Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Prof.

Mechanistic insights into the role of extracellular vesicles at the bacterial-host interface

Infection biology

Introduction: Bacterial and human cells produce extracellular vesicles (EVs) in response to diverse stimuli, e.g. toxins, oxidative stress, nutrient depletion, or high cell density. EVs function as transport systems for proteins, enzymes, DNA and RNA, thereby contributing to interkingdom communication. Several studies have characterized isolated EVs from both cell types under distinct conditions.Objectives: There is currently no platform that enables the simultaneous production of bacterial and host EVs within the same environment. We present a method for bacteria-host co-cultivation using the Transwell® insert system, which allows the exchange of solutes between the two cell types whilst preventing their direct contact.Methods: We investigated both Gram-positive and Gram-negative bacteria, harboring distinct pathogenicity traits: Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli and Neisseria meningitidis. Bacteria were co-incubated with peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. The co-cultivation allowed recovery of the shared supernatant and further isolation and characterization of the mixed pool of bacterial and host extracellular vesicles (mix-EVs).Results: Dynamic light scattering microscopy revealed a negative correlation between the bacterial concentration and the formation of mix-EVs. Exposing healthy human PBMCs to mix-EVs led to an increased expression of pro-inflammatory cytokines (e.g. IL-6, TNF-α) and several chemokines (e.g. MIP-1a, CCL2, CCL5). Additionally, the transcriptome profile of the PBMCs after exposure to mix-EVs revealed an enrichment of IL-1β transcripts, which was further confirmed at the protein level. By utilizing toll-like receptor pathway specific inhibitors, we could show that this increased expression of IL-1β is mediated by IRAK and TRAF6 related pathways.Conclusions: This work provides a new platform for the study of EVs at the pathogen-host interface and presents mechanistic insights into the effect of EVs on an infected host.

Poster

Prof. Dr.

Streptococcus agalactiae in elephants – a comparative genomic analysis

Streptococcus agalactiae (GBS) is a bacterium well-known for causing neonatal infections in humans and is also a major pathogen in the dairy industry, responsible bovine mastitis. While GBS research has largely focused on human and bovine infections, this pathogen has a broader host range, infecting various animals, including dolphins, camelids, rats, fish, seals, and elephants. However, studies of isolates from elephants have rarely been conducted. In recent years, our group hast occasionally isolated GBS from African and Asian elephants kept in various zoos in Germany and characterized with molecular methods. This study presents the first whole-genome sequencing analysis of GBS isolates from elephants, aiming to expand understanding of GBS infection mechanisms in this unique host.Next-generation-sequencing was conducted with 24 GBS isolates followed by comparative genomic analyses to determine the pan- and core genome and identify singleton gene. We searched for antimicrobial resistance genes and performed an in-depth analysis of the capsular polysaccharide (cps) locus. Additionally, we screened for mobile genetic elements and performed a genome-wide association study to identify gene enrichment specific to the elephant host group.Twelve isolates were assigned to multilocus sequence type 2019, while the remaining isolates represented a previously unidentified sequence type. All isolates carried the mprF gene, linked to host cell uptake and disease progression. Serotyping efforts revealed the only four isolates could be assigned to a serotype. Primers flanking the cps-locus were used to screen for this region. Only one isolate contained the full region, nine isolates lacked both primer sequences, while ten had a deletion within the region. No plasmids were identified in the isolates. However, several regions indicative of potential phage presence were detected. The marR gene was significantly associated with the elephant host, which plays a role in controlling the oxidative stress response. Two additional proteins (Maf family protein; acyl-CoA thioester hydrolase/BAAT C-terminal domain-containing protein) were uniquely associated with the elephant isolates, while one histidine kinase was found in all but one of the elephant isolates.Future studies should include additional isolates from elephants and other wildlife animals to better understand the pathogenic properties and potential zoonotic impact of this pathogen.

