Biology subjects

Couto, N.

Publications and source records attributed to Couto, N..

10 recordsLinked to original sources

bioRxiv2026

AMRgen: an R package for antimicrobial resistance genotype-phenotype analysis

Microbial whole-genome sequence data is now generated at scale, including to support antimicrobial resistance (AMR) surveillance and understand resistance mechanisms, yet analytical infrastructure for systematically linking AMR genotypes to measured phenotypes remains fragmented. Here we present AMRgen, an R package to support systematic AMR genotype-phenotype analysis. AMRgen imports and harmonises genotypic data from common bioinformatics tools, alongside phenotypic data from automated antimicrobial susceptibility testing instruments and public repositories. It supports common analyses linking data to reference distributions, modelling associations, quantifying concordance, and producing publication-ready visualisations including UpSet plots that jointly display genotypic marker combination frequencies and associated phenotypic distributions. We demonstrate AMRgens utility using publicly available surveillance data for World Health Organization priority AMR pathogens, Neisseria gonorrhoeae, Klebsiella pneumoniae, Escherichia coli and Salmonella enterica. AMRgen, available free and open-source at https://AMRgen.org, provides a reproducible end-to-end foundation for genotype-phenotype research in AMR genomics, clinical microbiology, and public health surveillance.

microbiology↗

bioRxiv2025

Monitoring of vaccine targets and interventions using global genome data: vaccines.watch

The expansion of pathogen genome sequencing into routine disease surveillance programmes is set to bring rapidly-growing volumes of increasingly structured data on a global scale. This has the potential to deliver exciting opportunities for accelerating vaccine development and monitoring. Here we present an interactive platform, vaccines.watch (https://vaccines.watch), which aims to support decision-making around vaccine formulations and roll-out by enabling interrogation of vaccine target diversity from global genome data. We have initially focused on targets included in existing or prospective multivalent polysaccharide- based vaccines for Streptococcus pneumoniae, Klebsiella pneumoniae (and related species) and Acinetobacter baumannii. The platform currently displays data for >100k high-quality genomes with geotemporal sampling information (post-2010), with new genomes assembled, analysed and incorporated on an ongoing basis (every 4 hours) as public data are newly deposited. Crucially, users can view vaccine target information in the broader context of genotypic variants (e.g. sequence types) and antimicrobial resistance markers. The platform also enables users to review the composite serotypes of pneumococcal vaccine formulations among the available genomes. For example, using data in vaccines.watch from 3 June 2025, we observed that serotypes included in the PCV13 and PCV21 formulations accounted for 36.2% (11,907/32,918) and 87.4% (28,764/32,918) of global public genomes, respectively. The platform also enables continuous review of the global genomic landscape of the included pathogens, enabling identification of gaps (e.g. in geographic coverage) that should be targeted with increased genomic surveillance. Indeed we demonstrate that substantial geographic gaps remain in the coverage of available genomes, with over half of countries contributing no genomes for each of the three pathogens. However, while caution in interpretation is important, as global representativeness of genome data grows, vaccines.watch is positioned to support different stages of the vaccine pipeline, from selection of target antigens to post-rollout monitoring of population changes.

