Biology subjects

Middelboe, M.

Publications and source records attributed to Middelboe, M..

9 recordsLinked to original sources

bioRxiv2026

Slow to Start, Free at Last: Dual Effects of Mucin on Escherichia coli Phage T4

Bacteriophages traversing the gastrointestinal tract are exposed to extreme physicochemical stresses that may rapidly compromise virion integrity and shape infection dynamics. While some phages bind to host-derived mucins at mucosal surfaces, the functional consequences of soluble mucin glycans for phage-host interactions remain incompletely understood. Here, we show that soluble mucin glycans exert dual effects on the Escherichia coli phage T4 by delaying infection initiation while simultaneously providing environmental virion stability. Mucin-coated T4 exhibits a lag in the onset of productive infection, consistent with transient steric occlusion from E. coli, yet without impairing overall phage progeny production once infection was established. We further show that E. coli can metabolize purified mucin, supporting a model in which dynamic remodeling of the mucin matrix gradually releases T4 and enables infection. Importantly, mucin coating substantially increases T4 survival under gastrointestinal-like stresses, including acidic pH and protease exposure. Moreover, we find that in a murine gut colonization model, a single oral dose of mucin-coated T4 displayed enhanced fecal persistence over a two-week period, which correlated with prolonged suppression of E. coli populations and delayed resolution of phage-associated functional shifts in the gut microbiome. Together, we find that that soluble mucin glycans actively shape T4 phage infection kinetics, virion stability, and ecological impact in the murine gut, and support mucin-based formulations as a strategy to extend the persistence and efficacy of orally delivered phages.

microbiology↗

bioRxiv2026

Predicting Phage Host Interactions Across Taxonomic Levels: A Systematic Review and Meta-Analysis for Microbial Ecology

The prediction of phage-host interactions is key for several applications in biotechnology, medicine, and microbial ecology. Wide studies in machine learning tools have allowed the exploration of these interactions across multiple taxonomic levels. A systematic review and meta-analysis were conducted on 570 records retrieved from PubMed, Scopus, and Web of Science. Eleven studies were selected for the meta-analysis, encompassing 61 datasets. Precision across taxonomic levels (Domain, Phylum, Class, Order, Family, Genus, Species) was evaluated for several prediction tools. Statistical tests, including the Shapiro-Wilk and ANOVA tests, were used. A mixed-effects meta-regression model was used to examine the impact of taxonomic subgroups on the prediction of the proportion of Correctly Predicted PHIs. The results indicated significant variability in the performance of prediction tools across taxonomic levels. Domain-level predictions exhibited near-perfect Proportion of Correctly Predicted PHIs (0.99), whereas finer resolutions (Family and Order) showed considerable variability, with average precision values of 0.682 and 0.775, respectively. The mixed-effects meta-regression analysis revealed that Family and Species taxonomic subgroups were associated with significant reductions in the prediction Proportion of Correctly Predicted PHIs with effect sizes of -0.1464 and -0.1944, respectively. Residual heterogeneity was negligible, indicating that the moderators adequately explained the variability in prediction precision. This study highlights the importance of selecting the appropriate prediction tool based on the desired taxonomic resolution. The findings emphasize the need for further refinement of prediction algorithms, particularly at the Family and Species levels, where tools exhibit the most variability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/721508v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@4105bforg.highwire.dtl.DTLVardef@e07c46org.highwire.dtl.DTLVardef@1ff139corg.highwire.dtl.DTLVardef@1608690_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Overview of the systematic review and meta-analysis framework evaluating ML-based phage-host interaction prediction tools across taxonomic levels. C_FIG

microbiology↗

bioRxiv2026

Infauna selectively enhance DNA virus diversity and activity in marine sediments

Viruses regulate microbial mortality and biogeochemical cycling in marine sediments; however, the ecological drivers of sediment viral communities remain unclear. Infauna, including sediment-dwelling meiofauna and macrofauna, are major ecosystem engineers that reshape sediment structures and microbial processes, but their influence on viruses is unknown. We combined infaunal gradient incubations with metagenomic and metatranscriptomic analyses to assess viral DNA and RNA responses. DNA viruses showed increased abundance (3-fold), diversity, richness, and transcriptional activity under higher infauna abundance conditions, whereas RNA viruses remained unaffected, revealing striking selectivity. This selectivity reflects an infauna-dependent component mediated by bacterial activity that cannot be explained by host abundance alone. Infection profiling revealed increased transcription of viral replication and structural genes, and lytic viruses under high infauna conditions. These findings establish infauna as a previously overlooked regulator of DNA virus dynamics, integrating viral ecology into faunal-microbial frameworks in benthic ecosystems and suggesting potential influences on geochemical cycles. TeaserInfauna selectively shape viral communities in marine sediments, revealing an overlooked effect on DNA viruses.

