Pneumonia is the leading infectious disease killer worldwide and commonly requires admission to critical care. Despite its prevalence, the underpinning biology of severe pneumonia remains incompletely understood. Here we perform multifaceted assessments of bronchoalveolar transcriptome, cytokines, microbiology, and clinical features to biologically characterise a cohort of patients with suspected severe pneumonia. Our data implicate three lung-restricted transcriptionally defined severe pneumonia endotypes (termed 'Pneumotypes' (Pn)). All three Pneumotypes have comparable clinical presentations and severity of respiratory failure but experience divergent outcomes. Pn1, the most common, is characterised by low alveolar cytokines, expanded tolerogenic macrophages and epithelial damage. Pn3 is characterised by immature neutrophil infiltration, IL-6-STAT3 activation and longer duration of mechanical ventilation. Pn2 displays the fastest resolution, exhibiting a balanced immune response and epithelial-endothelial repair signatures. We identify and validate mechanistically distinct phenotypes in the lungs of patients with suspected pneumonia and acute lung injury, implicating targets for personalised therapy.

Abstract Background Shigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo , to generate a “double-hit” Shigella glycoconjugate vaccine. Method Glycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. Results When co-produced in E. coli , PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella -specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. Conclusion A novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a “double-hit”, immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

Klebsiella pneumoniae has been traditionally considered an opportunistic pathogen, now otherwise healthy individuals commonly present with K. pneumoniae blood infections, which can result in serious complications such as liver abscesses and septic shock. Consequently, the bacterium has become a leading cause of human disease. Along with classical K. pneumoniae (cKp), which is an important reservoir of antimicrobial resistance (AMR) genes, hypervirulent K. pneumoniae (hvKp) is increasingly becoming of high clinical concern because it carries numerous virulence factors. A high proportion of bloodstream infections (BSIs) in Vietnam are associated with K. pneumoniae, with the epidemiology, population structures and transmission dynamics of hvKp being well described. However, little is known about how virulence-associated traits contribute to increased pathogenicity in this setting and which genetic determinants may be attributable to the virulence variation. In this cross-sectional study, we used a subset of clinical isolates to define the genomic structure and population of K. pneumoniae BSI isolates and additionally comprehensively characterised specific phenotypic features of hvKp associated with clinical manifestations. A genomic analysis of 83 random selected K. pneumoniae BSI isolates defined that lineage of KpI was the most common lineage (91.5%). The hypervirulent sequence type 23 (ST23) was the most prevalent (18.5%), followed by the multidrug resistance (MDR) lineages ST15 and ST17 (7.7% each). Specific hvKp ST23 phenotypic virulence assays showed considerable variation in virulence potential despite possessing a comparable compendium of virulence factors. Comprehensive analysis of genomic variations across the ST23 isolates revealed different genetic backgrounds and distinct mutations occurred on essential genes that might be involved the obsereved phenotypic diversity. These findings show the pathogenic potential of hvKp isolates from Vietnam, and also highlight the need for improved phenotype prediction to understand the clinical threat from strains in this groups.

This study explored the microevolution of Shigella sonnei in China, focusing on 281 isolates exhibiting coresistance to ceftriaxone and azithromycin (cefRaziR) and 99 ONPG-negative isolates. Phylogenetic analysis revealed that waterborne outbreak strains, characterized by multidrug resistance (MDR) and cefRaziR, clustered within the predominant domestic lineage I. In contrast, sporadic MDR strains harboring a wider array of antimicrobial resistance (AMR) genes were primarily associated with lineage II. The cefRaziR phenotype in lineage I was mediated by an IncB/O/K/Z plasmid carrying bla CTX-M-14, mphA, aac(3)-IId, dfrA17, aadA5, and sul1 genes. Lineage II strains acquired cefRaziR through a distinct IncFII plasmid possessing bla CTX-M-15, ermB, and mphA genes, and additionally carried a separate IncB/O/K/Z plasmid backbone with bla TEM-1, dfrA12, sul2, strA, strB, tet(A), and aac(3)-IId genes. Conversion to the ONPG-negative phenotype was linked to a deletion spanning approximately 10 kbp, which included two insertion sequences (IS1 and IS600), the mhpBAR operon, and the lacIZY operon. Genomic comparisons identified 66 SNPs and 9 accessory genes correlated with lineage II, and 23 SNPs with 9 accessory genes associated with ONPG-negative variants. Ongoing surveillance of S. sonnei epidemic clones is essential to elucidate their microevolution, track transmission, and assess public health implications.

