The Lancet Microbe

Shiga toxin-producing Escherichia coli (STEC) are an important public health concern due to their association with foodborne gastroenteritis and severe outcomes including haemolytic uraemic syndrome (HUS), particularly linked to the stx2a subtype of the Shiga toxin. We investigated the temporal dynamics and acquisition of stx2a among STEC isolates submitted to the United Kingdom Health Security Agency (UKHSA) between 2016 and 2024. 12,888 whole genome STEC sequences and associated metadata were analysed. 31.9% of STEC isolates harboured stx2a, spanning 78 O serogroups with a marked shift from STEC O157 to non-O157 serogroups over time. STEC O26:H11 and STEC O145:H28 were the primary drivers of observed increases, most commonly associated with stx2a alone or in combination with stx1a. The widespread and increasing presence of stx2a across the STEC population in England highlights an emerging public health risk and demonstrates the value of routine genomic surveillance in monitoring high-severity Shiga toxin subtypes.

Klebsiella pneumoniae (KP) isolates belonging to multi-locus sequence type 258 (ST258) are a frequent cause of hospital-associated outbreaks and display extensive multidrug resistance. The KP ST258 lineage consists of two genetically distinct clades, called Clade 1 and Clade 2. These two clades are genetically related to one another, but are historically distinguished by having different capsular polysaccharide types. While bacteria belonging to both clades are isolated from clinical infections, Clade 2 is isolated more frequently compared to Clade 1. To investigate drivers of this difference in clade prevalence, we collected 172 clinical KP ST258 isolates from patients at a single medical center. Clinical review showed that patients infected with Clade 2 isolates were more acutely ill than Clade 1-infected patients, despite having fewer comorbidities. We also found that Clade 2 isolates were more resistant to killing by human serum, despite binding more complement protein C3 than Clade 1 isolates. Additionally, mice infected with a Clade 2 isolate had increased bacterial dissemination from the lungs to the liver and spleen than mice infected with a Clade 1 isolate, and this dissemination required an intact capsule locus. Increased dissemination in mice was not due to differential serum killing, as mouse serum was unable to kill isolates of either clade, but dissemination was associated with decreased macrophage uptake of the Clade 2 isolate. Taken together, these data suggest that KP ST258 Clade 2 is more virulent than Clade 1, though the specific mechanisms at play appear to differ between mice and humans.

Third-generation cephalosporin (3GC) resistant Klebsiella pneumoniae are an increasing public health threat in Tunisia, yet there is limited data on the circulating lineages and antimicrobial resistance (AMR) determinants underlying this threat. Here, we employed whole-genome sequencing (WGS) in the Tunisian AMR surveillance system (TARSS) to characterize the 3GC resistance mechanisms, population structure, virulence, and transmission across three participating sentinel hospitals in Tunis and Ben Arous. We sequenced a balanced sample of stored 3GCR (3GC-resistant) isolates from blood and urine collected between 2018 and 2022. Of 322 sequenced isolates, 286 (89%) were confirmed as K. pneumoniae, representing 28.5% of all stored 3GCR isolates. The population structure was diverse (68 sublineages) and distinct between hospitals, although several globally distributed sublineages were detected across sites (SL383, SL101, SL307, SL15). Extended-spectrum {beta} - lactamases (ESBL) genes were detected in 77% of genomes, with blaCTX-M-15 (65.4%) and blaCTX-M-14 (8%) dominant at all sites and across diverse sublineages. AmpC genes occurred in 9%, and carbapenemase in 19.6% (blaOXA-48, 14.7%; blaNDM-5, 4.5%; blaNDM-1, 3.8%), with carbapenemases mainly observed amongst SL147 and SL383 at Hospital B (41.7%). Despite sequencing less than a third of the unique 3GCR infections in each hospital, we identified 24 probable nosocomial transmission clusters involving 64 isolates. Each cluster was restricted to a single hospital, although many were detected across multiple wards in the same hospital. The acquired virulence-associated locus (ICEKp) encoding yersiniabactin was common (48.6%). Hypervirulence-associated markers (encoding aerobactin, salmochelin, and/or hypermucoidy) were rare (8.7%) but increasing over time. These were mostly found in sublineages in which convergence of ESBL and hypervirulence has been reported in other settings (including SL147, SL101 and SL383), suggesting international dissemination of convergent strains. These findings show sustained ward-level nosocomial transmission of 3GCR K. pneumoniae lineages and site-specific differences in ESBL and carbapenemase burdens, which call for targeted infection prevention and control and for future routine integration of WGS into TARSS.

