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• Gladstone, Rebecca Ashley; Pesonen, Maiju; Pöntinen, Anna Kaarina; Mäklin, Tommi; MacAlasdair, Neil Zhang Nian Zhong & Thorpe, Harry [Show all 17 contributors for this article] (2026). Identification of transporter-dependent capsular loci associated with the invasive potential of Escherichia coli. Nature Microbiology. doi: 10.1038/s41564-026-02283-w. Full text in Research Archive Show summary

  Bacterial polysaccharide capsules contribute to antigenic diversity and immune evasion. Escherichia coli infections, including those caused by extraintestinal pathogenic E. coli (ExPEC), cause substantial antimicrobial resistance-associated morbidity and mortality. However, much-needed genotypic methods for E. coli capsule typing to aid epidemiological analysis and therapeutic design are lacking. Here we describe the curation of an in silico typing database for group 2 and 3 ATP-binding cassette transporter-dependent capsule (K) loci from 18,185 kps-positive E. coli genomes from all continents and its application to carriage and ExPEC disease cohorts. Capsules K1, K5 and K2 were the most common types in European BSIs, and together with K100 and K52 they were responsible for 58% of multidrug resistance, with differing associations with invasiveness. Homologous recombination, insertion sequences and plasmids were associated with capsular gene exchange. These findings improve understanding of capsule epidemiology and evolution to inform future diagnostic and therapeutic strategies to combat ExPEC infections.

• Ross, Theodor Anton; Janice, Jessin; Alonso, Sergio Arredondo; Löhr, Iren Høyland; Holsbø, Einar & Corander, Jukka [Show all 9 contributors for this article] (2025). Enterococcus lactis is ecologically and genetically distinct from the major opportunistic pathogen Enterococcus faecium. Microbial Genomics. 11(6), p. 1–14. doi: 10.1099/mgen.0.001420. Full text in Research Archive
• Alonso, Sergio Arredondo; Pöntinen, Anna Kaarina; Gama, João Alves; Gladstone, Rebecca Ashley; Harms, Klaus & Tonkin-Hill, Gerry [Show all 23 contributors for this article] (2025). Plasmid-driven strategies for clone success in Escherichia coli. Nature Communications. 16, p. 1–15. doi: 10.1038/s41467-025-57940-1. Full text in Research Archive
• Paganini, Julian A.; Kerkvliet, Jesse J.; Vader, Lisa; Plantinga, Nienke L.; Meneses, Rodrigo & Corander, Jukka [Show all 9 contributors for this article] (2024). PlasmidEC and gplas2: an optimized short-read approach to predict and reconstruct antibiotic resistance plasmids in Escherichia coli. Microbial Genomics. 10(2). doi: 10.1099/mgen.0.001193. Full text in Research Archive Show summary

  Accurate reconstruction of Escherichia coli antibiotic resistance gene (ARG) plasmids from Illumina sequencing data has proven to be a challenge with current bioinformatic tools. In this work, we present an improved method to reconstruct E. coli plasmids using short reads. We developed plasmidEC, an ensemble classifier that identifies plasmid-derived contigs by combining the output of three different binary classification tools. We showed that plasmidEC is especially suited to classify contigs derived from ARG plasmids with a high recall of 0.941. Additionally, we optimized gplas, a graph-based tool that bins plasmid-predicted contigs into distinct plasmid predictions. Gplas2 is more effective at recovering plasmids with large sequencing coverage variations and can be combined with the output of any binary classifier. The combination of plasmidEC with gplas2 showed a high completeness (median=0.818) and F1-Score (median=0.812) when reconstructing ARG plasmids and exceeded the binning capacity of the reference-based method MOB-suite. In the absence of long-read data, our method offers an excellent alternative to reconstruct ARG plasmids in E. coli.

• Kuronen, Juri; Horsfield, Samuel T.; Pöntinen, Anna Kaarina; Mallawaarachchi, Sudaraka; Alonso, Sergio Arredondo & Thorpe, Harry [Show all 14 contributors for this article] (2024). Pangenome-spanning epistasis and coselection analysis via de Bruijn graphs. Genome Research. ISSN 1088-9051. 34(7), p. 1081–1088. doi: 10.1101/gr.278485.123. Full text in Research Archive Show summary

  Studies of bacterial adaptation and evolution are hampered by the difficulty of measuring traits such as virulence, drug resistance, and transmissibility in large populations. In contrast, it is now feasible to obtain high-quality complete assemblies of many bacterial genomes thanks to scalable high-accuracy long-read sequencing technologies. To exploit this opportunity, we introduce a phenotype- and alignment-free method for discovering coselected and epistatically interacting genomic variation from genome assemblies covering both core and accessory parts of genomes. Our approach uses a compact colored de Bruijn graph to approximate the intragenome distances between pairs of loci for a collection of bacterial genomes to account for the impacts of linkage disequilibrium (LD). We demonstrate the versatility of our approach to efficiently identify associations between loci linked with drug resistance and adaptation to the hospital niche in the major human bacterial pathogens Streptococcus pneumoniae and Enterococcus faecalis.