Background Patients undergoing haematopoietic stem cell transplantation (HSCT) are highly susceptible to bloodstream infections (BSIs) caused by antimicrobial resistant (AMR) pathogens, yet the temporal dynamics and clinical relevance of intestinal AMR reservoirs remain poorly defined, particularly in high-burden settings. Methods We conducted prospective longitudinal surveillance of 81 HSCT recipients in India using targeted enrichment-based stool metagenomics, coupled with whole genome sequencing of bloodstream isolates and strain-level tracking. Results Across 252 samples, we identified a restructuring of the gut-associated resistome, characterised by depletion during conditioning and a marked post-engraftment expansion of non-efflux AMR determinants, including plasmid-borne carbapenemase and ESBL genes ( bla NDM, bla OXA). This expansion defined a previously underappreciated window of vulnerability during early immune recovery. Over 50% of patients harboured multidrug-resistant organisms, and carriage of bla NDM was associated with increased odds of subsequent infection. Strain-resolved analyses provided genomic evidence linking gut colonisation to bloodstream infection in a subset of cases, demonstrating the feasibility of non-invasive prediction of invasive disease. Patients with AMR-BSIs experienced high mortality (> 40%). Conclusions These findings establish that dynamic changes in the gut resistome following HSCT can identify periods of heightened infection risk and provide a foundation for predictive, genomics-guided surveillance and antimicrobial stewardship strategies in high-burden settings.

A study in the Neonatal Department of Maternity Hospital in HCM, Vietnam, in 2019 reported that the prevalence of Carbapenemase-producing Enterobacteriaceae (CPE) was 42.20% (n = 83). However, risk factors of CPE colonization and transmission were still an unsolved question. Hence, we implemented this study. A prospective study was conducted from April to July 2020 at the Childbirth Ward of Hung Vuong Hospital, where 359 pairs of mothers and their neonates participated in our research. We applied laboratory methods to confirm CPE colonization and its antibiotic resistance, including rectal swab tests, chromo-carba plates, MALDI-TOF method, antibiograms, and rep-PCR method. The 23.0 version of SPSS was a software to analyze personal characteristics, prevalence, and risk factors for CPE colonization. Adjusted odds ratio and 95% confidence interval were considered significant at P < 0.05. The results showed that the prevalence of CP E. coli transmission between mothers and neonates was 0.28% (1/359), confirmed by the rep-PCR method. The characteristics that reduced the CPE-colonization risks in mothers were the mother’s age (19-23 years old), vaginal delivery, mothers caring for neonates, skin-to-skin contact time, and breastfeeding. However, the risk factors that increased the CPE colonization in neonates were the NICU admission before fecal sampling and the number of vaginal examinations performed on mothers before delivery. Although the prevalence of mother-to-neonate CPE transmission was low, screening for CPE colonization at hospital admission, adhering to hand hygiene, and implementing aseptic medical practices are crucial standards for preventing and controlling CPE colonization in the healthcare sector.

Shigella sonnei is rapidly emerging as the dominant agent of shigellosis, an enteric disease responsible for a significant burden of morbidity and mortality worldwide. Whole-genome sequencing of S. sonnei isolated over the last three decades has revealed phylogenomic diversity within the population and the emergence of multiple lineages associated with distinct epidemiological patterns such as resistance to critical antimicrobials and/or transmission within different groups. However, most experimental work on S. sonnei biology and pathogenicity has focused on a single laboratory strain (53G), which is phylogenetically distant from currently circulating strains. Here, we introduce a set of phylogenetically diverse and epidemiologically relevant S. sonnei isolates made available through publicly accessible culture collections as a resource for laboratory science. We present their complete whole-genome sequences, including the pINV invasion plasmid (missing from a large proportion of public genome data due to loss during laboratory culture). Finally, the characterization and comparison of these complete genome sequences highlight evidence for ongoing adaptive evolution in S. sonnei, featuring the accumulation of insertion sequences, gene pseudogenization and structural variation.

