotype, specimen type, or ward. We analysed 25,888 genomes from 13,712 patients. Clonal clusters were defined by single-linkage clustering at ≤30 SNPs, based on within-patient distances and validated against inpatient movement records. Analyses were patient-based, with hospital comparisons using matched 18-month periods. Results. Clonal clusters included 14.1% of patients in Copenhagen and 16.5% in Hannover. Most clustered patients had no multidrug-resistant isolate (71.3% and 50.0%) and had no intensive care unit (ICU)-associated isolate (86.1% and 68.7%). Sequencing blood-culture isolates alone, the usual trigger for genomic investigation, would have identified only 14 of 900 clustered patients in Hannover (1.6%). Excluding colonisation screening would have missed 240 (26.7%). No targeted strategy detected more clustered patients than random sampling of the same number of isolates. Detection increased with sequencing effort without reaching a plateau. After matching periods and adjusting for ICU association, resistance, and specimen mix, clustering remained more common in Hannover (adjusted odds ratio 1.49, 95% CI 1.28–1.73), suggesting sampling explained much, but not all, of the difference. Conclusions. Most Gram-negative clusters involved non-MDR organisms on general wards and would have been missed by surveillance focused on resistance or intensive care. What is selected determines which patients can be detected; the number of isolates sequenced determines how many are found. The observed 14–16% is a lower bound. Surveillance should sample broadly and representatively, and report what was sequenced and how much.

Biofilm-associated diseases like peri-implant mucositis (PIM) and peri-implantitis (PI) are significant clinical challenges affecting millions of dental implant patients globally. Although studies have described the role of microbial, host, or environmental factors in disease development, their complex interplay, particularly during dysbiosis, remains poorly understood. This cross-sectional study characterized the microbiome composition and metatranscriptomes of 125 peri-implant biofilms from 48 individuals, uncovering molecular signatures linked to peri-implant health (PIH), PIM, and PI. Distinct variations were observed in biofilm amount, microbial composition and activity, phage populations, and host response. Biofilms were categorized into four community types (CTs) based on the bacterial transcriptional activity: one linked to PIH, one to PI, and two to PIM. PIH and PIM were primarily characterized by aerotolerant taxa with increased anabolic processes, while PI was dominated by obligate anaerobes with complex biofilm morphology. PIM samples, relative to PIH, were characterized by biofilm expansion with minimal functional changes, except for the Neisseria -rich PIM subtype showing higher pyruvate and lipoic acid metabolism. The phagome mirrored the bacterial compositional variations across disease states. Furthermore, human transcriptome responses varied, indicating increased keratinization in PIH, enhanced expression of ribosome components in PIM, and inflammatory signaling and hypoxia in PI. Additionally, we identified complex species–enzyme, phage–bacterium, and host–microbe associations within the peri-implant ecosystem. Our integrative multi-omics approach provides a comprehensive view of microbial, biochemical, host, and ecological factors associated with dysbiosis, offering novel insights into peri-implant disease dynamics. IMPORTANCE: Peri-implant mucositis and peri-implantitis are highly prevalent inflammatory conditions that compromise the long-term survival and success of dental implants, yet their underlying biological mechanisms are largely unresolved. The full-length 16S rRNA gene amplicon sequencing (full-16S) allows for high-resolution taxonomic profiling of peri-implant biofilms, thereby advancing our understanding of microbial composition across health and peri-implant diseases. The integration of metatranscriptomics, furthermore, captures actively transcribed genes within the biofilm and offers direct insights into microbial community functions and the broader molecular context of peri-implant dysbiosis. DNA- and RNA-derived abundances were strongly correlated, with only a few microbial classes showing moderate diagnosis-related differences after DNA-based normalization of transcriptional activity. In this study, we integrated full-16S with metatranscriptomic profiling to simultaneously assess microbial taxonomy, functional activity, phage dynamics, and host gene expression in peri-implant biofilms. Importantly, we provide a systems-level view and report previously undescribed associations between different molecular signatures in the peri-implant ecosystem.