• Arachchi, Sudaraka Mallawa; Tonkin-Hill, Gerry; Pöntinen, Anna Kaarina; Calland, Jessica Kate; Gladstone, Rebecca Ashley & Alonso, Sergio Arredondo [Show all 13 contributors for this article] (2024). Detecting co-selection through excess linkage disequilibrium in bacterial genomes. NAR Genomics and Bioinformatics. 6(2). doi: 10.1093/nargab/lqae061. Full text in Research Archive Show summary

  Population genomics has revolutionized our ability to study bacterial evolution by enabling data-driven discovery of the genetic architecture of trait variation. Genome-wide association studies (GWAS) have more recently become accompanied by genome-wide epistasis and co-selection (GWES) analysis, which offers a phenotype-free approach to generating hypotheses about selective processes that simultaneously impact multiple loci across the genome. However, existing GWES methods only consider associations between distant pairs of loci within the genome due to the strong impact of linkage-disequilibrium (LD) over short distances. Based on the general functional organisation of genomes it is nevertheless expected that majority of co-selection and epistasis will act within relatively short genomic proximity, on co-variation occurring within genes and their promoter regions, and within operons. Here, we introduce LDWeaver, which enables an exhaustive GWES across both short- and long-range LD, to disentangle likely neutral co-variation from selection. We demonstrate the ability of LDWeaver to efficiently generate hypotheses about co-selection using large genomic surveys of multiple major human bacterial pathogen species and validate several findings using functional annotation and phenotypic measurements. Our approach will facilitate the study of bacterial evolution in the light of rapidly expanding population genomic data.

• Thorpe, Harry; Pesonen, Maiju; Corbella, Marta; Pesonen, Henri; Gaiarsa, Stefano & Boinett, Christine J [Show all 28 contributors for this article] (2024). Pan-pathogen deep sequencing of nosocomial bacterial pathogens in Italy in spring 2020: a prospective cohort study. Lancet Microbe. 5(10). doi: 10.1016/S2666-5247(24)00113-7. Full text in Research Archive Show summary

  Background Nosocomial infections pose a considerable risk to patients who are susceptible, and this is particularly acute in intensive care units when hospital-associated bacteria are endemic. During the first wave of the COVID-19 pandemic, the surge of patients presented a significant obstacle to the effectiveness of infection control measures. We aimed to assess the risks and extent of nosocomial pathogen transmission under a high patient burden by designing a novel bacterial pan-pathogen deep-sequencing approach that could be integrated with standard clinical surveillance and diagnostics workflows. Methods We did a prospective cohort study in a region of northern Italy that was severely affected by the first wave of the COVID-19 pandemic. Inpatients on both ordinary and intensive care unit (ICU) wards at the San Matteo hospital, Pavia were sampled on multiple occasions to identify bacterial pathogens from respiratory, nasal, and rectal samples. Diagnostic samples collected between April 7 and May 10, 2020 were cultured on six different selective media designed to enrich for Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, and DNA from each plate with positive growth was deep sequenced en masse. We used mSWEEP and mGEMS to bin sequencing reads by sequence cluster for each species, followed by mapping with snippy to generate high quality alignments. Antimicrobial resistance genes were detected by use of ARIBA and CARD. Estimates of hospital transmission were obtained from pairwise bacterial single nucleotide polymorphism distances, partitioned by within-patient and between-patient samples. Finally, we compared the accuracy of our binned Acinetobacter baumannii genomes with those obtained by single colony whole-genome sequencing of isolates from the same hospital. Findings We recruited patients from March 1 to May 7, 2020. The pathogen population among the patients was large and diverse, with 2148 species detections overall among the 2418 sequenced samples from the 256 patients. In total, 55 sequence clusters from key pathogen species were detected at least five times. The antimicrobial resistance gene prevalence was correspondingly high, with key carbapenemase and extended spectrum ß-lactamase genes detected in at least 50 (40%) of 125 patients in ICUs. Using high-resolution mapping to infer transmission, we established that hospital transmission was likely to be a significant mode of acquisition for each of the pathogen species. Finally, comparison with single colony Acinetobacter baumannii genomes showed that the resolution offered by deep sequencing was equivalent to single-colony sequencing, with the additional benefit of detection of co-colonisation of highly similar strains. Interpretation Our study shows that a culture-based deep-sequencing approach is a possible route towards improving future pathogen surveillance and infection control at hospitals. Future studies should be designed to directly compare the accuracy, cost, and feasibility of culture-based deep sequencing with single colony whole-genome sequencing on a range of bacterial species. Funding Wellcome Trust, European Research Council, Academy of Finland Flagship program, Trond Mohn Foundation, and Research Council of Norway.

