Lars Jelsbak

Chronic wound infections are frequently associated with bacterial biofilms, which significantly reduce the efficacy of antimicrobial treatments. Conventional in vitro evaluation methods, such as disc diffusion assays and static biofilm models, lack physiological relevance, whereas more advanced wound models are often costly, complex, and difficult to reproduce. In this study, we present a cost-effective and easily applicable in vitro wound model based on gelatin–alginate hydrogels for the evaluation of topical antimicrobial therapies. Hydrogels composed of gelatin type A and sodium alginate were integrated into a transwell system and crosslinked using microbial transglutaminase and calcium ions. This configuration enables passive diffusion of simulated wound fluid from the basolateral compartment, thereby mimicking key aspects of the wound environment. The model supports the growth of Pseudomonas aeruginosa biofilms within a three-dimensional matrix. The hydrogel system supported bacterial growth and microcolony formation during 48 h of incubation at 37 °C. The system was applied in both biofilm inhibition and eradication studies using tobramycin, ε-poly-L-Lysine and the commercially available wound gel Prontosan^®. Viable bacteria were quantitatively recovered through hydrogel dissolution followed by serial dilution and count of colony-forming units. Overall, the developed model provides a simple and accessible platform for quantitative evaluation of antibiofilm treatments. While not intended to fully replicate the complexity of native tissue, this system offers a practical alternative to collagen-based models and helps bridge the gap between conventional in vitro assays and more advanced wound models.

Pseudomonas aeruginosa, a major opportunistic pathogen involved in cystic fibrosis and many nosocomial infections, exhibits strong competitive abilities in microbial communities. However, the mechanisms underlying its competitive success remain poorly understood. To investigate this, we introduced two P. aeruginosa strains—the moderately virulent PAO1 and the highly virulent PA14—into the THOR(The Hitchhikers Of the Rhizosphere) microbial model community, consisting of Pseudomonas koreensis, Flavobacterium johnsoniae and Bacillus cereus. Our results show that P. aeruginosa invasion significantly disrupts THOR biofilms, with greater reductions in biofilm biomass and shifts in microbial composition observed with PA14 compared to PAO1. This suggests that virulence traits expressed by P. aeruginosa play critical roles also in its ability to outcompete resident microbes. Additionally, we examined the competitive impact of an O-antigen-deficient P. aeruginosa strain, showing further reduced competitive ability with other community members. While our model system has limitations in replicating host-associated environments, it provides valuable insights into P. aeruginosa’s ecological strategies, with implications for understanding its competitive behaviour in community settings.

Natural products are central mediators of microbial interactions. However, once released into the environment, they also become available for neighboring microorganisms capable of degrading and modifying them through biotransformation. These biotransformations may fundamentally reshape metabolomes and influence community behavior, yet our understanding of these processes remains limited. Ribosomally synthesized peptides are particularly compelling in this context because their structural complexity and potent antimicrobial activity coexist with the potential to yield essential nutrients and reduced bioactivity through biotransformation. Identifying the pathways underlying these biotransformations is essential for understanding mechanisms that support microbial coexistence and nutrient recycling in soil microbiomes. Here, we used nisin as a model peptide to investigate biotransformation by soil bacteria. Selective isolation under nisin-rich, carbon-limited conditions yielded two Gram-negative isolates, Burkholderia stabilis and Pseudomonas fragi . Using growth assays and liquid chromatography–mass spectrometry, we found that both isolates grow in the presence of nisin while biotransforming and depleting the peptide. Burkholderia stabilis completely converted nisin through sequential cleavage of the C-terminus, hinge region and lanthionine ring C, whereas Pseudomonas fragi showed more limited processing restricted to the C-terminal region. Although these biotransformations dismantled structural features required for nisin’s antimicrobial activity, the intrinsic resistance of both isolates suggests a role beyond detoxification. We further detected nisin biosynthetic genes in the source environment, supporting nisin’s ecological relevance and suggesting that these bacteria may participate in its turnover in soil. Together, these findings reveal extensive microbial processing of nisin and support a role for antimicrobial peptide recycling in soil microbiomes. Importance: Natural products are often studied through the lens of their biological activities, but much less attention has been paid to what happens to these molecules after they enter complex microbial communities. Using the lantibiotic nisin as a model system, we show that soil bacteria can extensively biotransform and deplete an antimicrobial peptide through extracellular enzymatic activity. The presence of both nisin-producing and nisin-biotransforming microorganisms in the same soil environment suggests that antimicrobial peptides may be continuously produced and recycled in nature. Our findings highlight biotransformation as an important but underexplored process governing the persistence, turnover, and ecological roles of microbial natural products.