Transfer RNA (tRNA)-modifying enzymes are emerging as key regulators of bacterial physiology. MiaA, a tRNA isopentenyltransferase, is well studied in model organisms, but its role in the opportunistic pathogen Pseudomonas aeruginosa remains unclear. Using LC–MS, nanopore tRNA sequencing, as well as transcriptional, translational, and proteomic profiling, we mapped MiaA-dependent tRNA modifications and revealed unexpected effects of MiaA loss. Impaired translation of MiaA-sensitive codons reduced quorum-sensing-controlled virulence gene expression and attenuated pathogenicity in Galleria mellonella. Ribosome stalling at trp codons in miaA mutants overrides the attenuation-controlled repression of tryptophan biosynthesis, causing overproduction of tryptophan, along with upregulation of cognate tRNAs, thereby linking translation to global metabolic adaptation. MiaA is tightly regulated and is so central to bacterial physiology that its expression level correlates directly to virulence in clinical isolates, highlighting its role as a hub connecting translation, transcription, metabolism, and pathogenicity. These findings position MiaA as a key integrator of cellular processes critical for pathogen fitness and host interactions.

Background/Objectives: In Europe, Pseudomonas aeruginosa is the second most common cause of ventilator-associated pneumonia in intensive care units. Intrinsic antibiotic resistance and acquired carbapenemases can lead to high mortality. To guide more targeted antimicrobial therapy and adequate infection control measures, we performed a multicenter study on the prevalence and genetic basis of carbapenem resistance among P. aeruginosa (CR-PA) across 17 community hospitals in Austria. Methods: During a 3-month period, we collected 621 P. aeruginosa isolates from 560 patients. Antibiotic susceptibility testing was performed according to EUCAST guidelines, and all CR-PA isolates were subjected to whole genome sequencing. Results: Antibiotic susceptibility testing revealed carbapenem resistance in 5.41% (36/621) of the investigated P. aeruginosa isolates. Only 3 produced a carbapenemase (2 Verona Integron-encoded Metallo- ß-lactamases and 1 Imipenemase Metallo-ß-lactamase) and carried a carbapenemase-encoding gene. Among the studied P. aeruginosa isolates there was a high genetic diversity, excluding a single driving epidemic lineage in the included Austrian hospitals. Conclusions: The absence of interhospital clonal dominance suggests that carbapenem resistance emerged independently in different centers, likely driven by local antibiotic selection pressures rather than regional clonal spread.

RNA polymerase III (Pol III) transcribes cytosolic transfer RNAs (tRNAs) and other non-coding RNAs (ncRNAs) essential to cellular function. However, many aspects of Pol III transcription and processing, including RNA modifications, remain poorly understood, mainly due to a lack of available sensitive and systematic methods for their analysis. Here, we present DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs), a modified nanopore direct RNA sequencing approach and analysis framework that enables the specific and sensitive capture of pre-mature Pol III transcribed RNAs. Applying DRAP3R to distinct cell types, we identify previously unconfirmed tRNA genes and other novel Pol III transcribed RNAs, thus expanding the known Pol III transcriptome. Critically, DRAP3R also enables discrimination between co- and post-transcriptional RNA modifications such as pseudouridine (Ψ) and N ⁶ -methyladenosine (m ⁶ A) at single-nucleotide resolution across all examined transcript types and reveals differential Ψ installation patterns across tRNA isodecoders and other ncRNAs. Finally, applying DRAP3R to epithelial cells infected with Herpes Simplex Virus Type 1 reveals an extensive remodelling of both the Pol III transcriptome and epitranscriptome. Our findings thus establish DRAP3R as a powerful tool for systematically studying Pol III transcribed RNAs and their modifications in diverse cellular contexts.

Pseudomonas aeruginosa is a leading cause of chronic lung infections in cystic fibrosis (CF) patients. While bacteriophages hold potential as a treatment for antibiotic-resistant infections, the complex structure and heterogeneity of P. aeruginosa biofilms pose significant challenges to phage therapy. In this study, we investigate the adaptive evolution of the Pbunavirus phage PE1 to biofilms formed by a CF-derived P. aeruginosa isolate. Our findings reveal that biofilm-adapted PE1 mutants exhibit enhanced efficacy in controlling biofilms in vitro under conditions mimicking the CF lung environment. This improvement is attributed to the mutants' increased ability to recognize the diverse populations within the biofilm. Using a combination of cryo-EM, lipopolysaccharide (LPS) profiling, and adsorption assays, we demonstrate that mutations in tail fiber and baseplate genes of the phage improve adsorption and enable recognition of truncated LPS variants. This study highlights the critical role of biofilm heterogeneity in limiting phage effectiveness, identifies mechanisms to overcome this barrier, and pinpoints specific genomic targets for engineering phages tailored for therapeutic applications in CF patients.