Background: Antimicrobial resistance (AMR) is a global public health problem. Infections caused by extended-spectrum beta-lactamase (ESBL) and carbapenemase (CP) -producing Enterobacterales (E) threaten individuals and healthcare systems worldwide. Symptomatic infection caused by Enterobacterales is typically preceded by asymptomatic colonisation and often occurs in the most vulnerable individuals, thus interrupting asymptomatic transmission is desirable. The dominant transmission routes across the healthcare continuum including hospitals, intermediate care, and long-term care facilities are not well understood. Methods: Here we present a protocol describing a genomic surveillance framework developed for the Tracking Antimicrobial Resistance Across Care Settings (TRACS) Liverpool programme, which aims to identify critical ESBL-E transmission points in hospitals and care homes in Liverpool, UK. Our study integrates individual participant and healthcare facility data, validated standard operating procedures for taking and culturing stool, rectal, environmental, and staff samples, and genomic sequencing of ESBL-E, and statistical modelling approaches into a research framework for ESBL-E genomic surveillance. Discussion: There is a need for improved epidemiological and laboratory approaches to studying bacterial transmission. Drug-resistant enteric bacteria are a highly tractable marker of the movement of all enteric bacteria, and interventions designed to interrupt transmission of drug-resistant bacteria are expected to have a broader healthcare impact. This protocol provides a standardised, reproducible approach for identifying ESBL-E, tracking acquisition events, and linking clinical and environmental isolates through whole-genome sequencing.

Direct genome sequencing of Mycoplasma genitalium from clinical specimens is challenging due to the organisms low bacterial load. We developed and compared two DNA enrichment strategies--amplicon-based and hybridisation capture-based--coupled with next-generation sequencing, and assessed the suitability of DNA capture for whole-genome sequencing and high-resolution molecular typing. The enrichment approaches, RNA bait hybridisation and targeted sequence amplification, were combined with paired-end sequencing on the Illumina iSeq 100. Method performance was evaluated in 89 M. genitalium-positive specimens across five genomic loci: 23S and 16S rRNA, and parC and gyrA for macrolide, tetracycline, and fluoroquinolone resistance, respectively, and mgpB for phylogenetic analysis. Regarding antimicrobial resistance (AMR) determinants, the multiplex amplicon sequencing method demonstrated the highest sensitivity, at 93.3-98.9%. Although < 50% of samples were characterised using DNA capture, concordance between the two methods was excellent. Using DNA capture, 24.7% of specimens achieved a minimum coverage of 95% at 1x depth. Sequencing success was inversely correlated with human DNA contamination and the presence of low-quality reads. Whole-genome single-nucleotide polymorphism analysis provided higher discriminatory power than multi-locus sequence typing schemes and confirmed the clonality of multidrug-resistant M. genitalium strains belonging to the mgpB genotype 159. In conclusion, targeted amplicon-based enrichment is the most accurate and reliable approach for epidemiological studies focused on AMR and mgpB typing, whereas DNA capture is valuable for generating comprehensive genomic data from selected M. genitalium-positive specimens. Impact statementCulturing Mycoplasma genitalium from clinical specimens is challenging; consequently, epidemiological studies rely on molecular techniques. Whole-genome sequences have been obtained from a few M. genitalium clinical isolates, and direct sequencing from clinical specimens requires enrichment strategies to overcome the organisms low bacterial load. Consequently, published data on antimicrobial resistance (AMR) mechanisms and phylogenetic relationships among circulating strains remain incomplete. We demonstrated that targeted amplicon-based enrichment provided high-resolution AMR detection and highly sensitive mgpB genotyping, enabling the identification of minority variants. In contrast, hybridisation capture-based enrichment showed lower sensitivity but permitted successful WGS in a subset of M. genitalium-positive specimens and supported the development of new typing schemes. Although amplicon-based enrichment remains a reliable approach for epidemiological studies, strategic application of DNA capture facilitates the generation of comprehensive genomic data even though performance is reduced in the context of low bacterial loads, high host DNA contamination or suboptimal DNA quality.