genomics↗

bioRxiv2025

amr.watch - monitoring antimicrobial resistance trends from global genomics data

BackgroundWhole genome sequencing (WGS) is increasingly supporting routine pathogen surveillance at local and national levels, providing comparable data that can inform on the emergence and spread of antimicrobial resistance (AMR) globally. However, the potential for shared WGS data to guide interventions around AMR remains under-exploited, in part due to challenges in collating and transforming the growing volumes of data into timely insights. We present an interactive platform, amr.watch (https://amr.watch), that enables interrogation of AMR trends from public WGS data on an ongoing basis to support research and policy. MethodsThe amr.watch platform incorporates, analyses and visualises high-quality WGS data from WHO-defined priority bacterial pathogens. Analytics are performed using community-standard methods with bespoke species-specific curation of AMR mechanisms. FindingsBy 31 March 2025, amr.watch included data from 620,700 pathogen genomes with geotemporal information, with highly variable representation of different species and geographic regions. By integrating WGS data with sampling information, amr.watch enables users to assess geotemporal trends among genotypic variants (e.g. sequence types) and AMR mechanisms, with implications for interventions including antimicrobial prescribing and drug and vaccine development. Interpretationamr.watch is an information platform for scientists and policy-makers delivering ongoing situational awareness of AMR trends from genomic data. As broad adoption of WGS continues, amr.watch is positioned to monitor both pathogen populations and our global efforts in genomic surveillance, guiding control strategies tailored to each pathogens characteristics. FundingUK National Institute for Health Research & Gates Foundation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWhole genome sequencing (WGS) approaches enable us to track the spread of bacterial pathogens and antimicrobial resistance (AMR) with high resolution at local, national and global levels. To date, genomic studies assessing AMR dynamics have largely used retrospective data collected for specific research agendas. However, the growing volumes of publicly-shared WGS data, generated increasingly from routine surveillance, provide improved power to detect novel trends and guide interventions. Efforts to collate public bacterial genome data exist, such as AllTheBacteria, although these are aimed at the research community and do not facilitate data usage and interpretation by non-genomics experts, particularly those in public health. To our knowledge, no platforms exist for readily interrogating AMR dynamics from continuously updated public genome data across the array of different WHO priority bacterial pathogens. Of note, however, TyphiNet provides an interactive online dashboard for examining AMR trends exclusively from Salmonella Typhi using a periodically-updated data set from the Pathogenwatch platform. These findings are based on searching PubMed without language restrictions from Jan 1 2000 to December 31 2024, using terms related to "genomic surveillance" and "antimicrobial resistance". Added value of this studyWe have developed amr.watch which, to our knowledge, is the first platform that enables ongoing analysis and visualisation of AMR trends from public genome data across the spectrum of WHO priority bacterial pathogens via an accessible interface. Crucially, the platform incorporates processed genome data via a live always-on stream, enabling insights that are delayed only by the time to data deposition. We reviewed public genomes with available geotemporal information up until 31 March 2025, providing a contemporary global landscape of pathogen genome sequencing. While the number of genomes available annually grew over five-fold globally between 2010 and 2018, we also revealed the extent of differences in geographic representation, with 89.6% of genomes originating from high-income countries and 89 countries contributing no genome data from the priority pathogens. Implications of all the available evidenceRapid generation and sharing of global genome data enables us to more precisely track the spread and define the characteristics of contemporary circulating resistant pathogens. The amr.watch platform provides a solution for retrieving, curating and translating shared genomic data into relevant insights that are accessible and actionable by diverse stakeholders. It thereby forms a basis for monitoring progress in surveillance efforts, alerting on ongoing population changes and guiding enhanced precision for surveillance and interventional development. Additionally, our review of available bacterial genome data highlights the need for additional efforts to increase and sustain the implementation of genomic surveillance of AMR globally, and improve the timely sharing of WGS data and its associated metadata.

genomics↗

bioRxiv2024

Geographical distribution, disease association and diversity of Klebsiella pneumoniae KL and O antigens in India: roadmap for vaccine development