BACKGROUND:The spread of extensively drug-resistant (XDR) Salmonella Typhi variants in South Asia has severely limited treatment options for typhoid fever, a life-threatening systemic infection affecting millions. Azithromycin has become the last effective oral drug against XDR typhoid; however, despite increasing azithromycin resistance, limited data exists concerning the correlation between in vitro susceptibility and clinical treatment efficacy. METHODS:We developed a THP-1 macrophage infection model to evaluate time- and concentration-dependent azithromycin activity against intracellular and extracellular S. Typhi, including azithromycin-susceptible (MIC ≤16 mg/L) and azithromycin-resistant isolates (MIC ≥ 32 mg/L). The accumulation of azithromycin in THP-1 macrophages was quantified using LC-MS/MS. Pharmacological modelling was used to estimate key parameters (Emax, Emin, Cs, Csi), and compare azithromycin efficacy between intracellular and extracellular compartments. When available, in vitro findings were correlated with clinical treatment responses. FINDINGS:Azithromycin accumulated in THP-1 macrophages at concentrations 20-72 fold higher than in extracellular medium after 24 h. This intracellular accumulation was associated with concentration-dependent killing of S. Typhi in the intracellular assays, while only bacteriostatic effects were observed extracellularly. Importantly, pharmacological modelling data estimated that azithromycin concentrations ranging from 46.86 to 123.78 mg/L were sufficient to inhibit intracellular growth of azithromycin-susceptible isolates. In contrast, concentrations exceeding 460.63 mg/L were required to suppress intracellular growth of azithromycin-resistant isolates, substantially higher than the Cmax typically observed in human white blood cells (114.0-146.0 mg/L) following a 3 day course of oral azithromycin. Notably, among those with available data, three patients infected with azithromycin-resistant S. Typhi failed to respond clinically to azithromycin treatment. INTERPRETATION:We have established a THP-1 macrophage model to characterise the intracellular pharmacodynamics of azithromycin. Our findings indicate that azithromycin is likely ineffective for treating infections caused by azithromycin-resistant S. Typhi, warranting further clinical validation. FUNDING:Wellcome International Training Fellowship.

Resistance to the polymyxin antimicrobial colistin in Gram-negative bacteria is associated with a modification of the immunogenic lipid A moiety of the lipopolysaccharide (LPS). Chromosomal and plasmid-borne colistin resistance results in the addition of L-Ara4N and pEtN groups to lipopolysaccharide (LPS), respectively. Here, using THP-1 cells, we studied the impact of different LPS modifications of Klebsiella pneumoniae in stimulating host immune response. K. pneumoniae clinical isolates were screened for colistin resistance using broth microdilution (BMD) and the MALDIxin test. LPS was extracted from colistin-resistant isolates and used to stimulate differentiated THP-1 cells. Luminex cytokine assay measured the immune induction via a panel of proinflammatory cytokines. Out of a collection of 72 clinical K. pneumoniae, eight (11.1%) exhibited phenotypic colistin resistance with a minimum inhibitory concentration (MIC) of 8 to 64 mg/L. In total, five isolates possessed genes associated with polymyxin resistance; three isolates had a mutation in the pmrB gene, and two were mcr-8.1 positive. MALDIxin demonstrated that all eight phenotypic colistin-resistant isolates elaborated peaks at m/z 1,955 and m/z 2,193, indicating an L-Ara4N group of LPS modification. For two mcr-8.1 positive isolates, LPS had a pEtN group. The LPS modification positively correlated with colistin MIC (correlation coefficient, r= 0.6 and R2= 0.4). Compared to the native structure, LPS modification was associated with greater production of IL-1β, IL-6, and CXCL-8 (p<0.001). The pEtN-conjugated LPS triggered a significantly greater production of TNF-α, IL-6, and CXCL-8 compared to L-Ara4N (p<0.05). This study reveals that the colistin MIC value can significantly predict lipid A modification in clinical K. pneumoniae, and differences in resistance-mediated lipid A modification result in variation in the immunological response. This study highlights the potential of dynamic host-pathogen interaction in the context of colistin resistance.