BackgroundAchromobacter spp. are emerging opportunistic pathogens, associated with chronic infections, antimicrobial resistance, and poor clinical outcomes. The Danish epidemic strain (DES) of A. ruhlandii is highly drug-resistant and adapted to the cystic fibrosis (CF) airway, yet its evolutionary history and defining genomic features remain poorly understood. MethodsWe analysed genome and antibiotic susceptibility testing data for 58 longitudinally collected DES isolates sampled over 21 years at Rigshospitalet, Denmark. We combined these with 79 publicly available A. ruhlandii genomes and applied phylogenomics to infer DES emergence and transmission, and genome-wide association studies (GWAS) to identify lineage-specific and adaptive genomic features. ResultsDES forms a distinct monophyletic clade within A. ruhlandii, estimated to have emerged around 1990, with no evidence of dissemination beyond Denmark. GWAS identified key lineage-defining traits, including acquisition of large mobile genetic elements, plasmid integration events, and enrichment of resistance and iron acquisition genes. In addition, we detected other epidemic A. ruhlandii lineages with evidence of long-term persistence and inter-country spread, sharing similar genetic signatures of adaptation. ConclusionsThis study elucidates the genomic features associated with chronic infection and epidemic potential in A. ruhlandii. The DES lineage illustrates how extensive horizontal gene transfer, high intrinsic resistance potential, and enhanced host-adaptation traits, such as increased iron acquisition, can facilitate the emergence and persistence of successful epidemic lineages. These findings highlight shared evolutionary signatures of epidemic A. ruhlandii and underscore the need for continued genomic surveillance to detect and monitor emerging high-risk lineages in chronic infections.

Background: People living with HIV (PLHIV) in sub-Saharan Africa exhibit high rates of pneumococcal carriage compared to HIV-uninfected adults, despite antiretroviral therapy. We established a novel controlled human infection model of experimental pneumococcal carriage in people living with HIV to understand carriage dynamics in this at-risk population. Methods: Seventy-five virally suppressed and clinically stable PLHIV and 75 HIV-uninfected controls were inoculated with escalating doses of pneumococcus serotype 6B. Carriage acquisition and density were determined by microbiological culture of nasal wash samples collected before and up to 14 days after inoculation. Adverse events were identified by active and passive surveillance. Participant-reported acceptability was established using a Likert scale. Findings: No serious adverse events occurred. Mild adverse events were similar between groups (19% [14/75] in PLHIV, 13% [10/75] in HIV-uninfected; p=0.505). More than 90% of participants reported acceptability with all study procedures. Experimental carriage occurred in 21% (16/75) of PLHIV compared with 36% (27/75) of HIV-uninfected participants (adjusted odds ratio 0.39 [95% CI 0.16-0.91]). Among PLHIV without detectable cotrimoxazole, 28% (8/29) acquired experimental carriage. Carriage clearance rates were lower in PLHIV (hazard ratio 0.44 [95% CI 0.14-1.42]). Interpretation: In carefully selected PLHIV with effective viral suppression and clinical stability experimental pneumococcal carriage acquisition did not exceed that in HIV-uninfected adults, even after accounting for antibiotic use, natural pneumococcal co-colonisation, and sociodemographic differences. These findings suggest that high carriage prevalence in PLHIV in sub-Saharan Africa may be driven more by prolonged carriage duration than increased susceptibility to acquisition. This model provides a platform to investigate mechanisms underlying carriage susceptibility and impaired clearance in PLHIV and to evaluate interventions aimed at reducing the carriage burden in sub-Saharan Africa. Funding: Wellcome Trust

Conventional diagnostic methods (CDM) for Legionella preferentially detect L. pneumophila and frequently fail to identify non-pneumophila species (NPLS), obscuring the full clinical spectrum of infection and limiting surveillance accuracy. We analyzed plasma microbial cell-free DNA (mcfDNA) sequencing detections of Legionella spp. from a large clinical cohort tested between 2018 and 2024 and compared species distributions with culture and PCR confirmed cases reported in the most recent national surveillance datasets (2018-2021). To contextualize the clinical impact, we reviewed published reports in which mcfDNA sequencing was used to diagnose legionellosis (2021-2025) and evaluated real-world performance data from a hospital contributing 8.9% of detections within the cohort (Hospital A). mcfDNA sequencing identified proportionally fewer L. pneumophila, more NPLS, and fewer unresolved species than the CDC reports (all p<0.001). Among 15 publications describing 19 U.S. patients, 74% were immunocompromised and 79% had NPLS infections. Concordance between mcfDNA and CDM occurred in 31.6% of cases. At Hospital A with 36 detections, diagnosis was established by CDM alone in none, by both CDM and mcfDNA in 23.5%, and by mcfDNA alone in 76.5%, yielding an additive diagnostic value of 56.8% These findings suggest that plasma mcfDNA sequencing may improve detection of NPLS particularly in high-risk or diagnostically challenging patients and provide complementary data for both clinical diagnosis and epidemiologic surveillance.