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K. pneumoniae and its association with geographical distribution, disease severity and antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the Illumina platform, and genomic analysis was carried out using the Kleborate tool. KL64 (n=264/1072, 26%), KL51 (249/1072, 24%), KL2 (n=88/1072, 8%), O1/O2v1 (n=471/1072, 44%), O1/O2v2 (n=353/1072, 33%), and OL101 (n=66/1072, 6%) were the most prevalent serotypes. The study identified 119 different sequence types (STs) with varying serotypes, with KL64 being the most predominant in ST231 (26%). O serotypes were strongly linked with STs, with O1/O2v1 predominantly associated with ST231 (44%). Simpsons diversity index and Fishers exact test revealed higher serotype diversity in the north and east regions, along with intriguing associations between specific serotypes and resistance profiles. No significant association between KL or O types and disease severity was observed. Furthermore, we found no specific association of virulence factors with KL types or O antigen types (P>0.05). Conventionally described hypervirulent clones (i.e., KL1 and KL2) in India lacked typical virulent markers (i.e., aerobactin), contrasting with other regional serotypes. The cumulative distribution of KL and O serotypes suggests that future vaccines may have to include either [~]20 KL types or 4 O types to cover >85% of the carbapenemase-producing Indian K. pneumoniae population. The findings underscore the need for a vaccine with broad coverage to address the diverse landscape of K. pneumoniae strains in different regions of India. Understanding regional serotype dynamics is pivotal for targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse landscape of K. pneumoniae infections across India. Data SummaryO_LIAll the sequenced data has been submitted to the European Nucleotide Archive (ENA) under the Bioproject numbers PRJEB29740 and PRJEB50614. Run Accessions and Biosample numbers are provided in Supplementary Table 1 with corresponding metadata for each sample used in the study. C_LIO_LIThe Microreact link for the genomic analysis is provided (https://microreact.org/project/oqKM84GBszEPW9Emt2FKnP-klebsiella-pneumoniae-indian-serotypes). C_LIO_LIThe pipelines used in the study are published in gitlab (https://gitlab.com/cgps/ghru/pipelines). C_LIO_LIThe tools details and the implementation of the pipelines are described in protocols.io (https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bp2l6b11kgqe/v4). C_LIO_LIThe R scripts used with all the input files used for each script have been published in Fishare (https://doi.org/10.6084/m9.figshare.25414807.v1) C_LI Impact StatementKlebsiella pneumoniae produces polysaccharide capsules, which serve as both epidemiological markers and significant virulence factors. The increasing accessibility of whole genome sequencing has made it easier than ever to investigate this capsule diversity. This study is the first of its kind in India to comprehensively investigate the serotype diversity of K. pneumoniae strains and their association with disease severity, antimicrobial resistance/virulence patterns, and geographical distribution across various regions of the subcontinent. This multi-dimensional analysis not only provides valuable insights into the molecular epidemiology of K. pneumoniae in India but also offers crucial data for the development of targeted interventions, including vaccine formulations tailored to address the prevailing serotypes. These findings serve as a foundation for informed decision-making in the management and prevention of K. pneumoniae infections, ultimately contributing to improved public health outcomes in the region.

genomics↗

bioRxiv2024

Convergence and global epidemiology of Klebsiella pneumoniae plasmids harbouring the iuc3 virulence locus.

BackgroundKlebsiella pneumoniae (Kp) is an important pathogen of humans and animals, and recent reports of convergent strains that carry both virulence and antimicrobial resistance genes (ARGs) have raised serious public health concern. The plasmid-borne iuc locus, encoding the siderophore aerobactin, is a key virulence factor in this species. The variant iuc3 is associated with porcine and human clinical isolates and is carried by mostly uncharacterised IncF plasmids. MethodsWe used a combination of short-read and long-read sequencing to characterise IncFIB(K)/IncFII iuc3-carrying plasmids harboured by 79 Kp isolates and one K. oxytoca isolate recovered as part of two large One-Health studies in Italy (SpARK) and Thailand (OH-DART). Adding data from public repositories gave a combined dataset of 517 iuc3 isolates, and the plasmids were analysed using both clustering and phylogenetic methods. FindingsWe note seven large, convergent, plasmids from Thailand that have emerged through the hybridisation of co-circulating plasmids harbouring iuc3 and antimicrobial resistance genes (ARGs) encoding extended-spectrum beta-lactamases (ESBLs). We were also able to identify putative parental plasmids which were mostly associated with two neighbouring meat markets, as were the hybrid plasmids. Clustering and global phylogenetic analyses resolved an iuc3 plasmid sub-group circulating throughout Asia, with occasional examples in Europe and elsewhere. This variant carries multiple ARGs and is commonly harboured by clinical isolates, thus warranting targeted plasmid surveillance. InterpretationOur study reveals that plasmid hybridisation leading to the convergence of resistance and virulence traits may be very common, even in non-clinical ( One-Health) settings. Population-scale plasmid genomics makes it possible to identify putative parental plasmids, which will help to identify plasmid types that are most likely to hybridise, and what the selective consequences may be for the plasmid and host. A distinct iuc3 plasmid sub-variant is associated with clinical isolates in Asia which requires close monitoring. Research In ContextMultiple reports of convergent clones of Klebsiella pneumoniae that combine both hypervirulence and multidrug resistance (MDR-hvKp) have been published recently; a PubMed search in November 2023 using the key words convergence Klebsiella pneumoniae returned 143 papers, 99 of which were published from 2020 onwards. Our study demonstrates that the hybridisation of plasmids carrying AMR and virulence genes is a frequent, ongoing, process in natural populations. The subsequent transfer of plasmids conferring both traits is thus likely to be a key driver behind the spread of convergent strains. Our study also provides an exemplar of how hybrid assemblies can facilitate large-scale global genomic plasmid epidemiology. Evidence before the studyAlthough multiple recent reports highlight the emergence and spread of convergent Kp strains, the confluence of resistance and virulence genes within the same plasmid has not been studied at a population level, and putative parental plasmids are rarely identified. Moreover, there have been few high-resolution genomic epidemiology studies on closely related plasmids using both long and short-read data on a global scale. Added valueWe more than double the number of complete sequences available for plasmids harbouring iuc3 from 58 to 139 and provide evidence on the host lineages most likely to harbour these plasmids (e.g., ST35), and epidemiological source (e.g., pig, wild animal, human). Our comparative analysis of phylogenetic and clustering approaches will help to inform future plasmid epidemiological studies. ImplicationsThe hybridisation of plasmids harbouring virulence and resistance genes occurs frequently in natural populations, even within One-Health settings. However, the selective drivers (if any) and evolutionary consequences of this phenomenon are unclear. There is clear utility in generating closed plasmid genomes on a population scale, and targeted plasmid surveillance on a clinical sub-variant of iuc3 plasmids is warranted.