[This corrects the article DOI: 10.1371/journal.pntd.0013612.].

Background: Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are among the WHO's highest-priority antibiotic-resistant pathogens. Plasmids are the main drivers of ESBL dissemination, yet their reservoirs and transmission dynamics remain poorly understood in low- and middle-income countries such as Vietnam, where infections caused by ESBL-producing bacteria are prevalent. Methods: Here, we characterised the genetic structure of 68 ESBL-encoding conjugative plasmids isolated from the human gut microbiome (HGM) of healthy Vietnamese children. We further examined the extent of ESBL plasmid transfer between the HGM and human disease-causing Enterobacteriaceae pathogens (including Shigella sonnei, non-typhoidal Salmonella, extraintestinal pathogenic Escherichia coli ST131), as well as E. coli isolated from animals. Results: The dominant plasmid Inc groups found in cephalosporin-resistant human gut bacteria were IncF, IncB/O/K/Z and IncI1, carrying mainly blaCTX-M-14, blaCTX-M-15, blaCTX-M-27 and blaCTX-M-55. These plasmids from the HGM, rather than from animal E. coli, share higher genetic similarity to plasmids in human pathogens, suggesting that human gut is the main reservoir for clinically relevant ESBL plasmids. We also found that widespread ESBL plasmid variants exhibited higher conjugation frequencies, facilitating broader geographical and host dissemination. In contrast, less mobile plasmids persisted mainly through clonal expansion of their bacterial hosts. Conclusions: These findings highlight the central role of the human gut as a reservoir for ESBL plasmids and provide insights into the biological factors contributing to their successful spread among pathogenic Enterobacteriaceae. Our work underscores the need for targeted interventions to reduce colonization and transmission of ESBL-producing bacteria within the human gut. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Duy Thanh Pham is funded by Wellcome Trust International Training Fellowship (222983/Z/21/Z). Experimental work done in Christoph M. Tang's lab is funded by Wellcome Trust (221924/Z/20/Z). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Samples used in this study were collected from a longitudinal cohort study monitoring diarrhoeal disease episodes in children in Ho Chi Minh City (HCMC), Vietnam. Ethical approval for the study was granted by Oxford Tropical Research Ethics Committee (OxTREC number: 1058-13)32. Informed consent was obtained from all participants prior to enrolment. All procedures were conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data used in this work are publicly available in the European Nucleotide Archive (ENA) under project numbers PRJEB96742 (https://www.ebi.ac.uk/ena/browser/view/PRJEB96742)

Background: Typhoid and paratyphoid fevers represent a global health burden, especially in Southern Asia, exacerbated by the increase in antimicrobial resistance. While vaccines against Salmonella Typhi have been successfully introduced, a vaccine against S. Paratyphi A is not available, yet. Efforts to develop an effective vaccine targeting both Salmonella serovars are currently ongoing. GVGH is developing a bivalent vaccine constituted by the Vi-CRM197 typhoid conjugate vaccine (TCV), and the Salmonella Paratyphi A O-antigen (O:2), also conjugated to the CRM197 carrier protein (O:2-CRM197). In this work we have characterized a panel of S. Paratyphi A clinical isolates from endemic regions, differing in terms of their O:2 structural features. Methods: Rabbits were immunized with the S. Paratyphi A component of the vaccine candidate and the resulting sera were tested for their ability to bind and kill the isolates using flow cytometry and luminescence-based serum bactericidal assay (L-SBA). Results: The O:2-CRM197 glycoconjugate induced a functional immune response in rabbits, effectively binding and killing a diverse panel of clinical isolates. The sera demonstrated bactericidal activity independent of the O:2 structural variations, including differences in O-acetylation and glucosylation levels. Additionally, the study found that the O:2-CRM197 conjugate’s adsorption to Alhydrogel did not significantly impact its immunogenicity or bactericidal efficacy. Conclusions: The O:2-CRM197 component of the bivalent vaccine candidate shows promise in providing broad protection against S. Paratyphi A isolates, regardless of their O-antigen structural variations. The ongoing clinical studies on human sera are expected to confirm these results.

