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bioRxiv2025

Back-projection improves inference from sparsely sampled genomic surveillance data

Highly transmissible SARS-CoV-2 variants have emerged throughout the COVID-19 pandemic, driving new waves of infections. Genomic surveillance data can provide insights into the viruss evolution and biology. However, delayed and limited regional data can introduce biases in epidemiological models, potentially obscuring transmission patterns. To address this issue, we used a novel, variant-specific back-projection model to estimate a distribution of likely infection times from sample collection times. We combined this approach with epidemiological modeling to estimate selection for increased transmission in a way that accounts for the uncertainty in infection times. Tests in simulations demonstrated that our method can make the inference of selection more reliable. We also applied our approach to SARS-CoV-2 data, where it excelled in smoothing and extending data from geographic regions or times with poor sampling. Overall, our method can aid in the reliable identification of mutations and variants with higher transmission rates.

evolutionary biology↗

bioRxiv2025

Trypanosoma brucei from pigs in sleeping sickness foci from Cote Ivoire is structured into clonal and strongly subdivided populations

Human African trypanosomiasis (HAT), or sleeping sickness, is currently targeted for elimination. The etiologic agent of HAT is a trypanosome belonging to the species Trypanosoma brucei (Tb) s.l., a unicellular parasite transmitted by tsetse flies. Tb s.l. consists of three subspecies: T. b. brucei (Tbb), T. b. gambiense (Tbg) and T. b. rhodesiense (Tbr). These subspecies are morphologically indistinguishable and classified according to host in which they are found, type of disease and geographical distribution. During the last few decades, there has been considerable effort to genetically characterize Tb s.l. isolated from domestic and wild animals in order to better evaluate the impact of animal reservoirs on the epidemiology of HAT. To assess genetic diversity of Tb s.l. strains circulating in three endemic or historical HAT foci in Cote Ivoire, we conducted a population genetics study of these parasites. Biological and isolated stock samples collected from pigs and reference stocks were tested with the primers of the Trypanosoma gambiense-specific-glycoprotein gene (TgsGP) and were genetically characterized with eighteen microsatellite primers. TgsGP positive samples did not fit into Tbg as regard to their microsatellite profile. We also found that in Ivoirian foci, Tbb populations (animal trypanosomes) were structured as several strongly isolated units that propagate clonally. This is in variance with other published data on that subspecies. This study confirms the need to develop better tools to explore the relationships between Tbb and Tbg and to study the epidemiological role of potential animal reservoir for Tbg.

genetics↗

bioRxiv2025

Evaluating a SNP calling pipeline for Mycobacterium leprae

Mycobacterium leprae is the causative agent of leprosy, a persisting disease characterised by skin lesions and peripheral nerve numbness. Attempts to sequence the genome have been challenged by its inability to be cultured on artificial media. Advances in DNA extraction and sequencing technologies have enabled molecular epidemiology to be introduced for M. leprae. However, a validated SNP calling pipeline does not currently exist. A ground truth of pairwise SNP differences was first created using Minimap2 and three complete genomes of M. leprae. Using simulated reads from each of these genomes and the others as a reference, we evaluated the precision and recall of short-read SNP calling using Snippy. Approximately 80% of SNPs were called with false positives only due to ambiguous bases in certain genomes. Repeat region masking was found to be unnecessary for M. leprae SNP calling, unlike for M. tuberculosis. We find that SNP calling from short reads is robust and highly accurate from M. leprae, showing promise as a tool for molecular epidemiology studies to increase case detection and inform leprosy control strategies.

genomics↗

bioRxiv2025

Controlling Banana Bunchy Top Disease in Benin: crop protection strategies with socioeconomic perspectives

