Biology subjects

Clokie, M. R. J.

Publications and source records attributed to Clokie, M. R. J..

6 recordsLinked to original sources

bioRxiv2025

Effect of human urinary microenvironment and fluid flow on antibiotic and phage therapy efficacy against uropathogenic Escherichia coli

Urinary tract infections (UTI) remain a major global health burden, with high recurrence despite antibiotic treatment. The escalating prevalence of antimicrobial resistance further compromises therapeutic efficacy, contributing to an estimated 260,000 deaths annually. Conventional in vitro susceptibility assays often fail to predict clinical outcomes, underscoring the urgent need for physiologically relevant infection models. Here, we examined how microenvironmental complexity shapes uropathogenic Escherichia coli (UPEC) responses to antibiotics and bacteriophages using: human urine, a three-dimensional urothelial microtissue model (3D-UHU), and a novel mesofluidic system (P-FLO) that introduces physiologically relevant flow dynamics to the 3D-UHU. P-FLO was engineered from cost-effective 3D-printed components compatible with standard Transwell systems. Among the antibiotics tested, nitrofurantoin exhibited the greatest potency in minimum inhibitory concentration assays, but it failed to fully eradicate infection within the more physiological 3D-UHU model. A bacteriophage cocktail (LCPR1) showed markedly reduced activity in urine compared with nutrient-rich media, highlighting the influence of infection-site conditions. In contrast, in 3D-UHU, LCRP1 modulated host responses without reducing bacterial burden. Combination therapy (nitrofurantoin + LCPR1) eliminated planktonic bacteria under static conditions but offered no added benefit against adherent or intracellular populations relative to antibiotic monotherapy. Incorporating flow revealed additional layers of complexity, where shear stress induced bacterial elongation and attachment and altered drug performance, diminishing the efficacy of nitrofurantoin and combination therapy against planktonic populations despite increased drug exposure. Together, these findings demonstrate that the bladder microenvironment and its mechanical forces modulate host-pathogen interactions and profoundly influence UPEC infection dynamics and therapeutic outcomes, emphasizing the need for advanced, physiologically informed models to guide treatment strategies in the post-antibiotic era.

microbiology↗

bioRxiv2025

Hidden in plain sight: Bacterial genomes reveal thousands of lytic phages with therapeutic potential

Phages are typically classified as temperate, integrating into host genomes, or lytic, replicating and killing bacteria. Lytic phages are not expected in bacterial genome sequences, yet our analysis of 3.6 million bacterial assemblies from 1,226 species revealed >100,000 complete lytic phage genomes, which we term Bacterial Assembly-associated Phage Sequences (BAPS). This represents a [~]five-fold increase in the number of phages with known hosts and fundamentally reshapes our understanding of phage biology. Identifying BAPS has enabled the discovery of entirely novel phage clusters, including clusters distantly related to Salmonella Goslarviruses in E. coli, and Shigella, while significantly expanding known genera such as Seoulvirus (from 16 to >300 members). Notably, close relatives of therapeutic lytic phages were also detected, suggesting clinical isolate sequencing unknowingly archives viable phage candidates. The discovery of complete, lytic phage genomes within bacterial assemblies challenges long-standing assumptions and reveals a vast, untapped reservoir of phages.

microbiology↗

bioRxiv2025

In-vivo Efficacy of a Phage Cocktail Therapy that Targets ESBL-producing Klebsiella Urinary Tract Infections Clinical Isolates

Uropathogenic Klebsiella pneumoniae strains, are of major concern of with respect to antimicrobial resistance, which has sparked interest in phage therapy as an alternative, or compliment to antibiotics. However, very limited in vivo data on phage therapy safety and efficacy for treating UTIs is available. To address this, we developed a model to test these parameters for a phage cocktail optimised for Extended Spectrum Beta-Lactamases (ESBL)-producing Klebsiella UTI strains. Female C57BL/6J mice were infected with K. pneumoniae Top52 then received a single dose of the purified therapeutic phages, with observations over seven days. Results showed a significant reduction of bacterial burden in urine, bladder, and kidney samples starting from 4 h post-treatment until Day 7. Phage treatment also reduced local inflammation within the bladder. Additionally, cytokine profile demonstrated an increase in anti-inflammatory IL-10 and a decrease in pro-inflammatory IL-6, indicating a synergy between phage and the host immune response.