Background: The National incidence of bloodstream infection (BSI) caused by Klebsiella Pneumoniae increased during the Covid-19 pandemic in the United Kingdom, whilst we observed an increase in BSI caused by Gram-negative bacteria in our adult Intensive Care Unit (ICU). Methods: We audited all Gram-negative bacterial BSIs between 13th April 2020 and 25th December 2021 in our ICU. In total, 41 organisms underwent antimicrobial susceptibility testing and genome sequencing. Notable organisms isolated included 16 Klebsiella spp., 4 E. coli, and 4 Pseudomonas spp. Results: Overall, we observed a low prevalence of multi-drug resistant (MDR) organisms causing BSI on our unit. A fifth (4/20) of E. coli and Klebsiella spp. isolates carried ESBL or AmpC genes and a single P. monteilli carried the IMP-1 gene. At least 3/16 (19%) BSI with Klebsiella spp. were likely associated with transmission between patients in nearby beds. These transmission events were associated with two hypervirulent K. pneumoniae (ST412 and ST86) and a K. aerogenes ∗002b. Half of all K. pneumoniae associated with BSI were hypervirulent, having K2 or K57 capsule type and the presence of iuc, iro, Rmp genes. Discussion: Hypervirulent K. pneumoniae is an emerging problem, capable of causing a severe, disseminated infection. We suggest risk factors for transmission may include shared equipment, environmental and PPE contamination, and failure of effective hand hygiene. With COVID-19 infection endemic, if SARS-CoV-2 patients require cohort nursing in bays with sessional use gowns, we recommend these are short-sleeved, to facilitate effective hand hygiene. We argue strict Infection Control policy, including enhanced cleaning, is critical to reduce transmission of hypervirulent K. pneumoniae and advocate for enhanced national surveillance systems.

BACKGROUND Ciprofloxacin resistant Klebsiella pneumoniae is common or emerging in many geographies, and knowledge of local resistance rates is important for empirical therapy. Whilst there are known K. pneumoniae ciprofloxacin resistance determinants, there is a lack of systematic data on the effect of determinants, alone and in combination, and there are no publicly accessible tools for predicting resistance from whole genome sequence data. METHODS The KlebNET-GSP AMR Genotype-Phenotype Group aggregated a matched genotype-phenotype dataset of n=12,167 K. pneumoniae species complex ( Kp SC) isolates from 27 countries between 2001-2021. We developed a rules-based classifier to predict ciprofloxacin resistance by categorizing the number of quinolone resistance determining regions mutations in gyrA and parC , the number of plasmid-mediated quinolone resistance genes, and the presence/absence of aac(6ʹ)-Ib-cr (which can acetylate ciprofloxacin). Predictive performance was assessed using the discovery dataset, for which we re-phenotyped discrepant isolates; and validated using externally contributed datasets (n=7,030 Kp SC isolates). RESULTS The rules-based classifier predicted R vs S/I with categorical agreement, sensitivity, and specificity >96%, and major/very major error rates <4%. Performance was similar across diverse Kp SC sources (human, animal, other), species, and intra-species lineages. External validation of the classifier yielded overall 93.12% categorical agreement [95% confidence interval (CI), 92.50-93.74%], 8.65% major errors [95% CI, 7.34-9.97%], and 6.20% very major errors [95% CI, 5.51-6.90%]. We implemented the classifier in Kleborate, a command-line tool that is integrated into the Pathogenwatch web platform. Using this to assess the global distribution of ciprofloxacin resistance determinants in Kp SC genomes available in Pathogenwatch (n=31,319, from 109 countries between years 2000-2023), we observed a significant positive association between national quinolone consumption rates and predicted ciprofloxacin resistance (R2=0.20, p=0.004). CONCLUSIONS Ciprofloxacin resistance phenotypes can be reasonably predicted from genotypes, which is sufficient for informing surveillance. However, unexplained resistance remains and accuracy is insufficient for clinical applications. We demonstrate the value of aggregating genotype-phenotype data to explore resistance mechanisms and develop predictors, but highlight complexities in combining phenotype data from different assays and standards. ### Competing Interest Statement The authors have declared no competing interest. Bill & Melinda Gates Foundation, INV025280, INV077266 Wellcome Trust, 226432/Z/22/Z European Union‘s Horizon 2020 Research and Innovation Programme, 773830 Trond Mohn Foundation, TMF2019TMT03 SARA project (Surveillance of Antimicrobial Resistance in Africa, through a grant from the French Ministry of Europe and Foreign Affairs within the Fonds de solidarité pour les projets innovants (FSPI) Academy of Medical Sciences Health Foundation UK Vietnam MRC Newton Fund, MR/N029399/1 RG83380 National Institute for Health and Care Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance, NIHR207397