Microbial interactions can profoundly influence the activity of bioactive metabolites in plant-associated communities. De Rop et al. show that community members enzymatically degrade cyclic lipopeptides (CLiPs), metabolites that act both as virulence factors in plant pathogens and antimicrobial agents in biological control (J. De Rop, D. Prasad, N. Geudens, L. Zhou, et al., Appl Environ Microbiol 92:e01524-25, 2026, https://doi.org/10.1128/aem.01524-25 ). By neutralizing these molecules, microbes determine whether CLiP-mediated activities persist or collapse, revealing that metabolite function depends on community-mediated turnover and highlighting an underappreciated ecological layer that shapes plant disease and biocontrol outcomes.

The global burden of human disease caused by nontuberculous mycobacteria (NTM) is steadily increasing. Using our unique historical collection of freeze-dried mycobacterial isolates at the Statens Serum Institut, Copenhagen, we investigated long-term trends in NTM clinical relevance and species distribution in Denmark over the past seven decades. A total of 666 historical NTM isolates from human samples collected between 1948 and 1978 were compared with 1,820 contemporary isolates identified during routine diagnostics from 2013 to 2022. Species identification was performed for all isolates using the GenoType CM and AS line-probe assays. Among historical isolates, 16 different species were identified, with the majority classified as Mycobacterium sp. (32.1%, n = 214). In contrast, recent isolates were dominated by Mycobacterium avium (39.6%, n = 721), while Mycobacterium sp. accounted for only 7.8% ( n = 142). Furthermore, notable changes in prevalence over time at the single species level were observed for M. avium and Mycobacterium abscessus . Further genomic analyses of historical Mycobacterium sp. isolates revealed several uncommon or previously unrecognized species rarely detected in current diagnostic settings. In conclusion, our findings suggest a significant shift in the spectrum of species responsible for human NTM infections in Denmark over the past seven decades, with possible implications for diagnostic strategies, clinical management, and public health surveillance. IMPORTANCE: Infections caused by nontuberculous mycobacteria (NTM) are increasingly recognized worldwide, yet the long-term epidemiology of these human pathogens remains poorly understood. By leveraging a unique historical collection of mycobacterial isolates, this study suggests a shift in the species spectrum responsible for NTM infections in Denmark over the past seven decades, with Mycobacterium avium now predominating. Sequencing of historical isolates further uncovered several rare or previously unrecognized Mycobacterium species, illustrating the dynamic nature of NTM epidemiology over time. These findings underscore the need for sustained surveillance, precise species-level identification, and heightened clinical awareness to guide effective management of NTM infections amid a continually evolving disease landscape.

Root-associated microbiomes significantly influence plant growth and resilience through intricate chemical dialogues mediated by plant- and microbe-derived specialized metabolites. These metabolites play pivotal roles in shaping the assembly, dynamics, and ecological functions of soil microbiomes. Despite advances in in vitro and DNA sequencing studies, a comprehensive understanding of in situ chemical signaling within plant and soil microbiomes remains elusive due to experimental constraints. To address this gap, we developed and tuned a set of five whole-cell biosensors in Escherichia coli for spatiotemporal, nondisruptive detection of biologically relevant specialized metabolites, including 2,4-diacetylphloroglucinol, pyoluteorin, tetracycline, salicylic acid, and naringenin. Four of these biosensors were successfully adapted to the soil-compatible Pseudomonas putida KT2440 Δall-Φ strain. Additionally, the four sensors were shown to respond to their cognate ligand in a nonsterile soil extract medium containing a diverse microbiome extracted from soil. By employing genetic memory devices with DNA barcodes for readouts, our approach provides a scalable platform for sensing additional specialized metabolites in the future. This work demonstrates the potential of biosensor technologies to unravel the complex chemical interactions driving soil microbiome ecology, with implications for sustainable agricultural practices.

