Biology subjects

Park de la Torriente, A.

Publications and source records attributed to Park de la Torriente, A..

3 recordsLinked to original sources

bioRxiv2026

Analysis of a detoxified Escherichia coli strain for bacteriophage production

Phage therapeutics are re-emerging as adjuncts or alternatives to antibiotics and their clinical translation will be enhanced with production methods that minimise downstream processing. We evaluated whether an endotoxin-reduced E. coli strain developed for production of recombinant proteins, ClearColi(R), can serve as a useful, safe phage production host without compromising yield and whether targeted receptor complementation can expand its utility. The parent strain BL21(DE3), and its lipid A modified derivative, ClearColi(R), were compared with respect to infection and generation of phage. Across a panel of 31 phage, a similar host range was observed between BL21(DE3) and ClearColi(R). To expand host range ompC was genetically engineered into the chromosome of ClearColi(R), thereby adding OmpC-dependent phage to its production capacity. Production metrics were broadly comparable between the hosts; efficiency of plating and final titres for representative phage were not significantly different; burst size varied by phage but without consistent host bias. Endotoxin activity in ClearColi(R)-propagated lysates was reduced by over 1000-fold relative to BL21(DE3), reaching the low hundreds of endotoxin units (EU) versus hundreds of thousands for BL21(DE3). Intravesical administration of ClearColi(R)-derived phage (LUC4) into pigs elicited no clinical abnormalities and no significant increases in circulating cytokines up to 48 hours after administration. ClearColi(R) allows efficient production of diverse phage with low endotoxin, reducing the requirement for downstream processing. Although its minimal LPS reduces its capacity for producing some LPS-dependent phage and its growth is slower than BL21(DE3), requiring optimisation for maximal phage titre, the safety and simplified manufacturing process support further development of endotoxin modified strains for phage production. Impact statementAntibiotic resistance is a current global problem and treatments based on phage and phage products already have a proven track record with particular bacterial infections, especially in the urinary tract. While progress is being made on in vitro phage synthesis, large scale bacteriophage preparations require a bacterial host for production, consequently toxic components in the initial lysate need to be removed or significantly diluted for safe clinical use. This is a study of the potential to utilise an endotoxin-reduced E. coli strain, ClearColi(R), to produce safer phage therapeutics. Such endotoxin modified strains should minimise the processing steps required and reduce overall production costs of a phage preparation. The research demonstrates that the endotoxin-reduced strain was able to produce a wide range of phage and for studied examples at phage titres equivalent to the more toxic parent strain. We also show that the strain can be modified to increase its host range and confirm the very low endotoxicity of basic phage lysates produced by the strain. Replicating this process to engineer additional low-toxicity bacterial production strains will accelerate the development of safer, more cost-effective phage therapeutics.

microbiology↗

bioRxiv2026

Quorum sensing and capsule expression enable subpopulation evasion of phage killing in Escherichia coli ST131: Implications for targeted therapy

Escherichia coli ST131 is a globally disseminated multidrug-resistant lineage frequently associated with recalcitrant urinary tract infections (UTIs) and bacteraemia. While bacteriophages offer a promising alternative treatment to antibiotics, their efficacy is often limited in physiologically relevant conditions in comparison to laboratory media. In this study, we have investigated the mechanisms by which the representative ST131 strain, EC958, evades elimination by a model phage, LUC4. We observed that in the urine environment, EC958 can transiently resist phage infection by a density dependent mechanism and by the production of protective polysaccharides. Based on this understanding, we developed a phage treatment strategy that can sterilise an EC958 culture in urine-based medium, even at high bacterial densities. The rational design of the successful phage therapy strategy utilises a tailored phage cocktail containing phage that encode depolymerase enzymes to degrade bacterial surface carbohydrates and the targeting of multiple receptors to prevent the emergence of fixed genetic resistant mutants. We found the addition of specific carbon sources renders the bacteria more susceptible to phage infection. By combining these findings with a simulated bladder wash to model voiding, we successfully achieved elimination of EC958 cultures in a urine environment. This study provides a framework for overcoming both fixed and reversible phage resistance, offering a translatable strategy for effectively treating urinary tract infections with phage. Author SummaryIn this study, we investigated how bacterial populations can overcome a phage infection. Phage are viruses that naturally kill bacteria and provide an alternative treatment to antibiotics. We focussed on a particularly aggressive and antibiotic resistant strain of E. coli, EC958, which belongs to a group of E. coli strains that are a leading cause of urinary tract infections and life-threatening bloodstream infections worldwide. We found that in a simulated bladder environment, these bacteria do not rely on genetic mutations to survive but they employ a range of hide and seek strategies. We showed that bacteria can coat themselves in a protective layer to block the phage and use social signalling to enter a dormant state when cell density is high. When they are in this sleep-like state the phage cannot successfully infect. To overcome these bacterial defences, we developed a treatment strategy combining effective phage with specific naturally occurring additives, that trick the bacteria into waking up and becoming vulnerable again to phage infection. By also simulating a clinical bladder wash to reduce bacterial numbers and therefore reduce social-signalling, we were able to eliminate the bacterial population. Our findings suggest that by understanding bacterial strategies we can design more effective and personalised phage therapies to treat bacterial infections.

microbiology↗

bioRxiv2023

Predictive phage therapy for Escherichia coli urinary tract infections: cocktail selection for therapy based on machine learning models

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning models (MLM). For this purpose, MLM were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli (E. coli) associated with urinary tract infections. This is an important common infection in humans and companion animals from which multi-drug resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage were measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy. Significance StatementWith the growing challenge of antimicrobial resistance there is an urgency for alternative treatments for common bacterial diseases including urinary tract infections (UTIs). Escherichia coli is the main causative agent of UTIs in both humans and companion animals with multidrug resistant strains such as the globally disseminated ST131 becoming more common. Bacteriophage (phage) are natural predators of bacteria and potentially an alternative therapy. However, a major barrier for phage therapy is the specificity of phage on target bacteria and therefore difficulty efficiently selecting the appropriate phage. Here, we demonstrate a genomics driven approach using machine learning prediction models combined with phage activity clustering to select phage cocktails based only on the genome sequence of the infecting bacterial strain.

microbiology↗