Extraintestinal infections caused by Enterobacteriaceae represent a global concern, further exacerbated by the growing prevalence of antimicrobial resistance (AMR). Among these, invasive nontyphoidal Salmonella (iNTS) infections have become increasingly challenging to manage, and their global spread remains poorly understood. Here we compiled 1,115 patient records and generated a comprehensive genomic dataset on iNTS. Age and sex emerged as significant risk factors, with Salmonella Enteritidis identified as a major cause. We observed serovar-specific AMR patterns, with notable resistance to fluoroquinolones and third-generation cephalosporins. A global phylogenomic analysis of Enteritidis revealed three distinct clades, highlighting the accumulation of AMR determinants during its international spread. Importantly, our genomic and transmission analyses suggest that iNTS infections may involve human-to-human transmission, with diarrheal patients acting as potential intermediaries, deviating from typical zoonotic pathways. Collectively, our newly generated cohort and iNTS genomic dataset provide a framework for precise local iNTS burden and underscore emerging transmission trends.

Shigellosis is a leading cause of diarrhoeal deaths worldwide, with Shigella sonnei increasingly implicated as a dominant agent. S. sonnei is divided into five monophyletic lineages all sharing a single O-antigen, yet most contemporary infections are caused by just a few clonal sub-lineages within globally dominant Lineage 3 that are quite distinct from the widely used Lineage 2 laboratory strain 53G. Factors underlying the success of these globally dominant lineages remain poorly understood in part due to a lack of complete genome sequences and available animal models. Here, we utilise a novel reference collection of representative Lineage 1, 2 and 3 isolates with complete genome sequences, and find that epidemiologically successful S. sonnei harbour fewer genes encoding putative immunogenic components whilst key virulence-associated regions (such as the type three secretion system and O-antigen) remain highly conserved. Using a zebrafish infection model, we discover that Lineage 3 isolates are most virulent, driven by significantly increased dissemination and a greater neutrophil response. We show that Lineage 3 isolates have increased tolerance to complement-mediated killing and acidic conditions alongside upregulated expression of group four capsule synthesis genes. Consistent with these observations, infection of primary human neutrophils revealed that Lineage 3 isolates are more tolerant of phagosomal killing. Together, our findings link the epidemiological success of S. sonnei to heightened virulence and stress tolerance and highlight zebrafish as a valuable platform to illuminate factors underlying establishment of Shigella epidemiological success. ### Competing Interest Statement The authors have declared no competing interest.