microbiology↗

bioRxiv2025

Structure of NO16, a marine non-tailed vibriophage with a global distribution

Non-tailed phages remain an underexplored group in marine environments, as tailed phages have dominated sequence and culture collections. However, recent surveys indicate that non-tailed phages might be more abundant than tailed phages, and with different effects on microbial mortality and gene transfer. Here, we report the structural characterization of bacteriophage NO16, a non-tailed vibriophage, one of the simplest members of the Double Jelly Roll (DJR) lineage. Mass spectrometry analyses indicate that the virion is composed of at least nine different proteins. NO16 has a pseudoT = 21 capsid, similar to other phages in the lineage but differs in the organization of minor capsid proteins, mainly in terms of membrane-capsid contacts. The DJR major capsid protein GP19 is stabilized by a cation in the base as previously observed in corticovirus PM2, and by strong electrostatic interactions between monomers. Notably, localized reconstruction reveals a symmetry mismatch at the vertex, where two trimeric GP13 spikes are anchored to a pentamer of the penton base GP14. GP13 presents carbohydrate-binding modules and putative glycosylase activity, indicating implications for host entry. Finally, we use structural and functional predictions for NO16 non-structural proteins to propose a complete atlas of the NO16 infectious cycle.

microbiology↗

bioRxiv2025

Synergistic effects of commensals and phage predation in combating pathogen infections: a shrimp model

Colonization resistance is a fundamental mechanism by which microbiomes suppress pathogen invasion, yet the factors governing its efficacy remain unclear. By constructing a defined microbial consortium and employing in vivo models, we provide evidence supporting that phage-mediated pathogen suppression interacts with competitive dynamics within microbial communities to enhance colonization resistance. While individual species conferred limited protection, combining key taxa with phage treatment extensively improved pathogen exclusion. Phage-induced pathogen killing amplified nutrient competition among commensals, however, the timing intervention emerged as a crucial determinant of efficacy. Guided by these ecological principles, we formulated a minimal, synergistic consortium that robustly enhanced colonization resistance in vivo. These findings advance our understanding of microbiome-driven pathogen resistance and provide a strategic framework for designing protective microbial consortia with potential applications in human and animal health.

microbiology↗

bioRxiv2024

Spatial heterogeneity influences phage-host co-existence dynamics and phage resistance development in Vibrio anguillarum

Exposure of bacterial populations to phage infection pressure is a significant driver of phage-host co-evolution and diversification. However, the impact of the spatial organization of bacterial population co-existence and co-evolution dynamics remains poorly understood. Here, we investigated how the spatial structure of the host population affects phage-host interactions by co-cultivating a Vibrio anguillarum strain with the lytic T4-like vibriophage KVP40 under conditions that resulted in either a homogenous, well-mixed population or a heterogeneous population with bacterial aggregates. We observed markedly different temporal dynamics associated with the two population structures over a 30-day adaptive laboratory evolution experiment. Phage and host dynamics suggested that phage-sensitive subpopulations retained in the aggregates substantially prolonged the coexistence of phages and bacteria, relative to the homogenous environment. However, the limited phage propagation on the sensitive subpopulations was insufficient for host range expansion to occur. In contrast, daily supplementation of sensitive host bacteria in parallel experiments readily led to the emergence of phage mutants with enhanced infective capability and an expanded host range. These results underscore the importance of bacterial spatial organization in determining the outcome of phage attacks and highlight the complex interplay between genomic processes and ecological conditions in driving evolutionary innovations.

microbiology↗

bioRxiv2024

Diversification of Pseudomonas aeruginosa biofilm populations under repeated phage exposures decreases the efficacy of the treatment

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

microbiology↗

bioRxiv2020

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and Type IX Secretion System

Increasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence. ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

microbiology↗

bioRxiv2020

Prevalence of genetically similar Flavobacterium columnare phages across aquaculture environments reveals a strong potential for pathogen control

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 228 bacterial isolates demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential to be used in phage therapy. Significance StatementBacteriophages were discovered already over a century ago, and used widely in treatment of bacterial diseases before the era of antibiotics. Due to harmful effects of antibiotic leakage into environment, aquaculture is a potential target for phage therapy. However, the development of efficient phage therapy approach requires detailed characterization of bacterial pathogen virulence and phage host range. Here, we describe phage-bacterium interactions in the fish pathogen Flavobacterium columnare. We found that genetically similar phages are found from different fish farms, and their infectivity cluster according to genetic group of bacteria. In addition, phages were able to infect bacterial hosts from other farms, which is a preferable trait considering phage therapy approach. However, the most recently isolated phages had broader host range than the previously isolated phages, suggesting a response in the phage community to evolution of resistance in the bacteria. These results show that designing phage therapy for aquaculture (and other) systems needs consideration of both temporal and geographical aspects of the phage-bacterium interaction.

microbiology↗