SummaryO_LIThis study combines satellite mapping, field surveillance, farmer surveys, and disease modelling to assess BBTV dynamics across Benin. It provides the first national-scale map of banana cultivation in Benin and identifies high-risk areas for targeted intervention. C_LIO_LIThe study highlights the dominance of informal networks for exchange of planting material ([~]80% of farmers), limited farmer awareness of BBTV ([~]60% unaware), and low recognition of symptoms or transmission routes, all of which contribute to the spread and persistence of disease. C_LIO_LIAlthough women represented only a quarter of respondents, they demonstrated comparable levels of market orientation and access to planting materials and land. The findings call for spatially targeted, socially informed BBTV management strategies that integrate clean planting material systems, farmer education, and accessible control methods. C_LIO_LIBBTV was detected in 140 out of 747 fields surveyed, with the highest prevalence in the humid southern regions. No cases were found in the northern Sudanian zone, reflecting known agroecological patterns of disease distribution. C_LIO_LIModelling of three control strategies - decapitation, uprooting, and injection - revealed injection as most effective nationally (70% reduction in disease incidence), while uprooting was most effective in vulnerable communities with high disease pressure. C_LIO_LIThe study reveals an interconnected system in which socioeconomic vulnerability, informal seed networks, limited disease awareness, and spatial clustering of banana cultivation collectively drive BBTV dynamics in Benin, highlighting the need for epidemiologically effective, socially informed, and spatially targeted interventions. C_LI Societal Impact StatementBanana and plantain are critical for food security and income generation in West Africa, particularly for smallholder farmers in countries such as Benin. However, banana bunchy top virus (BBTV) poses a major threat to sustainable production. This study integrates high-resolution remote sensing, field epidemiology, mathematical modelling, and socioeconomic analysis to improve understanding of the spread of BBTV and to evaluate the effectiveness of different disease control strategies. Our findings reveal that BBTV risk is highest in southern Benin, where socioeconomic vulnerability is also greatest. Low disease awareness, limited adoption of effective control methods and informal exchange of planting material exacerbate this risk. By identifying priority areas and strategies tailored to local social, economic, and agroecological contexts, this research offers a roadmap for designing targeted, sustainable BBTV management programs. These insights can support smallholder resilience, reduce disease burden, and safeguard banana-based livelihoods across sub-Saharan Africa.

plant biology↗

bioRxiv2025

A colorimetric method for detecting virulent bacteriophage to Vibrio cholerae in fecal and environmental samples

BackgroundV. cholerae are often infected with vibriophages and these phages may be found associated with V. cholerae bacteria or may be detected independently from the bacteria, especially in environmental water samples. When detected, vibriophages serve as a surrogate for detection of V. cholerae and they also have an important role in the ecology of V. cholerae. Vibriophages can be detected using plaque assays or PCR, but these methods are time-consuming and require specialized laboratory resources. MethodologyTo address limitations of other methods, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled up to quickly and easily to screen a large number of samples. The assay uses resazurin and a bacterium, AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns color from blue to pink when added to a culture broth with growing bacteria. When a bacteria-free test sample, such as a Millipore filtered wastewater sample containing vibriophages, is added to a culture broth with AC6169, the bacteria will be lysed by the phages and will not grow, and the color of the broth will remain blue. However, if there are no phages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink. We developed the assay using ICP1 spiked samples of environmental water, stool and frozen bile peptone and found it to be sensitive with a limit of detection of 4 to 40 plaque forming units/ml. ConclusionThis colorimetric assay promises to provide a convenient method to detect vibriophages on a larger scale than was possible earlier to better understand their role as a surrogate for detecting V. cholerae and to better understand their role in the pathogenesis, ecology, and epidemiology of cholera. Author SummaryWe developed a simple and inexpensive colorimetric assay to detect virulent vibriophage in samples of wastewater and stool. The assay uses resazurin, a blue dye and a unique bacterium, AC6169, that is known to be susceptible to vibriophages. Bacteria-free samples (e.g. Millipore filtrates) suspected of having vibriophage are incubated for 2 or 3 hours in a broth with AC6169, following which resazurin is added. After a further 30-minute incubation, the color of the broth is observed. If the sample contains vibriophage, the broth will remain blue, but if there are no vibriophage, the broth will turn pink. The change in color from blue to pink is due to the metabolic reaction from the growing bacteria. When vibriophage are present, they lyse the bacterium AC6169, thus preventing the color change, but if vibriophage are not present, the bacteria multiply rapidly, and the metabolic reaction causes the broth to change color to pink. Because the assay procedure is inexpensive and simple, laboratories in cholera endemic areas should find it convenient for their epidemiologic and clinical surveillance activities.

microbiology↗

bioRxiv2025

High-Resolution Detection of Hidden Antibiotic Resistance with the Dilution-and-Delay (DnD) Susceptibility Assay

