The Lancet Microbe

BackgroundRespiratory metagenomics (RMg) needs evaluation in a pilot service setting to determine utility and inform implementation into clinical practice. MethodsFeasibility, performance and clinical impacts on antimicrobial prescribing and infection control were recorded during a pilot RMg service for patients with suspected lower respiratory tract infection (LRTI) on two general and one specialist respiratory intensive care units (ICU) at Guys & St Thomas NHS foundation Trust, London. ResultsRMg was performed on 128 samples from 87 patients during the first 15-weeks providing same-day results for 110 samples (86%) with median turnaround time of 6.7hrs (IQR 6.1-7.5 hrs). RMg was 92% sensitive and 82% specific for clinically-relevant pathogens compared with routine testing. 48% of RMg results informed antimicrobial prescribing changes (22% escalation; 26% de-escalation) with escalation based on speciation in 20/24 cases and detection of acquired-resistance genes in 4/24 cases. Fastidious or unexpected organisms were reported in 21 samples including anaerobes (n=12), Mycobacterium tuberculosis, Tropheryma whipplei, cytomegalovirus and Legionella pneumophila ST1326, which was subsequently isolated from the bed-side water outlet. Application to consecutive severe community-acquired LRTI cases identified Staphylococcus aureus (two with SCCmec and three with luk F/S virulence determinants), Streptococcus pyogenes (emm1-M1uk clone), S. dysgalactiae subspecies equisimilis (STG62647A) and Aspergillus fumigatus with multiple treatments and public-health impacts. ConclusionsRMg provides frequent diverse benefits for treatment, infection control and public health. The combination of rapid comprehensive results, alongside revealing and characterising a hidden burden of infections makes the case for expediting routine service implementation.

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.