Shigella sonnei is a human-adapted enteric pathogen with a very low infectious dose and increasing antimicrobial resistance. In high-income settings, transmission is multimodal including sporadic cases/outbreaks associated with food and travel, as well as sustained transmission among sexual networks of men who have sex with men (MSM). Whole-genome sequencing (WGS) now underpins national shigellosis surveillance in the United Kingdom. Hence, consistent, communicable genotyping is essential for case linkage and trend detection across heterogeneous transmission modes. Here, we evaluate the performance of WGS genotyping approaches for granulating outbreaks of S. sonnei shigellosis, particularly considering differential performance in dense sexual transmission where highly clonal MSM-associated sublineages pose distinct clustering challenges. Specifically, we compare performance of the current practice approach (10 SNP-distance clustering based on SNP address [t10]), allele-based methods (EnteroBase cgMLST/HierCC [HC5]), a pathogen-specific genotyping scheme (sonneityper), and two k-mer based approaches (PopPUNK and KPop), on a bona fide UK surveillance dataset (n = 3,639 isolates from between 2016 and 2022), and stratify analyses by demographics (i.e. presumptive MSM [pMSM] versus non-pMSM). Comparison metrics indicate that t10 clustering method groups data more broadly than HC5, and k-mer-based methods may capture genetic variation independent from SNP or allele-based approaches. Clusters derived from k-mer-based methods offer similar resolution to HC5 and reflect different demographics, but had unconvincing utility for this pathogen. These findings suggest a transmission context-aware surveillance workflow for shigellosis in high income settings: anchor routine communication on a portable allele-based backbone and augment with more granular, complementary methods (e.g., k-mer-based micro-partitioning or phylogenetic analysis) in comparatively low genomic-density regions of population structure (e.g., pMSM transmission lineages) to stabilise clusters and reduce artefactual chaining.

Aminoglycoside drugs, amikacin, streptomycin, and amikacin liposome inhalation suspension are crucial for treating refractory Mycobacterium avium-intracellulare complex pulmonary disease. In Mycobacterium tuberculosis, cross-resistance occurs between amikacin and kanamycin, but not between amikacin and streptomycin in genetic drug susceptibility testing. However, the occurrence of cross-resistance among aminoglycosides remains unclear in M. avium-intracellulare complex. We aimed to evaluate cross-resistance among aminoglycosides to determine whether streptomycin or kanamycin remains effective after the development of amikacin resistance. This single-center retrospective study included 20 patients with amikacin-resistant M. avium-intracellulare complex harboring rrs mutations. Paired analyses of streptomycin and kanamycin minimum inhibitory concentration values before and after amikacin resistance development were performed. In addition, streptomycin- and kanamycin-resistant strains were generated in vitro and resistance-associated mutations were identified using whole-genome sequencing. No significant increase was observed in streptomycin minimum inhibitory concentration values following amikacin resistance. In contrast, kanamycin values uniformly increased to >256 g/mL after the acquisition of amikacin resistance. Furthermore, amikacin- and kanamycin-resistant isolates shared mutations at position 1408 in the rrs gene, whereas streptomycin-resistant isolates exhibited mutations at position 20 in the rrs gene. These results suggest that amikacin and kanamycin exhibit cross-resistance in M. avium-intracellulare complex, whereas amikacin and streptomycin may not. Two cases in our cohort in which streptomycin treatment was effective after the acquisition of amikacin resistance further support these findings. In conclusion, streptomycin may be a potential therapeutic alternative for amikacin-resistant M. avium-intracellulare complex pulmonary disease. Future studies correlating streptomycin minimum inhibitory concentration values with clinical outcomes are required.

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of [&ge;]2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance.