In Vitro Synergy of Colistin in combination with Farnesyltransferase inhibitors against ESKAPE Bacteria

IntroductionThe emergence of antibiotic resistance is steadily increasing. One way to partially overcome these problems is to test already approved drugs (drug repurposing). Farnesyltransferase inhibitors (FTIs), primarily used in cancer therapy, have shown antimicrobial effects against some gram-positive bacteria [1]. To explore their impact on other gram-positive as well as gram-negative bacteria and assess potential synergy, FTIs are tested with colistin, which is known to disrupt the outer membrane. This study includes not only the key ESKAPE pathogen panel but also additional bacterial strains to gain a broad understanding of differential bacterial responses to FTIs. This approach aims to better understand and categorize FTIs" properties and identify dose-dependent synergistic effects relevant for the treatment of resistant bacteria.Materials & MethodsTen bacterial strains were tested to evaluate potential synergy between colistin and various drugs, focusing on FTIs and the ESKAPE panel. Antimicrobial susceptibilities were determined by broth microdilution. Synergy was assessed using a checkerboard assay to calculate ∑FIC values.ResultsWhen combined with sub-MIC colistin, FTIs inhibited the growth of gram-negative bacteria. Tipifarnib, an imidazole-containing heterocyclic FTI, demonstrated stronger effects on gram-negative bacteria than lonafarnib, a non-peptidic tricyclic FTI. Additionally, the peptidomimetic FTIs B581 and FTI-277 inhibited the growth of gram-negative bacteria in combination with colistin but showed no effect on the gram-positive strains tested. In contrast, bempedoic acid and αHFP, which both target the mevalonate pathway, exhibited no inhibitory activity.DiscussionFTIs inhibited both gram-positive and gram-negative bacteria when combined with sub-MIC colistin, potentially reducing the required colistin dosage and thus minimizing side effects. This combination may also target colistin-resistant bacteria carrying the mcr-1 gene, the most prevalent and well-characterised colistin resistance gene. The mechanism of action likely differs from the eukaryotic pathway. The species-dependent effects suggest FTIs disrupt multiple biosynthetic pathways. Further studies will be conducted to clarify the mechanisms.[1] Weber et al. 2019 Front. Microbiol. 12, 628283

Microorganisms represent a dormant phenotype during long-term starvation in tap water

The expression of the dormancy state is a widespread mechanism of microorganisms to swerve environmental stress situations. Dormant cells assumed by low metabolism activity indicated by a lack of multiplication under these conditions.We investigated the transfer of faecal, skin and infectious pathogens as well as environmental microorganisms to this dormant state by starvation and long-term persistence in tap water, and we analysed long-term survival and re-cultivation.Bacteria were adapted to tap water by pre-cultivation in Luria both producing high bacterial masses followed by addition of the nine-fold sterile tap water volume and ongoing incubation for further 72h at room temperature. After harvesting and suspension in tap water, cells rotated at low speed to minimize biofilm formation for up to 12 weeks. Re-growth verified long-term viability, and the cell number was determined weekly by counting the colony forming units (CFU/mL) in dilution series.During a 6-week incubation period, the survival rate decreased less than 2 log10 levels for Pseudomonas aeruginosa, Bacillus subtilis, E. coli and the enterococcal species E. faecium and E. faecalis. A stronger drop in the cell counts was observed up to week 12. The survival curve of the human colonizers Staphylococcus aureus and S. epidermidis decreased more rapidly over time. No colonies came up after 4 weeks. S. lentus and S. vitulinus, isolated from the environment, were more robust within a 6-week period showing only a slight decrease by few log10 levels in the cell counts. Overall, no increase in cell numbers was determined.Many microorganisms are able to express the dormancy state and to survive in tap water over weeks. Despite the stress conditions caused by lack of food in tap water, the bacteria showed a dormant state with a good re-cultivation rate. The ability of bacterial long-term survival displays a major infection risk, because water reservoirs can play a role as source for contamination and subsequent infections. The long-term existence of dormant bacteria cells in tap water should be viewed critically in order to maintain health and hygiene.