Rising rates of antibiotic treatment failure highlight the complexity of resistance mechanisms. While conventional, genetically encoded resistance is well established, recent studies have uncovered widespread non-canonical mechanisms driven by phenotypically insensitive subpopulations hiding in seemingly susceptible populations, such as heteroresistance, persistence or adaptive resistance. These variants are not only clinically significant but also act as evolutionary precursors to fully resistant populations, rendering infections increasingly difficult to control. Yet standard antibiotic susceptibility tests lack the resolution and dynamic range needed to detect this wide spectrum of resistance mechanisms and their evolutionary progression, often misguiding clinical decisions, obscuring mechanisms of failure, and underestimating the true epidemiology of antibiotic resistance. To address these limitations, we introduce the Dilution-and-Delay (DnD) assay--a practical, high-resolution approach that implements two basic principles of bacterial growth in the antibiotic susceptibility testing format. Using well-defined synthetic communities and strains, we demonstrate that the DnD assay quantifies viable cells over more than eight orders of magnitude and detects rare antibiotic-insensitive cells at frequencies as low as 1 in 100 million. It additionally reports the standardized MIC as a secondary output, thereby capturing the average antibiotic response of the majority population and heterogeneity of minority subpopulations within a single assay. We scaled this approach for high-throughput application, classifying [~]120 previously uncharacterized clinical isolates of Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii--across five antibiotics. This robust, quantitative, and scalable platform opens the door to next-generation antibiotic susceptibility testing, with broad utility in basic research, clinical diagnostics, and epidemiological surveillance. This improved testing will guide evidence-based clinical decisions and prevent the rapid evolution of antibiotic resistance, thereby counteracting the rising rate of treatment failure. SignificanceAntibiotic treatment failure due to inappropriate prescription remains a critical threat to global health. It also accelerates the evolution of resistance, making infection increasingly difficult to control. A key underlying issue is that current antibiotic susceptibility testing lacks the resolution and dynamic range needed to detect a wide range of resistance mechanisms and their evolutionary progression, leading to misinformed clinical decisions. While population analysis profiling (PAP) offers sufficient accuracy, it is too labor-intensive for routine use. Single-cell approaches are technically demanding and resource-intensive. Here, we present a susceptibility testing strategy that preserves the operational efficiency and compatibility of conventional workflows, while dramatically extending their resolution and dynamic range. Our approach is scalable, quantitative, and well-suited for high-throughput applications in both research and clinical settings--closing a critical diagnostic gap in the fight against antibiotic treatment failure.

microbiology↗

bioRxiv2025

Whole genome sequencing reveals a novel Renibacterium salmoninarum lineage and suggests geographic endemism combined with anthropogenic spread in the North-East Atlantic area

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD) and has been isolated from northern European salmonid farms since the 1960s. The bacterium has been detected only sporadically in Norway during the last decades, but the country experienced several geographically spread outbreaks since December 2022. The phylogenomic relationships of R. salmoninarum isolates associated with the epizootics ongoing on the West Coast and in Mid-Norway were explored using a whole genome sequencing approach. A broad overview of the phylogeography of this pathogen was gained through sequencing and analysis of a collection of isolates from Norway (n=67), Iceland (n=12), Denmark (n=12), and the Faroe Islands (n=1), along with a collection of 109 publicly available sequences. We identified two distinct contemporary clades of R. salmoninarum causing BKD in Norway in the period 2022-2024. Both clades belong to the expanding, aquaculture-associated Lineage 1. The epidemiological picture appears consistent with contemporary aquaculture operations, raising questions on the effectiveness or practice of current biosecurity practices towards R. salmoninarum. Our work also describes a hitherto undescribed lineage (Lineage 3), predominantly from Iceland, where BKD is considered endemic. The detection of endemic reservoirs of R. salmoninarum in European water systems underscores the potential for ongoing pathogen circulation independent of acute outbreaks. This finding emphasizes the importance of further investigation of the mechanisms of pathogen persistence, particularly within environments related to aquaculture, where chronic infection reservoirs could compromise disease management and biosecurity. IMPORTANCERenibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a chronic salmonid pathogen that often presents subclinically, complicating detection and control. Effective management of BKD is therefore crucial for sustainable aquaculture and wild fish conservation. Recognizing the need for comprehensive sampling, previous studies have underscored the importance of a global isolate collection to better understand the epidemiology of the pathogen. While the slow genetic evolution and highly conserved genome of the bacterium have reduced the resolution of conventional typing methods in the past, applying whole genome sequencing has enabled its molecular tracing at the outbreak level. Here, we present the largest phylogenomic analysis of R. salmoninarum to date, including sequences from Denmark, Iceland, and the Faroe Islands for the first time. This expanded dataset elucidates past and present movements of the pathogen in European waters, providing actionable insights to support development of targeted BKD management and biosecurity strategies.