Serologic surveillance of at-risk populations can be used to directly estimate the incidence of typhoidal Salmonella infection across a variety of settings, including those without access to facility-based blood-culture surveillance. We collected paired blood samples approximately three months apart from an age-stratified random sample of healthy children and adults in Bangladesh, Malawi, and Nepal as part of the Strategic Typhoid Alliance Across Asia and Africa (STRATAA) study. We used a multiplex bead assay to measure the concentration of IgG antibodies against seven Salmonella typhi/paratyphi antigens (CdtB, FliC, HlyE, LPSO2, LPSO9, Vi, and YncE) in each sample and identified recently infected participants by fitting a regression mixture model to the change in IgG concentration between participants' samples. We estimated the seroincidence of infection in a Bayesian framework for each study site, age group, and antigen target. Finally, we compared the seroincidence estimates with crude and adjusted estimates of clinical incidence based on blood-culture surveillance. Seroincidence estimates were significantly higher than enteric fever incidence across all study sites, age groups, and antigen targets, even after adjusting for underreporting (median ratio: 24.2, interquartile range: 11.4-58.9). Seroincidence consistently peaked in the 0-4-year age group and declined moderately between children and adults (33% to 58% decline in HlyE seroincidence between the 5-9 and 30 + year old age groups), while enteric fever incidence peaked in older children and fell sharply in adults (71% to 95% decline in adjusted clinical incidence). Seroincidence estimates based on the FliC, YncE, and HlyE antigens individually had the strongest correlation with observed enteric fever incidence across age groups and study sites (r = 0.72, 0.69, and 0.63, respectively). These findings suggest that in endemic settings, both children and adults are frequently infected by typhoidal Salmonella serotypes, although only a fraction of these infections present as clinically identifiable enteric fever cases.

Antimicrobial resistance (AMR) has become a global health crisis, creating an urgent need for rapid and accurate bacterial identification to guide appropriate antibiotic therapy. Automated segmentation of bacteria in microscopic images enables quantitative analysis of cellular morphology and spatial patterns, offering valuable cues for early species recognition. Such image-based insights can accelerate diagnostic decisions and promote rational antibiotic use, ultimately mitigating the impact of AMR. Hence, we present a significantly expanded dataset of E. coli fluorescence images, extending that of Dat et al. (Duong et al., 2023) with additional samples and refined annotations to better support bacterial segmentation research. We extensively re-examine several popular segmentation models using standardised COCO metrics (mAP, mAP(0.50), mAP(0.75), mAP(small)) and find that methods originally designed for semantic segmentation struggling with instance-level tasks. On the other hand, two-stage detectors, especially Cascade Mask R-CNN (Vasconcelos et al., 2018) and its Hybrid Task Cascade (HTC) (Chen et al., 2019) variant, perform best when paired with modern backbone networks. In particular, our framework introduces a novel synergy between the ConvNeXtV2-Tiny backbone and the HTC architecture, enhanced by the CBAM (Kweon et al., 2018) attention mechanism (reduction ratio of eight), to achieve superior feature refinement and instance delineation. This configuration sets a new performance benchmark, achieving mAP(0.50 )= 0.940, mAP(0.75 )= 0.882, mAP(small )= 0.788, and mAP(0.50:0.95 )= 0.787, and $mAP_{0.50:0.95} = 0.787$, and directly supports the development of data-driven, clinically relevant microbial analysis systems that can guide evidence-based and timely antimicrobial treatment strategies.

Humans and animals are ubiquitously colonized by Enterobacteriaceae, a bacterial family that contains both commensals and clinically significant pathogens. Here, we report Enterobacteriaceae megaplasmids of up to 1.58 Mbp in length in infant and adult guts, and other microbiomes. Of 19 complete plasmid genomes, one was reconstructed from an E. coli isolate; others were linked to species of Citrobacter and Enterobacter via analysis of genome modification patterns. The detection of related plasmids in different Enterobacteriaceae, conjugation machinery, and more diverse modified motifs in certain plasmids compared to hosts suggests that these elements are self-transmissible, with a broad host range. The plasmids encode multi-drug efflux systems and potential secreted effectors. Up to 208 tRNAs are encoded and include sequence variants that may counter tRNA-centric defense mechanisms. Overall, the vast megaplasmid coding capacity may broaden host range, increase competitiveness, control invasion by other elements, and counter programmed cell death.