Identification of novel molecular targets for inhibition of E. faecalis biofilms

Enterococcus faecalis, a common gut commensal and opportunistic pathogen, is a major cause of hospital-acquired infections. Its ability to form biofilms enhances antibiotic tolerance and persistence, making infections difficult to treat (de la Fuente-Núñez et al., 2013). Given that 80% of infections are associated with biofilms (Park and Sauer, 2022), there is a critical need for new therapeutic approaches.The primary objective of our research is to identify new biofilm inhibitors for E. faecalis through two complementary approaches. First, an automated high-throughput (HTP) screening process is being developed, integrating machine learning to accelerate drug discovery. Data from biofilm screening assays will be leveraged to train machine learning models, streamlining the identification of promising compounds by predicting their efficacy more efficiently.The second approach focuses on discovering novel drug targets through comparative transcriptomics paired with proteomics, comparing biofilm and planktonic states of the bug. Identified targets will be analyzed using protein chemistry techniques, followed by rational drug design to develop therapeutic compounds aimed at disrupting biofilm formation.Preliminary results from transcriptomic and proteomic analyses, which reveal differential expression patterns of the planktonic and biofilm state, that may serve as potential drug targets, will be presented. Early-stage evaluations of the automated biofilm assay will also be shown.By connecting HTP screening with machine learning, the drug discovery process becomes iterative, where each method refines the other, potentially improving the quality of outcomes.Since no biofilm treatments have advanced beyond clinical trials, novel drug targets are essential. An integrated omics approach, overcoming the limitations of single-omics, can reveal new therapeutic pathways.---de la Fuente-Núñez, C. et al. (2013) "Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies", Current Opinion in Microbiology, 16(5), pp. 580–589. https://doi.org/10.1016/j.mib.2013.06.013.Park, S. and Sauer, K. (2022) "Controlling Biofilm Development Through Cyclic di-GMP Signaling", in A. Filloux and J.-L. Ramos (eds) Pseudomonas aeruginosa. Cham: Springer International Publishing (Advances in Experimental Medicine and Biology), pp. 69–94. https://doi.org/10.1007/978-3-031-08491-1_3.

Abstract author

Prof

Understanding how biofilms develop on catheters in liver transplant patients

In liver transplant patients, catheters facilitate bile drainage but also provide surfaces for bacterial biofilm formation, elevating infection risks, particularly under immunosuppressant therapy. This pilot study investigated surgical and microbiological factors influencing biofilm formation and infection risk in liver transplantation. Results showed that in surgery duct-to-duct reconstruction lowers infection risk relative to biliodigestive anastomosis. This can be correlated to biofilm formation on biliary catheters associated with duct-to-duct reconstructions correlated with higher bacterial loads and adverse outcome as cholangitis and hospital stay. Microbiome analysis of the catheter biofilms revealed a bile duct microbiome composition similar to the gut, suggesting an ascending infection pathway. Notably, Enterobacteriaceae constituted 90–95%, whereas Enterococcaceae represented only 5–10%, contrasting with the usual 50/50 distribution observed in standard cases of cholangitis.To assess the pathogenic potential of bile duct microbiome, live bacteria were isolated from catheters, identified through genetic analyses and antibiotic resistance analysis. Although many isolates harbored antibiotic resistance genes, most did not show phenotypic resistance, suggesting that biofilm formation, rather than antibiotic resistance, may be a primary pathogenic factor. Using these isolates, we developed an in vitro model for biofilm formation on catheters. Single-species, dual-species, and complex microbiome biofilms were profiled using 16S rRNA sequencing, and live-dead cell ratios evaluated through LIVE/DEAD staining in confocal laser scanning microscopy (CLSM) and CFU enumeration. Enterobacteriaceae consistently formed dense biofilms with high viable cell content, while Enterococcaceae biofilms were sparse to nonexistent. Dual-species biofilms and microbiome biofilms were thicker and exhibited higher survival ratios. As biofilm-forming bile duct microbiome bacteria can disperse into the abdominal cavity due to bile leakage post-operation, polymicrobial biofilms potentially spreading across various catheters and can include nosocomial bacteria, posing considerable challenges in clinical settings. These findings provide valuable insights into biofilm dynamics and may inform strategies for treatment or prevention of infection.