Root-associated microbiomes significantly influence plant growth and resilience through intricate chemical dialogues mediated by plant- and microbe-derived specialized metabolites. These metabolites play pivotal roles in shaping the assembly, dynamics, and ecological functions of soil microbiomes. Despite advances in in vitro and DNA sequencing studies, a comprehensive understanding of in situ chemical signaling within plant and soil microbiomes remains elusive due to experimental constraints. To address this gap, we developed and tuned a set of five whole-cell biosensors in Escherichia coli for spatiotemporal, non-disruptive detection of biologically relevant specialized metabolites, including 2,4-diacetylphloroglucinol, pyoluteorin, tetracycline, salicylic acid, and naringenin. Four of these biosensors were successfully adapted to the soil-compatible Pseudomonas putida KT2440 Δall-Φ strain, enabling the detection of transient bioavailable signals central to plant-microbe interactions. Additionally, the four sensors were shown to respond to their cognate ligand in a non-sterile soil extract medium containing the diverse microbiome found in soil. By employing genetic memory devices with DNA barcodes for readouts, our approach provides a scalable platform for sensing additional specialized metabolites in the future. This work demonstrates the potential of real-time biosensor technologies to unravel the complex chemical interactions driving soil microbiome ecology, with implications for sustainable agricultural practices.

Antimicrobial combination therapy is widely used to combat Mycobacterium tuberculosis ( Mtb ), yet resistance rates continue to rise. Mutator strains, with defects in DNA repair genes, are a known source of resistance in other bacterial infections, but their global role in Mtb remains unclear. Here, we provide a comprehensive overview of the contribution of single nucleotide polymorphisms (SNPs) in DNA Repair, Replication, and Recombination (3R) genes to resistance in Mtb . Through large-scale bioinformatics analysis of 53,589 whole-genomes, we identified 18 novel SNPs linked to genotypic drug resistance in 3R genes, covering 12.5% of clinical isolates/strains with available genome sequences. Notably, a number of the detected SNPs were positively selected during Mtb evolution. Experimental mutation frequency tests further indicated functional defects of these sequence variants in key DNA repair pathways. Our findings indicate that the role of mutability in 3R genes plays a significant role in Mtb resistance at the global level.

Evolution of the highly successful and multidrug resistant clone ST111 in Pseudomonas aeruginosa involves serotype switching from O-antigen O4 to O12. How expression of a different O-antigen serotype alters pathogen physiology to enable global dissemination of this high-risk clone-type is not understood. Here, we engineered isogenic laboratory and clinical P. aeruginosa strains that express the different O-antigen gene clusters to assess the correlation of structural differences of O4 and O12 O-antigens to pathogen-relevant phenotypic traits. We show that serotype O12 is associated with enhanced adhesion, type IV pili dependent twitching motility, and tolerance to host defense molecules and serum. Moreover, we find that serotype O4 is less virulent compared to O12 in an acute murine pneumonia infection in terms of both colonization and survival rate. Finally, we find that these O-antigen effects may be explained by specific biophysical properties of the serotype repeat unit found in O4 and O12, and by differences in membrane stability between O4 and O12 expressing cells. The results demonstrate that differences in O-antigen sugar composition can affect P. aeruginosa pathogenicity traits, and provide a better understanding of the potential selective advantages that underlie serotype switching and emergence of serotype O12 ST111.