Julian Parkhill

Bacterial genetic variation contributes to the outcome of infection, but its relationship with host immune responses is rarely investigated in the same patients, limiting the biological interpretation of pathogen-associated disease severity. Here, we integrated pathogen genomics, clinical outcomes and host transcriptomics to investigate host-pathogen interactions in melioidosis, a frequently fatal infection caused by Burkholderia pseudomallei . We analysed prospective clinical data from 2,142 patients, whole-genome sequences from 2,101 infecting isolates and 646 paired host blood transcriptomes across five cohorts collected between 2012 and 2023. Infections were typically dominated by a single strain, with limited within-host diversification, supporting the use of an infecting isolate genome to investigate pathogen- associated clinical outcomes. Bacterial genome-wide association studies identified one locus associated with binary mortality at genome-wide significance. Incorporating patient age, sex, body mass index and Charlson Comorbidity Index increased this to four, while modelling time to death as a continuous phenotype identified seven loci. Integration with paired host transcriptomes linked mortality-associated bacterial genetic variation to imputed signatures of increased immature neutrophils and reduced neutrophil degranulation. An intergenic region upstream of the polyketide synthesis locus BPSS1006 - BPSS1011 was additionally associated with downregulation of host genes involved in myeloid-cell signalling and cell proliferation pathways. These findings connect naturally occurring B. pseudomallei genomic variation associated with mortality to distinct host immune signatures, providing insight into pathogen- host interactions underlying severe melioidosis and a framework for investigating the contribution of pathogen genetic diversity to clinical outcomes.

Staphylococcus aureus is a major human pathogen and commensal of the nose and other body sites. S. aureus colonisation (carriage) increases the risk of infection, so identifying carriers is central to infection control; however, few studies have combined robust methods with sufficient statistical power to determine the risk factors for carriage. We recruited over 22,000 adults into the CARRIAGE cohort study, who each returned self-taken nasal swabs and a questionnaire weekly for three weeks. We show that in a multivariable analysis with correction for multiple testing that the only factors significantly associated with persistent S. aureus carriage were younger age (with carriage rates declining after 60), male sex, higher BMI, chronic skin conditions, and antibiotic use in the prior month. Many previously proposed factors, including smoking and livestock contact, were not significantly associated. Overall, we demonstrate that only a limited number of risk factors influence S. aureus colonisation

Escherichia coli sequence type (ST) 131 is the most widely studied genetic lineage of the species to date, originally identified in the early 2000s as an increasingly common cause of human urinary tract and bloodstream infections worldwide. This lineage is subdivided into four extant main subclades A, B, C1 and C2 that exhibit distinct features in terms of invasiveness, antibiotic resistance and transmissibility. However, the evolutionary pathway from the generally susceptible ST131-B to the drug-resistant ST131-C clades remains poorly mapped. To fill this knowledge gap, we analysed in detail human clinical isolates obtained in Vietnam, designated as belonging to the generally neglected minor clade ST131-B0. We sequenced them using both short- and long-read technology and combined these data with a recently published high-resolution genomic collection to provide further insight into the evolutionary process and its timeline. Extensive genomic analyses established ST131-B0 as an intermediary progenitor in the evolutionary path leading from ST131-B to the ST131-C clades, associated with multiple type I pili switches, as well as the loss and gain of specific chromosomal genes representing diverse core functions such as metabolism, transcription, DNA binding and type II toxin–antitoxin systems. Furthermore, all Vietnamese isolates of ST131-B0 unprecedentedly harboured bla _CTX-M genes encoding extended-spectrum β-lactamases, a trait dominant in ST131-C clades and not previously observed in ST131-B0. Our study supports the hypothesis that the ST131-C clades have gradually evolved from ST131-B by reducing the host range with better adaptation to colonizing humans under selective conditions.

Hantaviruses are emerging zoonotic viruses that infect humans with high fatality rates. We currently have a poor understanding of the processes that contribute to hantavirus mutagenesis and therefore the potential for viral adaptation. To understand the drivers of mutations in hantaviruses, we calculated and compared mutational spectra across 14 divergent hantavirus species. We found that mutagens that are conserved across host species have a major impact on hantaviruses mutagenesis, contributing to at least six of the 12 possible mutation types. We found differences in mutational signatures across hantaviruses, some of which may be due to variation in host mutagen levels that are known to influence somatic mutations. However, we could find no evidence for differential mutational signatures between viruses associated with distinct diseases in humans and no evidence of tissue tropism differences between reservoir hosts. Using a novel method to quantify the impact of ZAP-mediated selection, we found that ZAP greatly influences mutational patterns in transition mutation types across hantaviruses. We also find evidence that APOBEC family proteins drive mutagenesis in all hantaviruses, with the likely exception of Nova virus. Our results therefore provide strong support that mutagens and host antiviral proteins are major contributors to hantavirus mutagenesis, thereby generating the genetic diversity necessary for viral adaptation.