PD Dr.

A widespread SCCmec-located gene cluster protects MRSA against toxic polysulfide

Colonization of the human body presents challenges for opportunistic pathogens, which must overcome host defense mechanisms, antimicrobial compounds, and competition with the commensal microbiota. One of these opportunistic pathogens is Staphylococcus aureus (S. aureus), with methicillin-resistant S. aureus (MRSA) strains being one of the most common antibiotic-resistant nosocomial pathogens, responsible for millions of life-threatening infections worldwide8. S. aureus often colonizes mucosal surfaces of the human body and is therefore exposed to environmental stress factors, including hydrogen sulfide (H2S). H2S is ubiquitous in the human body, and while low concentrations act as a signaling molecule, high concentrations are toxic. The elevated H2S levels are primarily a consequence of bacterial sulfur compound metabolism. On mucosal surfaces, such as the nose and gut, H₂S concentrations can reach up to 0.4 mM9 and between 0.3–3.4 mM10,11, respectively, due to the degradation of sulfated sugar residues and l-cysteine from mucin12. Under oxic conditions, which prevail on mucosal surfaces13, H₂S oxidizes into highly reactive polysulfides, creating another toxic stressor for the bacteria. While it is known that S. aureus protects itself from H₂S via its cst gene cluster (tauE, cstR, cstA, cstB, sqr)14, the detoxification of polysulfides is virtually unknown. We showed that S. aureus uses an SQR-independent variant of the cst pathway to detoxify polysulfides. Furthermore, our research revealed that the cst gene cluster is heterogeneously distributed in staphylococcal genomes, with multiple clinically relevant SCCmec types introducing an additional cst (cst2, without sqr) into the genomes of MRSA strains. Phenotypic analyses of a wide range of clinical and laboratory-derived strains demonstrated that the additional cst2 confers high polysulfide tolerance to MRSA, providing a significant fitness advantage in polysulfide-rich environments that results in the displacement of MSSA strains in direct intraspecies competition.8 Murray et al. (2022): in: The Lancet 399, 629–6559 Ikeda et al. (2019): in: Molecules (Basel, Switzerland) 2410 Braccia et al. (2021): in: Frontiers in microbiology 12: 70558311 Dordević et al. (2021): in: Journal of advanced research 27: 55–6912 Stümmer et al. (2023): in: Antioxidants (Basel, Switzerland) 1213 Espey (2013): in: Free radical biology & medicine 55: 130–14014 Shen, et al. (2015): in: Biochemistry 54 29, 4542–4554

Active downregulation of the type III secretion system at higher local cell densities promotes Yersinia replication and dissemination

The Type III secretion system (T3SS) is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. T3SS secretion is tightly regulated and accompanied by a phenomenon called secretion-associated growth inhibition (SAGI). Since actively secreting bacteria cannot grow and divide, a non-uniform distribution of T3SS expression is conceivably beneficial during infection. Yersinia enterocolitica, a main T3SS model organism that uses the system to evade the host immune response, had so far been found to uniformly express and assemble T3SSs, which are then activated by target cell contact. However, in this study, we found that Yersinia actively suppress T3SS expression, assembly and activity at higher cell densities, such as inside microcolonies. This effect is highly specific to the T3SS, reversible, and distinct from stationary phase adaptation. It is conferred by the T3SS transcriptional activator VirF, which is downregulated at the higher cell densities and whose in trans expression restores T3SS assembly and activity. Transcript analysis showed that this effect is mediated by increased levels of the regulatory RNAs CsrBC, which sequester the regulatory protein CsrA and destabilize the virF transcript. Cell adhesion, an essential trait for Yersinia virulence, linked to T3SS secretion and regulated by VirF also showed to be affected by local higher cell densities. The concomitant downregulation of the VirF-dependent adhesin YadA led to a drastic reduction in bacterial cell adhesion. We propose that this active suppression of T3SS secretion and cell attachment at higher local bacterial densities promotes a switch during Yersinia infection from a T3SS-active colonization stage to a bacterial replication and dissemination phase.

