Biology subjects

Gally, D. L.

Publications and source records attributed to Gally, D. L..

10 recordsLinked to original sources

bioRxiv2026

Integrating bacterial and viral genomic information enhances and extends the machine learning predictions of bacteriophage activity against pathogenic Escherichia coli

The use of bacteriophage (phage) to treat bacterial infections is undergoing a resurgence due to the rise of antibiotic resistance. Pathogenic Escherichia coli, including extraintestinal pathotypes such as uropathogenic E. coli (UPEC), pose a substantial clinical burden with the challenge of multi-drug resistance strains, making it important to progress alternative treatment options such as those based on phage. One major hurdle is selecting effective phage combinations against an infecting strain while accounting for the multiple mechanisms that determine phage susceptibility and bacterial resistance. We previously analysed over 9000 interactions between 31 phage and 314 sequenced E. coli and built machine learning models to predict phage activity against "unseen" E. coli based on these scores and bacterial genome sequence data. However, this approach did not leverage phage gene content, limiting its ability to generalise across phage. Here, we advance this framework by combining bacterial and phage pangenomes into a single predictive model, allowing gene content across related phage as well as bacteria to be incorporated as machine learning features. This unified approach increased predictive accuracy relative to the single-phage models and produced a single model capable of predicting previously unseen phage-E. coli interactions. In leave-phage-out analyses, it also predicted activity for phages whose interaction data were entirely excluded from training. The genomic features most consistently contributing to prediction included bacterial determinants of surface recognition and anti-phage defence, alongside phage-associated features, indicating that this combined model, termed "PanPhage", captures genetic information relevant to multiple stages of the phage-host interaction.

microbiology↗

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↗

bioRxiv2025

Phage-encoded sRNA counteracts xenogenic silencing in pathogenic E. coli

Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogenic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet to be expressed they must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in multiple copies (up to nine) in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes and is enriched in E. coli strains that carry the locus of enterocyte effacement (LEE). We show that HnrS directly base-pairs with the ribosome-binding site of the hha mRNA, repressing its translation and thereby reducing Hha-enhanced H-NS silencing. This counter-silencing de-represses the LEE type III secretion system and concomitantly represses motility. Transcriptomic profiling further revealed that HnrS indirectly activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways that contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that prophage-encoded, multicopy hnrS provides a counter-silencing mechanism that reduces Hha-H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the core H-NS regulon. HnrS illustrates how mobile genetic elements deploy sRNAs to counteract xenogenic silencing and promote virulence gene expression, enhancing colonisation of the host. Importance statementHorizontally acquired genes are often silenced to protect bacterial genomes, but this defence also limits the expression of new traits. We identify a prophage-encoded small RNA, HnrS, that counteracts this restriction by repressing the xenogenic silencer Hha, lifting Hha-H-NS-mediated repression of virulence and metabolic genes. HnrS activates the locus of enterocyte effacement, nitrate/nitrite respiration, and nitric oxide resistance--pathways that help E. coli survive and colonise the inflamed gut. Our findings reveal an RNA-based counter-silencing mechanism encoded by mobile genetic elements, showing how phages can reprogram bacterial regulatory networks to promote adaptation and pathogenicity.

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↗

bioRxiv2020

Prophage-dependent recombination drives genome structural variation and phenotypic heterogeneity in Escherichia coli O157:H7