Background: Shigella is a leading cause of childhood diarrhea in low- and middle-income countries and is increasingly resistant to first-line antibiotics. We conducted a surveillance study to determine the incidence, genomic characteristics, and AMR profiles of Shigella infections in children under five with moderate to severe diarrhea (MSD) in Lusaka, Zambia. Methods: Between 15 September 2020 and 30 November 2021, a prospective cohort study of 1,400 children under five was enrolled during a community census in a peri-urban setting and passively followed for 9.5 months for MSD. During enrollment, socio-demographic data were collected using electronic questionnaires, while clinical data were collected through the DHIS platform. The main outcome, Shigella in diarrheal stool in under 5 children, was detected using culture and Loop-mediated Isothermal Amplification (LAMP) targeting the ipaH gene. Cox proportional hazards models were used to assess the incidence and risk factors of Shigella (ipaH) infections. Whole-genome sequencing (WGS) was used to characterize the genomic diversity and antimicrobial resistance genes, complemented by phenotypic antibiotic susceptibility testing. Results: There were 230 first episodes of Shigella over a follow-up time of 9,581.7 child-months, yielding an incidence of 24.0 (95% CI 21.1-27.3) cases per 1,000 child-months, with the highest incidence among 2 to 3-year-olds. The key risk factors identified were the water source (p=0.025) and age group (p=0.014). Genotypic characterization revealed 10 S. flexneri, 9 S. sonnei, and 3 S. boydii. The S. sonnei isolates formed two clusters, differing in virulence factors and plasmid profiles, indicating two possible circulating strains. Shigella isolates exhibited phenotypic and genotypic multidrug resistance, including against trimethoprim, aminoglycosides, and beta-lactams. Plasmid-mediated quinolone resistance (qnrS1) was identified in four S. flexneri isolates, with these genes located on the IncFIB(K) plasmid, highlighting the potential for horizontal transmission and spread of quinolone resistance in this region. No phenotypic and genotypic resistance to macrolides, the first-line treatment for Shigella in Zambia, was observed. Interpretation: We report a high burden of Shigella with multidrug resistance, including resistance to fluoroquinolones. These findings highlight the increasing resistance of Shigella to first-line antibiotics and underscore the importance of developing safe and effective vaccines, improving WASH conditions, and ongoing AMR surveillance. Funding: The EDCTP2 program, supported by the European Union, the Faculty for the Future Foundation (FFTF), the Netherlands Organization for Health Research and Development (ZonMw), and Health-Holland AMR-Global, Gloria, and Track-AMR.

Infections caused by carbapenem-producing Enterobacterales (CPEs) are a persistent and growing threat in healthcare settings. Yet, current infection prevention and control (IPC) surveillance methods, which largely rely on the spatial and temporal proximity of patients, often misattribute or miss infection transmission events. Here, we develop and retrospectively evaluate an integrated methodology that combines analyses of ward-level patient movement data and whole-genome sequencing (WGS) data analyses, providing measures of bacterial and plasmid similarity. Specifically, we evaluate this methodology across two datasets: a CPE outbreak of diverse carbapenem types (103 genomes, January 2021 to March 2021) and an Imipenem-Hydrolysing beta-lactamase-positive CPE outbreak (82 genomes, June 2016 to October 2019), using standard clinical criteria and conservative genomic thresholds to quantify how often current IPC surveillance methods correctly identify genomically confirmed transmission events. Findings show that, across 3,423 patient contact-genome pairs, current IPC surveillance methods detected only 20.5% of genomically confirmed transmission events whilst maintaining 98.5% specificity, with missed events arising from temporal, spatial, and cross-species, mechanistic blindspots. In contrast, WGS-enabled IPC surveillance methods provided a 25 to 47-day earlier detection window and, in a linked economic evaluation, delivered annualised savings of up to GBP 3.6 million, as well as a return on investment exceeding 2-fold in 7 of 8 cost scenarios. By operationalising high-throughput WGS data analysis with clinically relevant patient movement data, we evidence that it may be possible to disrupt and thereby mitigate the effects of AMR-driven CPE outbreaks, supporting investigations into the adoption of WGS-enabled IPC surveillance as a standard-of-care tool.

Background: Global syphilis rates have risen dramatically since the early 2000s. Genomes can be used to inform rational control and intervention strategies by enhancing surveillance and ensuring vaccines have broad global utility. However, although >1000 Treponema pallidum genomes are now available from high-income countries, genomic data from Africa remain limited. Methods: We combined samples from 1198 participants recruited into a genital ulcer aetiology study in Botswana, Ghana, Uganda and Zimbabwe (collected 2022-2023) with 276 samples from national syphilis surveillance in South Africa to generate 147 novel African T. pallidum genomes (collected 2006-2023). Combining these with 167 publicly available African genomes and 1062 genomes from 24 non-African countries, we performed contextual population genomic analyses to understand the T. pallidum genomic diversity and transmission within and between African countries and the rest of the world. Findings: Contrasting with previous studies showing global circulation of highly similar T. pallidum, we found remarkable diversity amongst African T. pallidum. Of 56 sublineages, 20 were exclusively found amongst 6 African countries, 31 were found amongst 24 non-African countries, and 5 were found in both. Sublineage sharing between Africa and the rest of the world was rare, with 83.8% of African syphilis caused by locally circulating sublineages. Only 20.1% of African syphilis was resistant to macrolides (global average = 68.6%); where resistance occurred, this was strongly linked to introduction of global sublineages into Africa. Interpretation: African T. pallidum is characterised by locally circulating strains not found globally. Since sublineage sharing between countries is low, cataloguing African T. pallidum diversity will require intense local sampling in many countries. These findings will inform ongoing strategies for genomic surveillance and vaccine design, whilst contributing to our understanding of the spread of antimicrobial resistance in Africa, enabling refined treatment guidelines based on local data. Funding: Wellcome and the Gates Foundation.