genomics↗

bioRxiv2025

Balancing disease control efficacy, resistance durability, and economic outcomes: Insights from a participatory modeling study integrating grapevine cultivar deployment timing with fungicide treatments

1In this study, we developed, assessed and compared strategies for deploying disease-resistant grapevine cultivars in partnership with a wine cooperative in South-West France. In four collaborative workshops, cooperative managers and multidisciplinary researchers jointly designed six feasible deployment strategies. These scenarios, implemented within real vineyards, involve planting resistant cultivars (RC) in vineyards (i) more than 30 years old, (ii) on 3.3% of the older plots each year or (iii) on plots located in no-treatment zone (due to proximity to watercourses or human dwellings), with and without a fixed maximum percentage of resistant cultivars in the vineyard. The strategies were evaluated with the landsepi model, which simulates the spread and evolution of pathogens across agricultural landscapes in response to host resistance deployment. The assessment criteria included downy mildew control efficacy, resistance durability, decrease in fungicide use, and economic benefits for individual vineyards and the cooperative. These scenarios led us to study more specifically the effects of two little-studied factors: (i) the presence or absence of fungicide applications and (ii) the massive or progressive introduction of resistant cultivars. Fungicide application substantially decreases the risk of resistance breakdown and complements the epidemiological control provided by resistant cultivars, particularly when the fitness costs of virulence are low. The probability of pathogens adapted to RC becoming established was generally lower with progressive than massive RC introduction; however, when establishment did occur, the time to establishment was shorter for progressive strategies at a cropping ratio [≥] 50%. Ultimately, the choice of RC introduction strategy had a minimal overall impact on disease control due to compensatory epidemiological and evolutionary processes, including dilution effects, and the probability and timing of resistance breakdown. These results are discussed in the broader framework of action-research approaches and the challenges and opportunities for resistant cultivar adoption by vine-growers. Our study highlights the potential of participatory approaches for developing practical, scientifically grounded strategies for sustainable viticulture.

evolutionary biology↗

bioRxiv2025

Numerical Solution to SIR Model using SBP-SAT operators

Accurate and stable numerical simulation of epidemic dynamics is essential for translating mathematical models into reliable computational tools for public health. The Susceptible-Infectious-Recovered (SIR) model remains a cornerstone of mathematical epidemiology, yet the robustness of its numerical treatment strongly influences predictive reliability in applications ranging from outbreak forecasting to intervention assessment. Here, we introduce a high-order Summation-By-Parts (SBP) framework with Simultaneous Approximation Terms (SAT) for the numerical solution of the SIR system. Unlike standard integrators, the SBP-SAT method enforces stability at the discrete level while retaining high accuracy, offering a principled approach for handling nonlinear epidemic dynamics. We validate the scheme against the analytical solution of the classical SIR model, demonstrating both accuracy and robustness. By situating SBP-SAT methods within the growing landscape of numerical approaches for SIR-type models--including Runge-Kutta, finite difference, and fractional-order solvers--this work establishes SBP-SAT as a powerful and generalizable alternative for computational epidemiology. Beyond the classical SIR model, these results pave the way for applying SBP-SAT schemes to more complex epidemic models where stability and accuracy are critical, thereby advancing the methodological foundations of computational biology.

ecology↗

bioRxiv2025

Machine learning based lineage prediction from AMR phenotypes for Escherichia coli ST131 clade C surveillance across infection types