Overcoming resistance via reactivation – Mode of action of vancomycin conjugates

Glycopeptide antibiotics (GPAs) are drugs of last resort to treat infections caused by (multi-) resistant Gram-positive pathogens. They inhibit peptidoglycan biosynthesis mainly by binding to the D-alanyl-D-alanine terminus of lipid II peptide stem, thereby blocking transglycosylation and transpeptidation reactions in a sterical manner. Vancomycin (VAN) and teicoplanin (TEIC) represent the prototype GPAs in clinical use today. However, widespread GPA resistance has become a serious threat to global health. Resistance resulting from alteration of the PGN precursor, e. g. by exchange of the terminal D-alanine to D-lactate or D-serine, is conferred by the acquisition of a van-resistance operon. Enterococci, which harbour a vanA or vanB cluster, are most significant in the clinical setting. Research is focused on structural modification of glycopeptide antibiotics to overcome resistance. Lead candidate FU002, a vancomycin-hexa-arginine derivative, with high potency against vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) was generated by conjugation of polycationic peptides to VAN. Cell-based and in vitro assay are used to elucidate the precise mechanism of action to build a rational basis for targeted structure optimization. Differences in potency are not based on interaction with a secondary binding site on lipid II and remain still elusive.

Assessing the direct antibacterial activities of synthetic host defense peptides

Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs) are effectors of innate immunity present in all forms of life and have co-evolved with bacteria for millions of years. AMPs bear great potential for future antibiotic therapy. Although bacteria are frequently exposed to these AMPs, emergence of resistance is rarely reported. Furthermore, they combine direct antimicrobial activities together with immunomodulatory properties boosting their effectiveness in infection control. Many HDPs are known to disturb bacterial membrane functions, but the specific mechanisms of action (MoA) are not fully understood. Artificially designed AMPs based on the Anti-Lipopolysaccharide factor (ALF) of Limulus polyphemus were supposed to bind free lipopolysaccharides from Gram-negative pathogens and prevent sepsis. Here, we show that synthetic anti-lipopolysaccharide peptides (SALPs) Pep19-2.5 and Pep19-4LF demonstrate promising antibacterial activity and that their MoA is more direct, likely involving the interaction with specific bacterial target molecules.

Unraveling bacterial-archaeal interactions within the human oral microbiome – insights into the etiology of periodontal disease

Methanogenic archaea produce methane as the metabolic end product for energy conservation. Among other methanogenic pathways, the ability to produce methane from CO2 and H2 in hydrogenotrophic methanogenesis is the most widespread. The hydrogenotrophic species Methanobrevibacter oralis is the predominant methanogen residing in human subgingival plaque. Its prevalence has recently been linked to periodontitis, indicating a pivotal role in oral health and disease. Previous studies yielded evidence of close interactions between M. oralis and various bacterial species, as they synergistically participate in the anaerobic degradation of organic compounds. Importantly, this syntrophic cross-feeding between methanogens and hydrogen-producing bacteria, referred to as interspecies hydrogen transfer, is suggested to indirectly promote pathological conditions because it facilitates the growth and colonization of secondary fermenting pathogens. However, much is still unknown about the complex microbial interplay between M. oralis and oral community members that contribute to dysbiosis and, ultimately, to the pathogenesis of periodontitis. A more profound knowledge of the role of M. oralis within the oral microbial community is required to gain deeper insight into the etiology and manifestation of this polymicrobial disease.In this context, we aim to gain information from three different perspectives. First, we will investigate the relationships (mutualism and competitiveness) between M. oralis and potential bacterial interaction partners through co-culturing and quantitative PCR (qPCR) techniques. Second, we will employ qPCR to quantify M. oralis in patient samples. Third, an additional information layer will be added by quantifying and visualizing M. oralis in an established biofilm model using qPCR and FISH. Ultimately, these findings will contribute to a greater understanding of the dynamic interplay between M. oralis and other oral microbes, offering valuable perspectives for developing potential therapeutic strategies against periodontal infections.