• Alonso, Sergio Arredondo; Gladstone, Rebecca Ashley; Pöntinen, Anna Kaarina; Gama, João Alves; Schürch, Anita C. & Lanza, Val F. [Show all 10 contributors for this article] (2023). Mge-cluster: a reference-free approach for typing bacterial plasmids. NAR Genomics and Bioinformatics. 5(3). doi: 10.1093/nargab/lqad066. Full text in Research Archive Show summary

  Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce mge-cluster that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. Mge-cluster platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen Escherichia coli, studying the prevalence of the colistin resistance gene mcr-1.1 within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment.

• Chaguza, Chrispin; Pöntinen, Anna Kaarina; Top, Janetta; Alonso, Sergio Arredondo; Freitas, Ana R. & Novais, Carla [Show all 12 contributors for this article] (2023). The population-level impact of Enterococcus faecalis genetics on intestinal colonization and extraintestinal infection. Microbiology spectrum. 11(6), p. e0020123–e0020123. doi: 10.1128/spectrum.00201-23. Full text in Research Archive Show summary

  Enterococcus faecalis is a commensal bacterium of the human gastrointestinal tract that causes opportunistic infections. The E. faecalis genetic changes associated with pathogenicity, particularly gut-to-bloodstream translocation, remain poorly understood. Here, we performed a genome-wide association study (GWAS) of 736 whole-genome sequences of fecal and bloodstream E. faecalis isolates from hospitalized and nonhospitalized individuals, respectively, to identify E. faecalis genetic signatures associated with the patient’s hospitalization status and body isolation source. We found that infection by hospitalization status and extraintestinal infection are heritable traits, with ~40% and ~30% of their variation explained by E. faecalis genetics, respectively. Furthermore, a GWAS using linear mixed models did not pinpoint any clear overrepresentation of individual genetic changes by hospitalization status or body isolation source after controlling for the population structure. However, we observed elevated signals in a genomic region containing a prophage element. However, the lineages themselves and their associated virulence factors and antibiotic resistance genes showed variable frequency among blood and fecal isolates and in hospitalized and nonhospitalized individuals. Altogether, our findings indicate that E. faecalis infection by hospitalization status and body sites is partially influenced by the overall genetic background of the isolates and antibiotic resistance patterns rather than genetic variation at individual loci, which suggests a greater role of other host and environmental factors and ultimately the opportunistic pathogenic lifestyle of this versatile host generalist bacterium.

• Tonkin-Hill, Gerry; Gladstone, Rebecca Ashley; Pöntinen, Anna Kaarina; Alonso, Sergio Arredondo; Bentley, Stephen D. & Corander, Jukka (2023). Robust analysis of prokaryotic pangenome gene gain and loss rates with Panstripe. Genome Research. ISSN 1088-9051. 33(1), p. 129–140. doi: 10.1101/gr.277340.122. Full text in Research Archive Show summary

  Horizontal gene transfer (HGT) plays a critical role in the evolution and diversification of many microbial species. The resulting dynamics of gene gain and loss can have important implications for the development of antibiotic resistance and the design of vaccine and drug interventions. Methods for the analysis of gene presence/absence patterns typically do not account for errors introduced in the automated annotation and clustering of gene sequences. In particular, methods adapted from ecological studies, including the pangenome gene accumulation curve, can be misleading as they may reflect the underlying diversity in the temporal sampling of genomes rather than a difference in the dynamics of HGT. Here, we introduce Panstripe, a method based on generalized linear regression that is robust to population structure, sampling bias, and errors in the predicted presence/absence of genes. We show using simulations that Panstripe can effectively identify differences in the rate and number of genes involved in HGT events, and illustrate its capability by analyzing several diverse bacterial genome data sets representing major human pathogens.

• Alonso, Sergio Arredondo; Blundell-Hunter, George; Fu, Zuyi; Gladstone, Rebecca Ashley; Fillol-Salom, Alfred & Loraine, Jessica [Show all 25 contributors for this article] (2023). Evolutionary and functional history of the Escherichia coli K1 capsule. Nature Communications. 14(1). doi: 10.1038/s41467-023-39052-w. Full text in Research Archive Show summary

  Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.

• Pöntinen, Anna Kaarina; Top, Janetta; Alonso, Sergio Arredondo; Tonkin-Hill, Gerry; Freitas, Ana R. & Novais, Carla [Show all 21 contributors for this article] (2021). Apparent nosocomial adaptation of Enterococcus faecalis predates the modern hospital era. Nature Communications. 12. doi: 10.1038/s41467-021-21749-5. Full text in Research Archive Show summary

  Abstract Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the “modern hospital” era, suggesting selection in another niche, and underlining the generalist nature of this nosocomial pathogen.