Colonization of the gastrointestinal (GI) tract by vancomycin-resistant Enterococcus faecium (VREfm) often precedes bloodstream infection and serves as a reservoir for onward patient transmission in healthcare settings. Routine clonal isolate-based sequencing often underestimates within-patient diversity, and can miss transmission involving low-abundance and co-colonizing strains. Here we applied culture-enriched metagenomic sequencing to matched GI tract and blood VREfm populations collected [&le;]14 days apart from 35 patients with positive VREfm blood cultures collected between 2020 and 2025 at a single hospital. GI populations exhibited greater within-patient diversity than bloodstream populations, including multi-strain colonization in five patients. Among single-strain populations, variant analysis suggested distinct environment-specific pressures between the GI tract and bloodstream environments. To assess transmission using culture-enriched metagenomic sequencing, we compared all 70 VREfm populations against 470 contemporary clinical VREfm isolate genomes collected from the same hospital and identified 19 putative transmission clusters, including six clusters involving multi-strain populations. Together, these results demonstrate how culture-enriched metagenomic sequencing improves resolution for assessing within-patient VREfm diversity and enhances the detection of transmission events that could be missed by clonal isolate-based surveillance. Impact StatementVREfm bloodstream infection is often seeded from bacteria colonizing the gut. The genetic diversity within gut and blood VREfm populations, and the role of this diversity in bacterial transmission, has been difficult to resolve as genomic surveillance typically relies on sequencing a clonal clinical isolate from each patient. Using culture-enriched metagenomic sequencing of matched GI tract and bloodstream VREfm populations from 35 patients at a single hospital, we found that the GI tract reservoir contained VREfm populations with greater strain and variant diversity than populations collected from the bloodstream. By integrating population sequencing with a large collection of VREfm clinical isolate genomes, we further demonstrate that different strains co-colonizing the GI tract of the same patient can reside in multiple putative transmission clusters, revealing potential transmission links that clone-based approaches are likely to miss. These findings demonstrate the potential utility of culture-enriched metagenomic sequencing for higher-resolution hospital surveillance of bacterial transmission. Applying this approach to other bacterial pathogens could improve our ability to detect and interpret transmission involving heterogeneous microbial populations that colonize and infect hospitalized patients. Data SummaryPatient demographic data and clinical characteristics can be found in Table S1 (online Supplementary Material). All sequencing data generated in this study has been deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA901969, with sample accession numbers listed in Table S2. Sequences used to construct the local reference strain database are available at NCBI BioProject PRJNA475751, with accession numbers listed in Table S3. Variants identified in single-strain blood and GI populations are listed in Table S4. Accession numbers for clinical isolate genomes included in transmission analyses are listed in Table S5. RepositoriesSequencing data generated in this study is deposited in the National Center for Biotechnology Information (NCBI) under BioProject PRJNA901969.