In low-resource settings, drinking water is susceptible to faecal contamination, but the epidemiological link between extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in humans and in drinking water is poorly understood. This study characterised E. coli from household drinking water and human clinical samples in a closed setting with the aim of investigating the directionality of ESBL-positive E. coli spread. Sixty E. coli isolates from 38 human clinical and 22 drinking water samples were assessed for resistance to 17 antibiotics and genome-sequenced using Oxford Nanopore long-read technology (ONT). An additional 255 E. coli genomes associated with human infections in the setting were retrieved from public sequence read archives and included in the study. Core-genome alignments were used to build maximum-likelihood phylogenetic trees. To identify transmission events, pairwise single-nucleotide polymorphism (SNP) distances were computed for all strains with a maximum inter-isolate distance of 25 SNPs, defining a transmission network. Escherichia coli strains obtained from human and drinking water sources bore similar resistance genes encoding resistance to tetracycline, aminoglycosides, ciprofloxacin, and β-lactams, including carbapenems, often encoded on plasmids. Screening all 316 isolates for resistance genes reveals that human-derived isolates bore the greatest burden of resistance genes. Evidence of recent E. coli transmission between drinking water sources and of strain sharing among human hosts was observed. Core genome analysis revealed that at least one pair of human and environmental isolates was within the SNP cutoff, implying that some E. coli causing human infection could be traced to drinking water sources.

The expanding global crisis of bacterial infections caused by antimicrobial-resistant pathogens has resulted in an urgent need for therapeutics. Previous efforts to target pathogen surface antigens with monoclonal antibodies (mAbs) have sought to activate the complement system, primarily through the antibody-dependent classical pathway. However, most mAbs have induced insufficient complement activation because pathogen evasion strategies disrupt the complex spatiotemporal requirements for activation of the classical pathway initiation complex C1. To address this, we developed a targeted complement activation therapy (T-CAT), which uses antibodies furnished with the enzymatic capability to initiate complement activation directly on the bacterial surface without involvement of the classical pathway initiation complex. We found that T-CAT mAbs directed against bacterial surface antigens can overcome the strategies that pathogens evolved to escape from classical pathway–mediated clearance by the immune system. We further demonstrated that T-CAT mAbs could safely and effectively be used to treat infectious disease in experimental murine models of sepsis and pneumonia caused by Klebsiella pneumoniae , Pseudomonas aeruginosa , Streptococcus pneumoniae , and Neisseria meningitidis . Together, these data highlight the potential for T-CAT as a next-generation mAb platform with broad applicability against diverse microbial species, including multidrug-resistant pathogens, without promoting drug resistance.

Ornithobacterium hominis is a recently described Gram-negative bacterium that colonizes the human nasopharynx and may be associated with poor upper respiratory tract health. Here, we describe the isolation of O. hominis from samples collected from a South African birth cohort, creating the first archive of cultured strains of the species from Africa. Sequenced genomes from this archive reveal that South African O. hominis is more similar to Australian strains than those from Southeast Asia and that it may share genes with other members of the microbiome that are relevant for virulence, colonization and antibiotic resistance. Leveraging existing microbiome data from the cohort, O. hominis was found to be closely associated with bacterial co-colonizers that are rare in non-carrier individuals, including Suttonella , Rappaport , Helcococcus , Lwoffella , Moraxella and Gracilibacteria . Their collective acquisition has a significant impact on the diversity of nasopharyngeal communities that contain O. hominis . Individuals who have not yet acquired O. hominis have a higher abundance of Lwoffella lincolnii than individuals who never acquire O. hominis , suggesting that this could be a precursor state for successful colonization.

Environmental exposure to Burkholderia pseudomallei , the causative agent of melioidosis, remains poorly characterised due to the low sensitivity of conventional detection methods. Here, we develop CRISPR-BEEPs, a sensitive and resource-efficient CRISPR-based assay, and evaluate its performance against conventional culture-based plate inspection using double-qPCR as the reference standard. CRISPR-BEEPs demonstrated higher sensitivity (93.5% vs 19.4%) and high specificity (100% vs 98.0%). We apply the assay to water samples from natural and piped sources across 15,118 km² in northeast Thailand, collected from or near the households of 439 participants with melioidosis. We compared these with households of 190 participants with other bacterial infections and 506 healthy control participants living in the same endemic region who had never developed melioidosis. CRISPR-BEEPs detects B. pseudomallei in 73.3% of groundwater, 32.9% of surface water, and 26.2% of piped water samples, with results comparable to double-qPCR. The improved sensitivity reveals a significant association between environmental detection within 10 km of households and melioidosis risk (OR 2.74; 95% CI:1.38-5.48), an association undetectable using conventional methods. These findings expose critical sanitation gaps and highlight the value of high-resolution environmental surveillance for disease prevention.

Streptococcus canis represents a major canine pathogen, accounting for 22.4% of streptococcal infections in dogs. However, despite its prevalence in veterinary medicine, the mechanisms underlying S. canis pathogenesis and survival remain poorly understood. Identifying targeted treatments against S. canis could help to reduce dysbiosis-related complications and minimize the selection of resistant neighbouring bacteria. In this study, we employed transposon-directed insertion-site sequencing for the first time to generate saturated mutant libraries of S. canis . By comparing three distinct strains, we defined the shared essential genome of this pathogen. We found that 90.4% of its essential genes are also present in the essential genomes of other related pyogenic streptococcal species, including Streptococcus pyogenes , Streptococcus agalactiae and Streptococcus equi subsp. equi , demonstrating the translational relevance of S. canis research to broader streptococcal biology. Notably, we identified two genes uniquely essential to S. canis at the terminal steps of glycolysis: ldh , which governs lactate metabolism, and pta , which catalyses the conversion of acetyl-CoA to acetyl phosphate in acetate metabolism. We propose that targeting these pathways may offer a novel, species-specific therapeutic strategy for treating S. canis infections.