2.Rising antimicrobial resistance (AMR) in Escherichia coli bloodstream infections (BSIs) in high-income settings has typically been dominated by one clone, the sequence type (ST) 131. More specifically, ST131 clade C (ST131-C) is associated with fluoroquinolone resistance and extended-spectrum {beta}-lactamases (ESBLs). Even though urinary tract infections (UTIs) are a known common precursor to BSIs, there is currently limited knowledge on the longitudinal prevalence of ST131-C in UTIs and, therefore, the temporal link between the two infection types. Leveraging available genomic and antimicrobial susceptibility test (AST) data for ciprofloxacin, gentamicin, and ceftazidime in 2790 E. coli BSI isolates, we trained random forest and Extreme Gradient Boosting (XGBoost) classifiers to predict if an E. coli isolate belongs to ST131-C using only AST data. These models were used to predict the yearly prevalence of ST131-C in 22942 UTI and 24866 BSI isolates from Norway. The XGBoost classifier achieved a prediction F1-score of over 70% on a highly unbalanced dataset where only 4.3% of the genomic BSI isolates belonged to ST131-C. The predicted prevalence of ST131-C in UTIs exhibited a similar annual trend to that of BSIs, with a stable infection burden for eight years after its rapid expansion, confirming that the persistence of ST131-C in BSIs is largely driven by ST131-C UTIs. However, a higher prevalence of ST131-C in BSIs ([~]7%) compared to UTIs ([~]4%) suggests a subsequent enrichment of ST131-C. Our study highlights how existing epidemiological knowledge can be supplemented by utilising extensive data from AMR surveillance efforts without genomic markers. 3. Impact statementThis study proposes a potential analysis method that leverages AST data, which is already regularly collected for AMR surveillance purposes. Using such data to approximate the population-wide prevalence of MDR clones, such as ST131-C, could allow for larger-scale, retrospective studies of its prevalence in a population than genomic-based methods at a significantly lower cost. Such a method could supplement existing knowledge and epidemiology study practices. We use the proposed method to find relationships between the prevalence of the important MDR E. coli clone ST131-C in UTIs and BSIs in Norway. These results suggest that monitoring and reducing MDR in UTIs could reduce the burden of this invasive clone in hard-to-treat BSIs. 4. Data summaryAll AST data, clonal information for isolates with genomic data, and code used in this study can be found in the following repository: https://github.com/theodorross/EColi-UTI-Predictions. Only the clone and published metadata information is shown for the UTI data shared by Handal, Kaspersen et al.

bioinformatics↗

bioRxiv2025

Mating behaviour and connectivity drive sporadic European bat lyssavirus 2 infections in Myotis myotis

Gaining insight into the transmission and persistence of zoonoses in wild reservoirs is essential for preventing outbreaks, assessing demographic impacts, and informing conservation strategies. Achieving this requires reliable estimates of key epidemiological parameters, which are governed by aspects of host ecology and behaviour that are notoriously difficult to quantify in many wild species. Taking advantage of a long term study of the ecology of five Greater mouse-eared bats (Myotis myotis) maternity colonies in France ([~]2700 marked individuals), we sought to characterise lyssavirus infection dynamics in a wild bat reservoir. Although no lyssavirus was isolated from any of the 422 saliva samples tested, we report the first indirect evidence of European bat lyssavirus 2 circulation in this species. Interestingly, seropositivity appeared sporadic within colonies (0-100% annually) and synchronized across them, with highest seroprevalence in 2015-2017, but remaining low otherwise. Fitting Bayesian compartmental epidemiological models, we found that shifts in EBLV-2 prevalence were primarily driven by mating season introduction, age-dependant variation in transmission, and frequency-dependent transmission dynamics. Lyssavirus outbreaks involve few deaths overall but can still yield high annual mortality rates. These findings underscore the value of integrating ecological and individual-level data with mechanistic modelling to enhance our understanding and management of zoonotic pathogens in wildlife.

ecology↗

bioRxiv2025

Direct nanopore sequencing of M. tuberculosis on sputa and rescue of suboptimal results to enhance transmission surveillance

Whole-genome sequencing (WGS) enhances precision in predicting antimicrobial resistance and tracking Mycobacterium tuberculosis (MTB) transmission. Due to MTBs slow-growing nature, genomic results are delayed; however, few efforts have sought to accelerate them by performing WGS directly on respiratory specimens. Most culture-free efforts have focused on accelerating resistance prediction. The present study provides further evidence to the only preceding study aiming to accelerate precise delineation of transmission, coupling culture-free WGS to a surveillance programme. Our study is distinguished from its predecessor by being the first to apply flexible nanopore sequencing to further accelerate the process. A total of 71 sputa were selected, in which we applied only a procedure to deplete human DNA, thus avoiding costly and cumbersome capture-bait alternatives. Optimal results (>90% genome covered, mean coverage >45x and >70% genome covered >20x) were obtained from 33.8% of cases, allowing the assignment to transmission clusters close to diagnosis of every new case. A further 12.6% of samples yielded suboptimal results (15.5%-90.92% at >10x), which were exploited through a rescue pipeline. This approach was based on identifying informative SNPs acting as markers for relevant transmission clusters in our population. The pipeline enabled pre-allocation of new cases to pre-existing clusters and, in some cases, precise genomic relationships with the preceding cases in the cluster. In summary, this study demonstrates that epidemiologically valuable information can be obtained directly from sputum in approximately half the samples analysed. It represents a new advancement in the pursuit of faster comparative genomics, with epidemiological purposes, at diagnosis.