Infection of cornea models with different Neisseria gonorrhoeae derivatives to study the importance of the Type IV pilus for bacterial adherence and infection outcome

Neisseria gonorrhoeae (Ngo) is a human obligate pathogen and is the main cause of ophthalmia neonatorum, a type of conjunctivitis that affects newborns, which untreated can lead to blindness (1). One of the most important virulence factors of Ngo is the Type IV pilus, filamentous protein structures that facilitate the adhesion of the bacteria to their target tissue.In this project, we studied the importance of the Type IV pilus in Ngo infection of cornea tissue models, derived either from cell lines or from primary cells (2). We infected the models with MS11 Ngo derivatives: MS11 F3 (Pili+, RecA+), MS11 N159 (Pili+, RecA-, Adh+cornea) and MS11 N191 (Pili+, RecA-, Adh-cornea). We observed that the bacterial adherence was highest for N159, which could be confirmed using fluorescence microscopy and SEM. However, no significant difference in the tissue integrity between the non-infected and infected models was detected. Cytotoxicity assessment by measuring LDH in the supernatant showed that the highest cytotoxicity was present after 72 h of infection with N159 Ngo. In the primary-cell-derived models the cytotoxicity was comparable between the different derivatives. Measuring of the cytokines showed that IL-8 was the most secreted cytokine; its concentration was the highest in N159-infected cell line-derived models after 72 h, suggesting a specific response that could be possibly pilus-dependent. However, there was no significant difference in the IL-8 secretion between primary-cell derived models infected with F3 and N159 after 72 h. To challenge the pilus relevance, the cell-line derived models were treated with trifluoperazine, a drug that has shown an effect on pilus retraction. We observed a reduction of more than 50% of the bacterial adherence when treated in an early stage of the infection. There was also a reduction of the secretion of IL-8, suggesting a possible decrease also on the severity of the infection outcome. These results show that cornea models can be used for studying Ngo infection, as well as the importance of the functional Type IV pilus and its role not only in bacterial adherence but also in the outcome of the infection.1. Tjia, K., van Putten, J., Pels, E., & Zanen, H. (1988). The interaction between Neisseria gonorrhoeae and the human cornea in organ culture. Ophtalmology, 341-3452. Lotz, C., et al. (2016). Alternative methods for the replacement of eye irritation testing. ALTEX - Alternatives to animal experimentation. 55-67

Jun.-Prof.

Glycopeptide antibiotics (GPAs) are drugs of last resort to treat infections caused by (multi-) resistant Gram-positive pathogens. They inhibit peptidoglycan biosynthesis mainly by binding to the D-alanyl-D-alanine terminus of lipid II peptide stem, thereby blocking transglycosylation and transpeptidation reactions in a sterical manner. Vancomycin (VAN) and teicoplanin (TEIC) represent the prototype GPAs in clinical use today. However, widespread GPA resistance has become a serious threat to global health. Resistance resulting from alteration of the PGN precursor, e. g. by exchange of the terminal D-alanine to D-lactate or D-serine, is conferred by the acquisition of a van-resistance operon. Enterococci, which harbour a vanA or vanB cluster, are most significant in the clinical setting. Research is focused on structural modification of glycopeptide antibiotics to overcome resistance. Lead candidate FU002, a vancomycin-hexa-arginine derivative, with high potency against vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) was generated by conjugation of polycationic peptides to VAN. Cell-based and in vitro assays are used to elucidate the precise mechanism of action to build a rational basis for targeted structure optimization.

Infection biology 2

Termin

Programm