genomics↗

bioRxiv2021

One Health or Three? Transmission modelling of Klebsiella isolates reveals ecological barriers to transmission between humans, animals and the environment

The Klebsiella group is highly diverse both genetically and ecologically, being commonly recovered from humans, livestock, plants, soil, water, and wild animals. Many species are opportunistic pathogens, and can harbour diverse classes of antimicrobial resistance (AMR) genes. K. pneumoniae is responsible for a high public-health burden, due in part to the rapid spread of health-care associated clones that are non-susceptible to carbapenems. Klebsiella thus represents a highly pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs. Here we report an analysis of 6548 samples and 3,482 genome sequences representing 15 Klebsiella species sampled over a 15-month period from a wide range of clinical, community, animal and environmental settings in and around the city of Pavia, in the northern Italian region of Lombardy. Despite carbapenem-resistant clones circulating at a high frequency in the hospitals, we find no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside of the clinical environment. The non-random distribution of species and strains across sources point to ecological barriers that are likely to limit AMR transmission. Although we find evidence for occasional transmission between settings, hierarchical modelling and intervention analysis suggests that direct transmission from the multiple non-human (animal and environmental) sources included in our sample accounts for less than 1% of hospital disease, with the vast majority of clinical cases originating from other humans.

microbiology↗

bioRxiv2020

Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

cell biology↗

bioRxiv2019

DEN-IM: Dengue Virus identification from shotgun and targeted metagenomics

Dengue virus (DENV) represents a public health and economic burden in affected countries. The availability of genomic data is key to understand viral evolution and dynamics, supporting improved control strategies. Currently, the use of High Throughput Sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR amplified viral sequences (targeted metagenomics), is the most informative approach to monitor the viral dissemination and genetic diversity.\n\nDespite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper reproducibility and comparison of results.\n\nHere we present DEN-IM, a one-stop, user-friendly, containerised and reproducible workflow for the analysis of DENV sequencing data, both from shotgun and targeted metagenomics approaches. It is able to infer DENV coding sequence (CDS), identify serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement of extensive bioinformatics expertise.\n\nUsing DEN-IM, we successfully analysed two DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples of varying serotype and genotype, including a spiked sample containing the existing four serotypes. The second dataset consisted of 106 targeted metagenomics samples of DENV 3 genotype III where DEN-IM allowed detection of the intra-genotype diversity.\n\nThe DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM.

bioinformatics↗

bioRxiv2019

Designing synthetic bacterial consortia for landfill leachate treatment based on community matrices and regression tree analysis