genomics↗

bioRxiv2025

Uncovering the Genomic Landscape of Mycobacterium bovis in Wales

Bovine tuberculosis (bTB), caused by the bacterium Mycobacterium bovis, is one of the most pressing animal health issues in Wales today. It negatively impacts cattle health, affects profitability and trade, and can decimate years of genetic improvement towards desirable production traits. It also places a significant burden on the health and well-being of affected farming families. Eradication of bTB requires the understanding of local transmission pathways to target effective disease control interventions. Traditional genotyping methods, such as spoligotyping and VNTR analysis, which have previously been used in Wales to understand the epidemiology of M. bovis, have lacked the discriminatory power to accurately identify local transmission pathways. Recently, whole-genome sequencing has replaced traditional genotyping methods for all M. bovis strains isolated from infected animals in Wales. In this study, we characterised the genomic diversity of M. bovis across Wales by analysing the genome sequence of all 379 M. bovis isolates obtained from culture-positive animals in Wales in 2021. Analyses uncovered three main clusters that are geographically distinct. A further three clusters containing fewer isolates were also geographically separated, two of which had particularly large SNP distances compared to the majority of other Welsh isolates, suggesting independent introductions of M. bovis strains that are not endemic to Wales. Fine-scale and epidemiologically relevant genetic structuring was identified within the six main clusters, indicating region-specific evolution, which can drive local disease dynamics. Finally, A number of SNPs in coding genes were identified that have the potential for important advantageous physiological consequences that may impact host-pathogen interactions and necessitate further investigation.

microbiology↗

bioRxiv2025

cgDist: An Enhanced Algorithm for Efficient Calculation of pairwise SNP and InDel differences from Core Genome Multilocus Sequence Typing

Bacterial genomic surveillance requires balancing computational efficiency with genetic resolution for effective outbreak investigation. Traditional cgMLST distance calculations treat all allelic differences as equivalent units, potentially obscuring nucleotide-level variation critical for source attribution. While SNP-based methods provide enhanced resolution, their computational requirements limit routine deployment in surveillance laboratories. We present cgDist, an algorithm that bridges this resolution gap by calculating nucleotide-level distances directly from cgMLST allelic profiles. The unified cache architecture stores comprehensive alignment statistics, enabling multiple distance calculation modes without redundant computation and supporting both dataset-specific and schema-complete cache generation. This design transforms genomic surveillance from batch processing to continuous streaming analysis, with cumulative performance benefits as laboratories accumulate alignment data. cgDist functions optimally as a precision "zoom lens" for the detailed investigation of clusters identified through initial cgMLST screening. Rather than restructuring entire population relationships, this targeted approach maximizes epidemiological insight precisely where enhanced resolution is most valuable. The algorithm ensures that cgDist distances are always greater than or equal to corresponding cgMLST distances, preserving epidemiological interpretability while adding critical genetic discrimination. The system includes integrated recombination detection capabilities that leverage cached alignment statistics to identify potential horizontal gene transfer events through mutation density analysis. This multi-scale analytical framework--from population screening through cluster zoom analysis to recombination detection--provides comprehensive surveillance capabilities within the computational constraints of routine public health practice.

bioinformatics↗

bioRxiv2025

Global Insights into Evolution and Adaptation in KPC and NDM Coproducing Carbapenem-Resistant Klebsiella pneumoniae.