The human zoonotic pathogen Escherichia coli O157 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157 serogroup and defines the phenotypic consequences of specific structural variants. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157 and by using both optical mapping and ONT long-read sequencing demonstrate that LCRs are generated in laboratory cultures started from a single colony and particular variants are selected during animal colonisation. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga toxin production, type 3 secretion and motility are affected by LCRs. In summary, E. coli O157 has acquired multiple prophage regions over time that act as genome engineers to continually produce structural variants of the genome. This structural variation is a form of epigenetic regulation that generates sub-population phenotypic heterogeneity with important implications for bacterial adaptation and survival. Author SummaryEscherichia coli has an open genome and has acquired genetic information over evolutionary time, often in the form of bacteriophages that integrate into the bacterial genome (prophages). E. coli O157 is a clonal serogroup that is found primarily in ruminants such as cattle but can cause life-threatening infections in humans. E. coli O157 isolates contain multiple prophages including those that encode Shiga-like toxins which are responsible for the more serious disease associated with human infections. We show in this study that many of these prophages exhibit large regions of sequence similarity that allow rearrangements to occur in the genome generating structural variants. These occur routinely during bacterial culture in the laboratory and the variants are detected during animal colonization. The variants generated can give the bacteria altered phenotypes, such as increased motility or toxin production which can be selected in specific environments and therefore represent a highly dynamic mechanism to generate variation in bacterial populations without a change in overall gene content.

microbiology↗

bioRxiv2020

A high-throughput genomic screen identifies a role for the plasmid-borne Type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions

Food-borne illness arising from Shiga-toxigenic Escherichia coli (STEC) is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. The initial stages of the interaction involve chemotaxis, attachment and potentially, responding to the early stages of microbe perception by the plant host. We used a high-throughput positive-selection approach to identify early interaction factors of E. coli O157:H7 isolate Sakai to spinach. A bacterial artificial chromosome (BAC) clone library was quantified by microarray hybridisation, and gene loci enrichment measured using a Bayesian hierarchical model. The screen of four successive rounds of short-term (2 hour) interaction with spinach roots produced in 115 CDS credible candidates, comprising seven contiguous genomic regions. Two candidate regions were selected for functional assessment: a chaperone-usher fimbrial gene cluster (loc6) and the pO157 plasmid-encoded type two secretion system (T2SS). Interaction of bacteria with spinach tissue was reduced in the absence of the pO157 plasmid, which was appeared to involve the T2SS EtpD secretin protein, whereas loss of loc6 did not impact interactions. The T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 {degrees}C, and etpD expressed in planta by E. coli Sakai on spinach plants. Thus, a whole genome screening approach using a combination of computational modelling and functional assays has identified a novel function for STEC T2SS in interactions with plant tissue.

microbiology↗

bioRxiv2020

Bacterial flagella disrupt host cell membranes and interact with cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of ligands and mechanisms of binding are still debated. In this study, the molecular basis of E. coli O157:H7 and S. Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. SEM revealed flagella disappearing into cellular surfaces and TEM of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6), and S. Typhimurium (P1 & P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerisation rates.

microbiology↗

bioRxiv2019

Comparison of Shiga toxin-encoding bacteriophages in highly pathogenic strains of Shiga toxin-producing Escherichia coli O157:H7 in the UK.