The performance of microbial communities exploited by industry are largely optimised by manipulating process parameters, such as flow rates, growth conditions, and reactor parameters. Conversely, the composition of microorganisms used are often viewed as a "black box". This is mostly due to the relatively high costs and technical expertise required to identify and quantify the microbial consortia, as well as limited tools to create functional assemblages. Unknown details about the interactions among species may impose a limit on how much microbial function can be optimised for industrial purposes. Here, a new workflow was developed for studying microbial consortia using high throughput, species and community specific measurements of growth rates and yields. Growth rate and yield among all single, pairwise, triple, quadruples, quintuple and sextuple combinations of six bacterial isolates on landfill leachate were evaluated. Additive, antagonistic (e.g. competitive) or synergistic (+/-) interactions can be inferred from the rate and yield data. We found that antagonistic interactions, which hinder growth and yield, were the dominant interaction type, with only a few synergistic interactions observed. Mixed effects models were used to investigate the relationship between interaction type and species richness (biodiversity). Community identity was found to be a more important factor in predicting yield determining interactions but not rate determining interactions. Species richness was a good predictor of rate determining interactions, with the most positive interactions happening at a low species richness. Regression tree analysis identified Lysinibacillus sp. as a keystone species, a genus previously associated with bioremediation. Its presence led to a drastic change in the function of the synthetic ecosystem, with both positive yield and rate determining interactions. We were able to infer interactions about specific pairs of species, and the competitive/synergistic tendencies of single species from only basic top-down growth measurements. In this way, we have demonstrated how factorial experiments using isolated microorganisms can be used to ultimately design synthetic consortia with desirable traits for industry. Funding statementThis research was funded by the EPSRC (Vacation Bursary Award), BBSRC (Impact Acceleration Award), Department of Chemical and Biological Engineering, The University of Sheffield (Summer Undergraduate Research Fellowship) and Viridor Ltd. Ethics statements(Authors are required to state the ethical considerations of their study in the manuscript, including for cases where the study was exempt from ethical approval procedures) Does the study presented in the manuscript involve human or animal subjects: No Data availability statementGenerated Statement: This manuscript contains previously unpublished data. The name of the repository and accession number are not available.

microbiology↗

bioRxiv2018

Unveiling Leishmania invasion of fibroblasts: calcium signaling, lysosome recruitment and exocytosis culminate with actin-independent invasion.

Intracellular parasites of the genus Leishmania are the causative agents of human leishmaniasis, a widespread emergent tropical disease. The parasite is transmitted by the bite of a hematophagous sandfly vector that inoculates motile flagellated promastigote forms into the dermis of the mammalian host. After inoculation, parasites are ultimately captured by macrophages and multiply as round-shaped amastigote forms. Macrophages seem not to be the first infected cells since parasites were observed invading neutrophils first whose leishmania-containing apoptotic bodies were latter captured by macrophages, thereby becoming infected. The fact that Leishmania spp are able to live and replicate inside immune phagocytic cells and that macrophages are the main cell type found infected in chronicity created the perception that Leishmania spp are passive players waiting to be captured by phagocytes. However, several groups have described the infection of non-phagocytic cells in vivo and in vitro. The objective of this work was to study the cellular mechanisms involved in the invasion of non-professional phagocytes by Leishmania. We show that promastigotes of L.amazonensis actively induces invasion in fibroblasts without cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Inside fibroblasts parasites transformed in amastigotes, remained viable for at least two weeks and re-transformed in promastigotes when returned to insect vector conditions. Similarly to what was observed for T. cruzi, infection involves calcium signaling, recruitment and exocytosis of lysosomes involved in plasma membrane repair and lysosome-triggered endocytosis. Conditions that alter lysosomal function such as cytochalasin-D and brefeldin-A treatment or the knockout of host cell lysosomal proteins LAMP-1 and 2 dramatically affected invasion. Likewise, triggering of lysosomal exocytosis and lysosome-dependent plasma membrane repair by low doses of streptolysin-O dramatically increased parasite entry. Together our results show that L.amazonensis promastigotes are able to take advantage of calcium-dependent lysosomal exocytosis and lysosome-induced endocytosis to invade and persist in non-phagocytic cells.\n\nAUTHOR SUMMARYIntracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting but overlooked finding on Leishmania infection is that non-phagocytic cells have also been found infected by amastigotes. Nevertheless, the mechanisms by which Leishmania invades non-phagocytic cells were not studied to date. Here we show that L. amazonensis can actively induce their own entry into fibroblasts independently of actin cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions such as calcium signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair and whose positioning and content interfere in invasion. Parasites were able to replicate and remained viable in fibroblasts, suggesting that cell invasion trough the mechanism demonstrated here could serve as a parasite hideout and reservoir, facilitating infection amplification and persistence.

cell biology↗