• Alonso, Sergio Arredondo; Top, Janetta; Corander, Jukka; Willems, Rob J. L. & Schürch, Anita C. (2021). Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals. Genome Medicine. 13(1), p. 1–18. doi: 10.1186/s13073-020-00825-3. Full text in Research Archive Show summary

  Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.

• Paganini, Julian A.; Plantinga, Nienke L.; Alonso, Sergio Arredondo; Willems, Rob & Schürch, Anita C. (2021). Recovering escherichia coli plasmids in the absence of long-read sequencing data. Microorganisms. 9(8). doi: 10.3390/microorganisms9081613. Full text in Research Archive Show summary

  The incidence of infections caused by multidrug-resistant E. coli strains has risen in the past years. Antibiotic resistance in E. coli is often mediated by acquisition and maintenance of plasmids. The study of E. coli plasmid epidemiology and genomics often requires long-read sequencing information, but recently a number of tools that allow plasmid prediction from short-read data have been developed. Here, we reviewed 25 available plasmid prediction tools and categorized them into binary plasmid/chromosome classification tools and plasmid reconstruction tools. We benchmarked six tools (MOB-suite, plasmidSPAdes, gplas, FishingForPlasmids, HyAsP and SCAPP) that aim to reliably reconstruct distinct plasmids, with a special focus on plasmids carrying antibiotic resistance genes (ARGs) such as extended-spectrum beta-lactamase genes. We found that two thirds (n = 425, 66.3%) of all plasmids were correctly reconstructed by at least one of the six tools, with a range of 92 (14.58%) to 317 (50.23%) correctly predicted plasmids. However, the majority of plasmids that carried antibiotic resistance genes (n = 85, 57.8%) could not be completely recovered as distinct plasmids by any of the tools. MOB-suite was the only tool that was able to correctly reconstruct the majority of plasmids (n = 317, 50.23%), and performed best at reconstructing large plasmids (n = 166, 46.37%) and ARG-plasmids (n = 41, 27.9%), but predictions frequently contained chromosome contamination (40%). In contrast, plasmidSPAdes reconstructed the highest fraction of plasmids smaller than 18 kbp (n = 168, 61.54%). Large ARG-plasmids, however, were frequently merged with sequences derived from distinct replicons. Available bioinformatic tools can provide valuable insight into E. coli plasmids, but also have important limitations. This work will serve as a guideline for selecting the most appropriate plasmid reconstruction tool for studies focusing on E. coli plasmids in the absence of long-read sequencing data.

• Alonso, Sergio Arredondo; Pöntinen, Anna Kaarina; Cleon, Francois Pierre Alexandre; Gladstone, Rebecca Ashley; Schürch, Anita C. & Johnsen, Pål Jarle [Show all 8 contributors for this article] (2021). A high-throughput multiplexing and selection strategy to complete bacterial genomes. GigaScience. 10(12), p. 1–13. doi: 10.1093/gigascience/giab079. Full text in Research Archive Show summary

  Background: Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results: Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions: The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

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• Mäklin, Tommi; Taira, Aurora; Alonso, Sergio Arredondo; Shao, Yan; Stratton, Michael R & Lawley, Trevor D [Show all 8 contributors for this article] (2025). Geographical variation in the incidence of colorectal cancer and urinary tract cancer is associated with population exposure to colibactin-producing Escherichia coli. Lancet Microbe. 6(5). doi: 10.1016/j.lanmic.2024.101015. Full text in Research Archive Show summary

  Biomedical research has implicated the bacterial metabolite colibactin as a causal risk factor for several cancer types, in particular, colorectal cancer. Colibactin has been known to drive tumorigenesis by inducing double-strand breaks in the DNA of epithelial cells exposed to colibactin-producing bacteria. Some phylogroup B2 Escherichia coli secrete colibactin during interbacterial warfare, concomitantly exposing the host to an increasing risk of DNA damage. This Personal View reviews the current knowledge about the cancer–colibactin interface and summarises metagenomics-based and population-genomics-based surveys to show that the prevalence of dominant colibactin-producing lineages of E coli varies considerably across geographical regions. The prevalence is further strongly associated with the age-standardised incidences of colorectal cancer, bladder cancer, and prostate cancer, suggesting that the degree of colibactin exposure in a population might contribute to the geographical variation of these cancers. Our observations provide a strong impetus for further research and the development of novel interventions to reduce the risks for colibactin-related cancers.

• Santos, Rodrigo; Alonso, Sergio; Cherry, Elizabeth & Sundnes, Joakim (2015). Simulations of Heart Function (editorial). Full text in Research Archive

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