ObjectivesThe worldwide dissemination of carbapenem-resistant Klebsiella pneumoniae co-producing KPC and NDM (KPC-NDM-CRKP) poses a substantial clinical challenge. MethodsThis study characterized 5 KPC-NDM-CRKP isolates from a tertiary hospital in southern China and integrated 460 global isolates genomes to demonstrate the population framework of this epidemic pathogen. Results5 clinical isolates were multidrug resistance, exhibiting high level resistance to first-line drugs including carbapenems and ceftazidime/avibactam. Globally, KPC-2-NDM-1 (50.3%) were the most common and an increasing trend of ST11-KL64 (20%) isolates carrying iucA and rmpA/A2. Resistance and virulence genes were positively associated with specific mobile elements including blaKPC-3 with Tn4401, iucABC with IncHI1B, iroBCDN with IncQ1. The phylogenetic analysis revealed distinct lineage-specific mutations and pan-genome enrichments synergistically promoted adaptive evolution: cluster 1 had membrane transport and secretion but enhanced immune evasion and horizontal gene transfer, cluster 3 adapted metabolically for nutrient-limited niches; cluster 4 reprogrammed metabolism for host adaptation, cluster 6 displayed membrane repair and reduced virulence (e.g., KC187). High-risk iucA/rmpA-positive isolates in cluster 2 acquired enhanced virulence and metabolic adaptive capacity through recombination hotspots (ABC transporters DdpABCD, efflux systems MdtABCD, metabolic enzymes XylB and MtlD, regulators BaeR and OmpR) and pan-genome enrichment (siderophore biosynthesis, toxin-antitoxin VapC and restriction-modification systems). Functionally, these traits correlated with high mucoviscosity, serum resistance, siderophore production and lower survival rates in G. mellonella that contribute to epidemiological success. ConclusionsThese findings demonstrated the discrete mutation, horizontal transfer, and accessory genome plasticity collectively drove the epidemiological success of KPC-NDM-CRKP.

microbiology↗

bioRxiv2025

Evaluating the Role of Anopheles Mosquitoes in the Global Spread of Arboviruses: A Review of Laboratory-Confirmed Viral Competence

Mosquito-borne diseases are a major global health concern, infecting up to 700 million people annually and causing over one million deaths. Of the several genera of biting mosquito, species of Anopheles are mostly studied for their ability (vector competence) to transmit Plasmodium protozoan parasites, some species of which cause malaria. More than 480 species of Anopheles have been described worldwide and about 70 of these are responsible for Plasmodium spp. transmission. However, the focus on Anopheles as vectors of Plasmodium has led to a relative lack of study about the ability of Anopheles to transmit viruses. Some Anopheles species have been experimentally confirmed as competentfor various arboviruses. In most cases they are secondary vectors, with relatively low competence, contributing to overall transmission while other species of mosquito or other vector are responsible for sustained transmission. Although secondary vectors may contribute less to transmission, they may play important epidemiological roles by extending transmission seasons and/or providing a means of virus overwintering. Here, we conducted an extensive review of scientific repositories, accessing a diverse range of journals and reports to build an exhaustive database of known Anopheles competence for arboviruses. Opinion pieces, modelling papers lacking experimental confirmation, and papers without vector competence data were excluded. In total, we reviewed 7,343 papers, and after exclusions, we retained 427 papers. We analysed these data, cross-referenced with ability to undergo diapause and geographical range. Our results show that some Anopheles spp. have the potential to be secondary vectors of some arboviruses, as well as undergoing diapause as adults in temperate regions of their range. Hence, we infer they could also play an epidemiologically significant role in the overwintering of mosquito-borne viruses. Author SummaryMosquito-borne diseases remain a major global health challenge, causing over one million deaths annually. Though Anopheles mosquitoes are widely studied for their spread of parasites that cause malaria, much less is known about their interaction with viruses. In this study we reviewed scientific articles to identify their role in the spread of mosquito-borne viruses. We examined studies that showed experimental evidence of transmission in the laboratory, and thus could potentially contribute to transmission as secondary vectors. Additionally, some Anopheles species in temperate regions can survive winter in a dormant state (diapause) as adults, which could enable viruses to persist between transmission seasons. Together, these results suggest that Anopheles mosquitoes may play a larger role in virus transmission than is currently appreciated, especially in temperate regions.

ecology↗

bioRxiv2025

Species identification and genotyping of Citrobacter spp. using genes with high nucleotide diversity.

