Biology subjects

Fitzgerald, J. R.

Publications and source records attributed to Fitzgerald, J. R..

8 recordsLinked to original sources

bioRxiv2026

A systematic comparison of tools for predicting antimicrobial resistance from nanopore sequence data

Antimicrobial resistance (AMR) presents a pressing need to ensure that the right antimicrobials are used to target the right microbes at the right time. Ideally, the appropriate antimicrobial is selected after patient samples have been cultured and assessed with antimicrobial sensitivity testing (AST). However, the time needed for culture-based diagnosis leads to immediate empirical treatment, often with broad-spectrum and/or high-tier antimicrobials. Direct nanopore metagenomic whole genome sequencing to identify pathogens and predict their antimicrobial resistance is a rapid and patient-side alternative. A limitation of this approach is potential inconsistencies in in silico predicted AMR phenotypes. Here, we benchmarked the current performance of in silico AMR prediction strategies for nanopore-generated long read data. Using nanopore data paired with AST phenotyping for 201 samples representing 27 bacterial species, we assessed the impact of basecalling mode, data volume, and assembly strategy, and compared the performance of eight in silico AMR prediction tools with seven AMR databases. We found that basecalling accuracy mode does not significantly affect the overall accuracy of in silico AMR predictions, but assembly strategy and data volume both do. Prediction tools using the ResFinder database scored best for balanced accuracy (0.80 {+/-} 0.02 for both ResFinder and ABRicate), whilst DeepARG scored best for sensitivity (0.65 {+/-} 0.03); predictions were more accurate for some antibiotic classes and genera than others. However, even the best performing in silico AMR prediction strategy missed some resistance identified by lab-based AST. We conclude therefore that, currently, in silico AMR prediction can supplement lab-based AST, but cannot yet replace it.

microbiology↗

bioRxiv2025

The cow udder is a potential mixing vessel for influenza A viruses

The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a "mixing vessel" for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells, along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains, including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland, suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary cells co-express -2,3 and -2,6 - linked sialic acids, and are susceptible to co-infection with human and avian IAVs. We conclude that the US cow influenza outbreak does not simply reflect a unique adaptation of the B3.13 genotype virus; rather, the bovine udder represents a permissive niche for IAV and a plausible site for reassortment, underscoring its potential role in generating novel influenza viruses with pandemic risk.

microbiology↗

bioRxiv2024

Bacterial metabolic remodelling by convergent evolution in response to host niche-dependent nutrient availability

New pathogens often arise after host jump events between species. However, our understanding of how bacterial pathogens pivot to distinct nutrient availabilities in a new host niche is limited. Staphylococcus aureus is a multi-host pathogen responsible for a global burden of disease in humans and farmed animals. Multiple human-to-bovine host switching events led to the emergence of S. aureus as a leading cause of intramammary infection in dairy cattle. Here, we employed ex vivo milk infections to investigate how bovine S. aureus has adapted to the dairy niche revealing metabolic remodelling including upregulation of genes for lactose utilisation and branched-chain amino acid biosynthesis in response to nutrient availability. Notably, infection of milk by bovine S. aureus results in a milk clotting phenotype associated with enhanced bacterial growth that is dependent on the protease aureolysin. The same adaptive phenotype has evolved convergently in different bovine S. aureus lineages via mutations in distinct regulatory gene loci that promote enhanced aureolysin expression. Taken together, we have dissected a key adaptive trait for a bacterial pathogen after a host-switch event, involving metabolic remodelling in response to the availability of nutrients. These findings highlight the remarkable evolutionary plasticity of S. aureus underpinning its multi-host species tropism.

microbiology↗

bioRxiv2023

Time-lapse mesoscopy of Candida albicans and Staphylococcus aureus dual-species biofilms reveals a structural role for the hyphae of C. albicans in biofilm formation

Polymicrobial infection with Candida albicans and Staphylococcus aureus may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors including metabolic processes and direct interaction of S. aureus with C. albicans hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, however the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of C. albicans/S. aureus whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form C. albicans in the biofilm founder-population profoundly affects the structure of the biofilm as it matures. Different sub-populations of C. albicans and S. aureus arise within each biofilm as a result of the different C. albicans morphotypes, resulting in distinct sub-regions. These data reveal that C. albicans cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection.