BackgroundPartial resistance to artemisinins (ART-R) has emerged in East Africa, associated with mutations in the Plasmodium falciparum kelch13 gene. It is currently unclear whether ART-R has implications for gametocyte production or for onward transmission to mosquitoes. MethodsIn a cohort of uncomplicated malaria patients attending Kalongo Hospital in northern Uganda, we quantified carriage of PfKelch13 mutant parasites by conventional sequencing and droplet digital PCR (ddPCR) for the C469Y and A675V mutations. Prevalence and density of gametocytes and ring-stage parasites were assessed by microscopy and quantitative reverse-transcriptase PCR (qRT-PCR). Lumefantrine concentrations, indicative of prior malaria treatment, were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. Transmission potential of wild-type and PfKelch13 mutant parasites was assessed by mosquito feeding assays and complemented with molecular characterization of parasites in wild-caught mosquitoes from household resting catches. FindingsWe enrolled 235 patients with symptomatic P. falciparum infection; PfKelch13 C469Y or A675V mutations were detected in 35.8% (78/218) of infections by sequencing and 59.1% (136/230) by ddPCR. Gametocyte carriage was 24.0% (56/233) by microscopy and 56.6% (133/235) by qRT-PCR and not associated with the abundance of PfKelch13 mutant parasites by ddPCR (p=0.603). Among a total of 227 mosquito feeds with patient whole blood, 1.4% (120/8745) of mosquitoes became infected. Mosquito infection rates were positively associated with gametocyte density ({beta} = 0.39, 95% CI = 0.23-0.59, p < 0.001) without an observed interaction with the abundance of PfKelch13 mutant parasites (p = 0.452). PfKelch13 C469Y or A675V mutations were detected in 40.1% (21/52) of malaria-infected bloodmeals of field-caught mosquitoes and in 28.0% (7/25) of sporozoite-positive mosquitoes. InterpretationWe conclude that pfkelch13 mutations are very common in patients in northern Uganda with uncomplicated malaria, mostly in multiclonal infections. We observed no evidence that ART-R affected gametocyte production or transmission to mosquitoes. FundingDutch Research Council (NWO) Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPartial resistance to artemisinins (ART-R) might enhance or inhibit gametocyte formation or transmission of malaria parasites to Anopheles mosquitoes. However, few studies directly assessed P. falciparum transmission potential in relation to sensitivity to ART-R or associated mutations. We searched PubMed on January 14th 2026, with no restrictions on publication date or language, for studies assessing gametocyte carriage or transmissibility to mosquitoes in uncomplicated malaria cases in relation to artemisinin resistance using the search terms ("uncomplicated malaria" OR "patient") AND ("gametocyte" OR "anopheles") AND (("artemisinin" AND "resistance") OR "pfkelch13" OR "kelch13"). From the 101 identified articles, the majority did not report on parasite resistance or resistance markers. Nineteen articles reported original patient data, with four additional studies examining parasite isolates for gametocyte production and transmissibility in vitro. In vitro studies all reported gametocyte formation in parasite isolates with pfkelch13 mutations and, for three studies where transmission was directly determined, evidence for successful mosquito infections from ART-R parasite isolates. None of these studies demonstrated consistent differences in gametocyte production or transmissibility between ART-R and sensitive parasite isolates. One clinical study from Thailand observed higher levels of gametocyte carriage during follow-up for infections with slow asexual blood-stage parasite clearance following artemisinin-combination therapy (ACT) and an increase in gametocyte carriage at clinical presentation over a period when the prevalence of ART-R was rising. A study with patient data from 7 Asian and 3 African countries reported higher proportions of pretreatment and post-treatment gametocytemia in patients with slow parasite clearance. Another study from Cambodia reported a higher prevalence of gametocytes at enrolment in areas affected by ART-R, but no association between gametocyte carriage and either individual-level parasite clearance or treatment failure. In contrast, a meta-analysis of ACT treatment efficacy in pregnant women observed no association between gametocyte carriage at baseline and treatment failure. Only one study, directly examined gametocyte carriage at clinical presentation with pfkelch13 mutations and found no association between gametocyte carriage and molecular markers of ART-R or ex vivo drug sensitivity in Cambodia. Added value of this studyWe performed a direct assessment of gametocyte production, carriage of mature gametocytes and transmission to Anopheles gambiae s.s. mosquitoes in relation to validated ART-R markers in patients presenting with uncomplicated P. falciparum malaria in an area affected by ART-R in Uganda. Our work demonstrates that parasites with the PfKelch13 C469Y and A675V mutations were very common, mostly in multiclonal infections. Gametocyte carriage and gametocyte commitment were similar between patients presenting with pure or predominantly wild-type infections and those presenting with PfKelch13 mutant infections. In feeding studies, mosquito infection rates were similar for patients with wild-type or PfKelch13 mutant infections; wild-caught mosquitoes confirmed ongoing transmission of parasites with molecular signatures of ART-R. Implications of all the available evidenceTaken together, there are no indications for altered gametocyte production or transmissibility to mosquitoes of infections with PfKelch13 C469Y and A675V. Future studies may examine impacts of other mutations in pfkelch13, study transmission potential in low endemic settings where monoclonal infections dominate and aim to understand how gametocyte clearance and post-treatment transmission potential may differ between ART-R and wild-type infections upon treatment.

Numerous factors may influence the optimal rollout of new gonococcal antibiotics. We compared eight rollout strategies using a gonorrhea transmission model and ranked strategies by the number of gonococcal infections and clinically useful antibiotic lifespan. Rankings were most sensitive to the starting ceftriaxone resistance prevalence and screening frequency.