2.Over the last 35 years in the UK the burden of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infection has, during different periods of time, been associated with five different sub-lineages (1983-1995: Ia, I/IIa and I/IIb, 1996-2014: Ic and 2015-2018: IIb). The acquisition of a stx2a-encoding bacteriophage by these five sub-lineages appears to have coincided with their respective emergences. Oxford Nanopore Technology (ONT) was used to sequence, characterise and compare the stx-encoding prophage harboured by each sub-lineage to investigate the integration of this key virulence factor. The stx2a-encoding prophage from each of the lineages causing clinical disease in the UK were all different, including the two UK sub-lineages (Ia and I/IIa) circulating concurrently and causing severe disease in the early 1980s. Comparisons between the stx2a-encoding prophage in sub-lineages I/IIb and IIb revealed similarity to the prophage commonly found to encode stx2c, and the same site of bacteriophage integration (sbcB) as stx2c encoding prophage. These data suggest independent acquisition of previously unobserved stx2a-encoding phage is more likely to have contributed to the emergence of STEC O157:H7 sub-lineages in the UK than intra-UK lineage to lineage phage transmission. In contrast, the stx2c-encoding prophage showed a high level of similarity across lineage and time, consistent with the model of stx2c being present in the common ancestor to extant STEC O157:H7 and maintained by vertical inheritance in the majority of the population. Studying the nature of the stx-encoding bacteriophage contributes to our understanding of the emergence of highly pathogenic strains of STEC O157:H7. 3. Impact statementThe application of ONT technology to sequence UK epidemic strains of STEC O157:H7 revealed stx2a-encoding prophage exhibit a high level of diversity. There was little evidence of geographical or temporal patterns of relatedness, or of intra-UK transmission of stx2a-encoding prophage between indigenous strains. The stx2a-encoding prophage in the UK lineages associated with severe disease appear to be acquired independently and most likely from different geographical and/or environmental sources. These data provide supporting evidence for the existence of a dynamic environmental reservoir of stx2a-encoding prophage that pose a threat public health due to their potential for integration into competent, indigenous sub-lineages of STEC O157:H7. We also provide further evidence that stx2c-encoding prophage exhibit a high level of similarity across lineage, geographical region and time, and have likely been maintained and inherited vertically. 4. Data summaryAll FASTQ files and assemblies of samples sequenced in this project were submitted to the National Centre for Biotechnology Information (NCBI). All data can be found under BioProject: PRJNA315192 - https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA315192. Strain specific details can be found in the methods section under data deposition. Publicly available data used in this project can be found via Table 1 and data bibliography. O_TBL View this table: org.highwire.dtl.DTLVardef@178103aorg.highwire.dtl.DTLVardef@1718d1corg.highwire.dtl.DTLVardef@1e7605forg.highwire.dtl.DTLVardef@1683451org.highwire.dtl.DTLVardef@42c400_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1 -C_FLOATNO O_TABLECAPTIONSummary of the PHE archived and publicly available strains used within this study, their Strain ID, lineage, phage type, Stx profile, assembly accession numbers and NCBI BioProject. C_TABLECAPTION C_TBL

genomics↗

bioRxiv2019

Resistance to change? The impact of group medication on AMR gene dynamics during commercial pig production

The anthropogenic selection of antimicrobial resistance (AMR) genes is under intense scrutiny, particularly in livestock production, where group antimicrobial administration is used to control disease. Whilst large epidemiological studies provide important data on the diversity and distribution of AMR genes, we have little insight into how group antimicrobial administration impacts AMR gene abundance and diversity within a system. Here, faecal microbiome and AMR gene dynamics were tracked for six months through a standard production cycle on a commercial pig unit. Our results demonstrate that specific AMR genes have reached an equilibrium across this farming system to the extent that the levels measured were maintained from suckling through to slaughter, despite increases in microbiome diversity in early development. These levels were not influenced by antibiotic use, either during the production cycle or following whole herd medication. Some AMR genes were found at levels higher than that of the bacterial 16S rRNA gene, indicating widespread distribution across the most common bacterial genera. The targeted AMR genes were also detected in nearby soil samples, several with decreasing abundance with increasing distance from the unit, demonstrating that the farm acts as a point source of AMR gene pollution. Metagenomic sequencing of a subset of samples identified 144 AMR genes, with higher gene diversity in the piglet samples compared to the sow samples. Critically, despite overwhelming and stable levels of resistance alleles against the main antimicrobials used on this farm, these compounds continue to control the bacterial pathogens responsible for production losses and compromised welfare.\n\nImportanceGroup antibiotic treatment has been used for decades to control bacterial diseases that reduce the productivity and compromise the welfare of livestock. Recent increases in antibiotic resistant infections in humans has resulted in concerns that antibiotic use in livestock may contribute to the development of untreatable bacterial infections in humans. There is however little understanding as to how the genes that bacteria require to become resistant to antibiotics respond during and after group antibiotic treatment of livestock, particularly in systems where high levels of antibiotics have been used for a prolonged period of time. We show that in such a system, levels of specific antibiotic resistance genes are high irrespective of group antibiotic treatments, whilst dramatic reductions in antibiotic use also fail to reduce the levels of these genes. These findings have important implications for public policy relating to the use of antibiotic in livestock farming.

microbiology↗