microbiology↗

bioRxiv2023

Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections

Antimicrobial resistance is one of the greatest current threats to human and animal health. There is an urgent need to ensure that antimicrobials are used appropriately to limit the emergence and impact of resistance. In the human and veterinary healthcare setting, traditional culture and antimicrobial sensitivity testing is typically conducted, requiring 48-72 h to identify appropriate antibiotics for treatment. In the meantime, broad-spectrum antimicrobials are often used, which may be ineffective or impact non-target commensal bacteria. Here, we present a rapid diagnostics pipeline, involving metagenomic Nanopore sequencing directly from clinical urine and skin samples of dogs. We have optimised this pipeline to be versatile and easily implementable in a clinical setting, with the potential for future adaptation to different sample types and animals. Using our approach, we can identify the bacterial pathogen present in a sample with 100% sensitivity within 5 hours. For urine samples, we can predict antibiotic sensitivity with up to 95% accuracy. However, skin swabs which exhibited lower bacterial abundance and higher host DNA, were less amenable and an additional host depletion step may be required prior to DNA extraction. In summary, our pipeline represents an important step towards the design of individually tailored veterinary treatment plans on the same day as presentation, facilitating effective use of antibiotics and promoting antimicrobial stewardship. Impact statementAntimicrobial resistance (AMR) is a major threat to veterinary and human healthcare. It is a one-health problem, as humans and dogs are in close contact, require similar antibiotics, and share bacterial pathogens and AMR genes. Limited treatments options due to AMR would have a catastrophic effect. The risk of infection would render much of modern healthcare (including critical care, orthopaedic and complex surgeries, implants and oncology) impossible. In addition, routine infections could become life threatening. It is therefore critical to preserve the efficacy of these drugs for the future. Inappropriate antimicrobial use is the single biggest factor driving AMR. Antimicrobial stewardship involves reducing antimicrobial use, using first-line narrow-spectrum drugs, and avoiding overly long treatment. Delays in culture-based diagnosis lead clinicians to speculatively use broad-spectrum antibiotics and prolong courses of treatment beyond clinical cure. Our rapid diagnostic approach will have a major impact in reducing, refining and replacing antibiotic use. This will advance antimicrobial stewardship in veterinary and human healthcare. Data summaryAll sequencing data mentioned in this work is available from NCBI, BioProject PRJNA925092, Biosamples SAMN32880396 to SAMN32880438, run accessions SRR23195371 to SRR23195413. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2022

Nanoscaled discovery of a shunt rifamycin from Salinispora arenicola using a three-colour GFP-tagged Staphylococcus aureus macrophage infection assay.

Antimicrobial resistance has emerged as an urgent global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by GFP-tagged Staphylococcus aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a novel, active rifamycin analogue. Our findings indicate a promising new approach to the identification of anti-microbial compounds with macrophage intracellular activity. The novel antibiotic identified here may represent a useful addition to our armoury in tackling the silent pandemic of antimicrobial resistance.

cell biology↗

bioRxiv2020

Massive genome decay and expansion of insertion sequences drive the evolution of a novel host-restricted bacterial pathogen

BackgroundThe emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as those associated with a switch in host or disease tropism are often accompanied by genetic adaptation. Staphylococcus aureus is a multi-host bacterial species comprising strains with distinct tropisms for human and livestock species. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for outbreaks of Morels disease, a lymphadenitis in small ruminants. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. ResultsEvolutionary genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages representing African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, several large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes resulting in a highly fastidious metabolism. Importantly, expansion of [~]87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, representing a novel concept of the IS regulon. ConclusionsOur findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of parallel mechanisms.

microbiology↗

bioRxiv2020

Population analysis of Legionella pneumophila reveals the basis of resistance to complement-mediated killing

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires disease. L. pneumophila is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 900 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We found that although some clones are more commonly associated with clinical infections, the capacity for human disease is representative of the breadth of species diversity. To investigate the bacterial genetic basis for human disease potential, we carried out a genome-wide association study that identified a single gene (lag-1), to be most strongly associated with clinical isolates. Molecular evolutionary analysis showed that lag-1, which encodes an O-acetyltransferase responsible for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. Functional analysis revealed a correlation between the presence of a functional lag-1 gene and resistance to killing in human serum and bovine broncho-alveolar lavage. In addition, L. pneumophila strains that express lag-1 escaped complement-mediated phagocytosis by neutrophils. Importantly, we discovered that the expression of lag-1 confers the capacity to evade complement-mediated killing by inhibiting deposition of classical pathway molecules on the bacterial surface. In summary, our combined population and functional analyses identified L. pneumophila genetic traits linked to human disease and revealed the molecular basis for resistance to complement-mediated killing, a previously elusive trait of direct relevance to human disease pathogenicity. SignificanceLegionella pneumophila is an environmental bacterium associated with a severe pneumonia known as Legionnaires disease. A small number of L. pneumophila clones are responsible for a large proportion of human infections suggesting they have enhanced pathogenicity. Here, we employed a large-scale population analysis to investigate the evolution of human pathogenicity and identified a single gene (lag-1) that was more frequently found in clinical isolates. Functional analysis revealed that the lag-1-encoded O-acetyltransferase, involved in modification of lipopolysaccharide, conferred resistance to the classical pathway of complement in human serum. These findings solve a long-standing mystery in the field regarding L. pneumophila resistance to serum killing, revealing a novel mechanism by which L. pneumophila may avoid immune defences during infection.

microbiology↗