Serological testing is important for assessing past exposure and immunity, but interpretation can be complicated by antibody cross-reactivity between closely related viruses. We assess this challenge for Usutu virus (USUV) and West Nile virus (WNV), flaviviruses that recently emerged in Europe. We analysed samples from wild blackbirds collected in the Netherlands between 2016-2022. Samples (N=1742) were screened using an NS1-based protein micro-array, with positives confirmed by Focus Reduction Neutralization Tests (FRNT). We jointly estimated seroprevalence and antibody responses by fitting a Bayesian latent-variable model to FRNT values. Estimates of homologous and cross-reactive antibody responses were used to improve interpretation of observed titres for serosurveillance. Estimated seroprevalence varied across time and regions between 4.9% (95%CrI 3.5-6.7) to 18.5% (95%CrI 14.9-22.7) for USUV and between 2.4% (95%CrI 1.3-3.8) to 6.4% (95%CrI 3.9-9.6) for WNV. These were 1.5 (USUV) to 2.4 times (WNV) higher than estimates based on the current threshold-based algorithm. USUV induced a higher antibody response and was more likely to induce a cross-reactive response than WNV. Our classification algorithm informed by these estimates showed high sensitivity (WNV: 0.88, USUV: 0.97) and specificity (both: >0.99). Our results illustrate how quantitative frameworks can improve serological interpretation in settings with co-circulating pathogens.

Extended-spectrum beta-lactamase-producing Enterobacterales such as Escherichia coli and Enterobacter hormaechei represent a growing public health challenge in clinical settings, particularly in low-and middle-income countries, due to the escalating threat of antimicrobial resistance (AMR). In this study, we aimed to identify the antibiotic resistance genes present in E. coli (n=4) and E. hormaechei (n=3) clinical isolates. Multidrug-resistant phenotypes were confirmed using disc diffusion assays against 20 antibiotics. Whole-genome sequencing of resistant isolates was performed using Oxford Nanopore Technologies. Genome assembly and analysis revealed high-risk clones, including sequence type (ST) 1193 in E. coli and ST78 in E. hormaechei. All E. coli isolates harbored the blaCTX-M gene in their chromosomes along with point mutations conferring resistance to fluoroquinolones, while E. hormaechei isolates encoded blaACT in their chromosomes. Additionally, both species carried plasmids with multiple antibiotic resistance genes, including blaOXA and blaTEM, co-located with metal resistance operons, indicating the potential for horizontal gene transfer. BLAST analysis revealed high sequence similarity between the plasmids identified in clinical isolates and those previously recovered from environmental sources, highlighting the role of environmental reservoirs in AMR dissemination. Notably, no carbapenem resistance genes were detected in any isolate. These findings underscore the growing threat posed by multidrug-resistant Enterobacterales in clinical settings and emphasize the urgent need for strengthened infection prevention and control measures to mitigate AMR spread.

BackgroundProlonged SARS-CoV-2 infection in immunocompromised individuals may accelerate virus evolution within the host, raising concerns about the virus evading immunity, developing resistance, and forming novel variants of concern. However, the determinants and public health implications of within-host viral evolution in this population remain incompletely understood. MethodsWe performed longitudinal analyses of SARS-CoV-2 genomes from 91 patients with COVID-19 who were classified as being severely or moderately immunocompromised. Using serial measurements of viral RNA loads and infectious titers, we modeled the shedding dynamics of the virus and stratified the infected cases by upper respiratory virus shedding duration to assess associations with within-host evolutionary dynamics. ResultsShedding modeling identified two profiles of shedding duration: intermediate and long. The long shedding profile (shedding lasting >21 days) was found in 14.8% of moderately immunocompromised cases and 72.1% of severely immunocompromised cases. Frequent single-nucleotide variants accumulated specifically in severely immunocompromised individuals with the long shedding phenotype, correlating positively with shedding duration. By contrast, mutations remained limited in moderately immunocompromised individuals with the long shedding phenotype and in severely immunocompromised individuals with the intermediate shedding phenotype. We identified mutations in the spike receptor-binding domain associated with monoclonal antibody resistance; however, we found no fitness-enhancing mutations for inter-host transmission, and antiviral drug resistance mutations were rare. Instead, mutations were introduced frequently and randomly across the entire viral genome. ConclusionsProlonged upper respiratory virus shedding exceeding 21 days combined with severe immunocompromise is a risk factor of the accumulation of within-host SARS-CoV-2 mutations. Although no variants of concern emerged, the introduction of genome-wide random mutations suggests that the risk for novel variant generation cannot be excluded. These findings highlight the need for intensive antiviral strategies to limit shedding duration to less than 21 days in severely immunocompromised patients, and for immunological investigations to elucidate the host factors underlying residual shedding control in those who achieve clearance within this threshold.