microbiology↗

bioRxiv2022

Defence systems provide synergistic anti-phage activity in E. coli

Bacterial defence against phage predation involves diverse defence systems acting both individually and concurrently, yet their interactions remain poorly understood. We investigated >100 defence systems in 42,925 bacterial genomes and identified numerous instances of their non-random co-occurrence and mutual exclusion. For several pairs of defence systems significantly co-occurring in Escherichia coli strains, we demonstrate synergistic anti-phage activity, including tmn synergising with defence systems containing sensory switch ATPase domains. Some of the defence system pairs that are mutually exclusive in E. coli significantly co-occur in other taxa, and one tested mutually exclusive pair showed synergy. These findings demonstrate compatibility and synergy between defence systems, allowing bacteria to adopt flexible strategies for phage defence, driven by specific environmental pressures. The evolution of bacterial immune repertoires seems to be shaped largely by selection for resistance to host-specific phages rather than by negative epistasis.

microbiology↗

bioRxiv2021

Identification of the receptor-binding protein of Clostridium difficile phage CDHS-1 reveals a new class of receptor-binding domains.

As natural bacterial predators, bacteriophages have the potential to be developed to tackle antimicrobial resistance, but our exploitation of them is limited by understanding their vast uncharacterised genetic diversity1,2. Fascinatingly, this genetic diversity reflects many ways that phages can make proteins, performing similar functions that together form the familiar phage particle. Critical to infection are phage receptor-binding proteins (RBPs) that bind bacterial receptors and initiate bacterial entry3. Here we identified and characterised Gp22, a novel RBP for phage CDHS-1 that infects pathogenic C. difficile, but that had no recognisable RBPs. We showed that Gp22 antibodies neutralised CDHS-1 infection and used immunogold-labelling and transmission electron microscopy to identify their location on the capsid. The Gp22 three-dimensional structure was resolved by X-ray crystallography revealing a new RBP class with an N-terminal L-shaped -helical superhelix domain and a C-terminal Mg2+-binding domain. The findings provide novel insights into C. difficile phage biology and phage-host interactions. This will facilitate optimal phage development and future engineering strategies4,5. Furthermore, the AlphaFold2-predicted Gp22 structure, which was strikingly accurate, paves the way for a structurome based transformation and guidance of future phage studies where many proteins lack sequence homology but have recognisable protein structures.

molecular biology↗

bioRxiv2018

Oscillating bacterial expression states generate herd immunity to viral infection

Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity, enabling individual populations to survive fluctuating, often antagonistic, selection pressures. As observed for adaptive immunity, hypermutation may facilitate survival of multiple, spatially-separated bacterial populations. We developed an oscillating prey assay to examine bacteriophage (phage) spread through populations of Haemophilus influenzae whose phage receptor gene, lic2A, is switched ON and OFF by mutations in a hypermutable tetranucleotide repeat tract. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%. In silico modelling indicated that phage extinction was interdependent on phage loss during transfer between populations and the frequency of resistant populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across multiple bacterial populations resulting in protective quarantining of some populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial herd immunity with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-sensitive sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions.\n\nImportanceHerd immunity is a survival strategy wherein populations are protected against invading pathogens by resistant individuals within the population acting as a barrier to spread of the infectious agent. Although, this concept is normally only applied to higher eukaryotes, prokaryotic organisms also face invasion by infectious agents, such as bacterial viruses, bacteriophage (phage). Here we use novel experimental approaches and mathematical modelling, to show that bacteria exhibit a form of herd immunity through stochastically generated resistant variants acting as barricades to phage predation of sensitive cells. With hypermutable loci found in many prokaryotic systems, this phenomenon may be widely applicable to phage-bacteria interactions and could even impact phage-driven evolution in bacteria.

microbiology↗