Citrobacter spp. are facultative anaerobic Gram-negative bacillus found in a wide range of habitats. Recently, there have been reports of an increasing number of cases of Citrobacter spp. being the cause of nosocomial infections, and of increasing highly antimicrobial-resistant (AMR) Citrobacter spp. In clinical laboratory testing, Citrobacter spp., such as C. freundii and C. braakii, are handled collectively as the C. freundii complex. This can be obstacle to collect information regarding species identification and genetic lineage which are necessary for estimating distribution mechanism and evaluating AMR degree for each Citrobacter species. This study investigated gene combinations that allow for Citrobacter spp. identification and genetic lineage estimation with as few genes as possible. Citrobacter genomes of 18 species were collected as many as possible from GenBank. After the gene annotation, nucleotide diversity of 1,113 genes contained in all 453 genomes was calculated. Genotypes of top seven highest nucleotide diversity (HND) genes were confirmed and investigated their distributions among Citrobacter genomes. Except for C. werkmanii and C. cronae, it was suggested that the genotype of the top HND gene, groups_3152, and combinations of the genotypes of the top two HND genes were a species- and genetic lineage-specific distribution, respectively. Considering that the genomes of C. werkmanii and C. cronae were highly similar and indistinguishable each other by conventional genotyping methods, it was suggested that the combination of the top two HND genes could be used to Citrobacter spp. identification and genetic lineage classification. Importance StatementMolecular epidemiology of individual Citrobacter species has not been widely performed, partly because Citrobacter spp. are handled as the C. freundii complex in the clinical testing and the multi-locus sequencing typing (MLST) has been performed targeting the entire genus Citrobacter. This study proposed a combination of two genes for identification of Citrobacter spp. and classify their genetic lineages. Currently, phylogenetic tree analysis has become possible using core-genome MLST and core-genome single nucleotide polymorphisms (SNPs) after whole genome sequencing (WGS) of bacterial isolates. But these phylogenetic analyses require a considerable amount of cost. In this regard, using two genes for Citrobacter species identification and genetic lineage classification reduces the required cost compared to WGS, encouraging its introduction into clinical testing and further performing molecular epidemiology of individual Citrobacter species.

genomics↗

bioRxiv2025

Deciphering the pangenome of the shellfish pathogen Vibrio europaeus: Evolutionary history and functional impact of core and accessory genes in aquaculture.

Vibrio europaeus is an important pathogen in shellfish aquaculture, yet its genomic diversity and adaptive potential remain poorly understood. Here, we present the first comprehensive analysis of the V. europaeus pangenome, integrating genomic data from all strains available to the date sequenced specifically for this study. Those were isolated from different aquaculture facilities (shellfish hatcheries) associated to mass mollusks mortalities from different geographical locations, years and host species. Our findings revealed an open pangenome with the 61% of the genes associated to the accessory genome that contributes to environmental and host adaptations. Phylogenomic analyses of the core-genome (39% of the pangenome size) allowed to evaluate the evolutionary history and intraspecific diversity of V. europaeus and revealed that Spanish strains displayed a much lower genetic variability than French, Chilean or American strains, probably due to a monophyletic radiation event. Functional annotation of core and accessory genes revealed the key virulence factors of the species while it also disclosed that those are located mainly into the core genes. The high number of anti-phage defense systems encoded in the accessory genome explained almost all the variability of the species. The results provide important insights into the evolutionary history and ecological versatility of V. europaeus, with potential implications for diagnostics, epidemiological surveillance, and disease management strategies in aquaculture. Impact statementThis study presents the first comprehensive pangenome analysis of Vibrio europaeus, an emergent pathogen responsible for severe economic losses in shellfish aquaculture, the second most important sector of global aquaculture. Here, we characterized for the first time the V. europaeus pangenome, integrating genomic data from all strains isolated to date, sequenced specifically for this study using NGS and/or third-generation (PacBio) technologies. This work achieved the most complete species pangenome to date and is among the first studies on aquaculture-related bacterial pathogens. Beyond a descriptive framework, the pangenome was critically examined to identify key traits, including virulence factors, secondary metabolite biosynthesis, and antimicrobial resistance genes, essential for host infection and adaptation. Moreover, the study of anti-phage defense systems was shown to account for much of the species genomic variability. The genomic resources and insights generated here substantially expand our understanding of V. europaeus biology and provide valuable information that can be applied for diagnostics, epidemiological surveillance, and sustainable management of this pathogen in aquaculture industry. Data summaryAll genome assemblies have been uploaded to the National Center for Biotechnology Information. The GenBank accession numbers for each of the 39 strains used in this study and detailed information can be found in Table S1. All bioinformatics tools used for comparative genomics have been listed in the Methods section including references, associated databases and analysis parameters.

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