BackgroundSalmonella Isangi is an under-characterised serovar repeatedly associated with antimicrobial resistant hospital infections. Outbreaks of extensively drug-resistant (XDR) Salmonella Isangi occurred in close succession within hospitals in Malawi and South Africa, prompting us to characterise the serovar using epidemiologic, phenotypic, and genomic perspectives. MethodsIn Malawi, we integrated hospital blood culture surveillance with environmental sampling from neonatal wards and urban waterways. In South Africa, we analysed isolates from five hospitals involved in a regional outbreak. We used whole genome sequencing (Illumina and MinION) to characterise AMR genes and plasmids, assessed biofilm formation, disinfectant susceptibility, in vivo virulence, and analysed all publicly available Salmonella Isangi genomes. Findings224 / 345 (65%) of genomes in the global collection belonged to Salmonella Isangi sequence type (ST) 335. Of these, 221 (99%) originated from Malawi and South Africa, including the isolates recovered from both outbreaks. 199 (89%) ST335 genomes carried determinants of resistance to fluoroquinolones and third-generation cephalosporins, consistent with an XDR profile. In Malawi, a single ST335 clade caused the outbreak and was simultaneously present in both the hospital environment and nearby rivers. Inter-hospital transmission of a separate ST335 clade sustained the outbreak in South Africa. Closely related Malawian and South African isolates carried distinct plasmids encoding similar resistance determinants; evidence from our study and public databases suggests gene transfer via a cointegrate intermediate. Five non-outbreak South African ST335 isolates harboured additional carbapenem and macrolide resistance genes. Phenotypically, Salmonella Isangi ST335 resembled Salmonella Typhimurium in biofilm formation and disinfectant tolerance but was less virulent in mice. InterpretationSalmonella Isangi ST335 combines a locally untreatable XDR profile with nosocomial transmission and environmental persistence, suggesting a high potential for future outbreaks. A distinct and potentially greater threat lies in the horizontal spread of its resistance determinants to Salmonella Typhimurium and Salmonella Enteritidis, the two dominant invasive serovars in the region. Strengthened surveillance, integrating phenotypic testing with targeted genomics, is urgently needed. Its absence in Malawi, in contrast to South Africa, underscores inequities in preparedness for emerging AMR threats. FundingThis work was supported by the Wellcome Trust through the Core Grant (206545/Z/17/Z) and the COVID-19 Sequencing Grant (220757/Z/20/Z) awarded to MLW. Additional support was provided by the Global Health Research Professorship to Melita Gordon from the UK National Institute for Health and Care Research (NIHR) (NIHR300039). Peter Johnston is funded by the Liverpool Clinical PhD Programme for Health Priorities in the Global South, supported by the Wellcome Trust (223502/Z/21/Z). For open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission. Whole-genome sequencing of Salmonella isolates from South Africa was made possible by support from the SEQAFRICA project which is funded by the Department of Health and Social Cares Fleming Fund using UK aid. The views expressed in this publication are those of the authors and not necessarily those of the UK Department of Health and Social Care or its Management Agent, Mott MacDonald. Analyses in this study were supported in part through use of software and workflows developed under National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) grant R21AI178369. The NIH had no role in study design, data collection, analysis/interpretation, or publication decisions. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Isangi is a recurrent cause of antimicrobial resistant hospital outbreaks. We searched PubMed for Salmonella Isangi and related synonyms (to February 23rd, 2026) and identified 39 articles. No prior studies have examined transmission routes or provided phenotypic characterisation beyond antimicrobial resistance testing. Outbreaks have been reported from five hospitals on three continents, as well as a foodborne outbreak in China. Two major sequence types (STs) consistently appeared: ST335 and ST216. ST216 was widely geographically distributed and recovered from a variety of animal, meat, and environmental sources. ST335 was primarily associated with human clinical cases. Added value of this studyThis investigation was motivated by an outbreak of extensively drug-resistant (XDR) Salmonella Isangi at a hospital in Malawi. The Malawian outbreak occurred shortly before a multi-centre nosocomial outbreak in South Africa, and we provide insights from both in our analysis. We combined local epidemiology, phenotypic analyses, and global genomic characterisation to deliver a comprehensive description of the serovar. Both outbreaks were caused by ST335, which is the dominant sequence type in South Africa and Malawi, but by distinguishable clades in each country. In Malawi, genetically indistinguishable isolates were simultaneously circulating among patients, the hospital environment, and rivers throughout Blantyre City. Transfer of patients between hospitals is likely to have sustained the outbreak in South Africa. Recombination through a cointegrate intermediate may explain why the same resistance determinants are carried on distinct plasmid backbones within the Malawian and South African ST335 clades. We identify five ST335 isolates in South Africa that were not related to either outbreak and which harbour carbapenem and macrolide resistance genes in addition to an XDR genotype. Implications of all available evidenceXDR Salmonella Isangi ST335 is a major threat in Malawi because effective therapy requires antibiotics that are seldom accessible in routine care. The ability of ST335 to transmit in hospitals and to persist in the environment may increase the risk of future outbreaks. Salmonella Isangi readily acquires and maintains antimicrobial resistance determinants through diverse plasmid backbones and recombination, raising concern for transfer to locally prevalent invasive Salmonella serovars. National genomic surveillance of the kind that exists in South Africa is essential to track and contain further resistance emergence, but such surveillance does not exist in Malawi. There is an urgent need to expand genomic surveillance in low-income countries if the threat posed by Salmonella Isangi and other pathogens that drive antimicrobial resistance is to be recognised early and effectively contained.

BackgroundEnteric fever is a serious public health concern. The causative agents, Salmonella enterica serovars Typhi and Paratyphi A, are frequently antimicrobial resistant (AMR), leading to limited treatment options and poorer clinical outcomes. We investigated the genomic epidemiology, resistance mechanisms and transmission dynamics of these pathogens at three urban sites in Africa and Asia. MethodsBacteria isolated from febrile children and adults at study sites in Dhaka, Kathmandu, and Blantyre were sequenced and AMR determinants identified. Phylogenomic analyses incorporating globally-representative genome data, and ancestral state reconstruction, were used to differentiate locally-circulating from imported pathogen variants. FindingsS. Paratyphi A was present in Dhaka and Kathmandu but not Blantyre. S. Typhi genotype 4.3.1 (H58) was common in all sites, but with different dominant variants (4.3.1.1.EA1 in Blantyre; 4.3.1.1 in Dhaka; 4.3.1.2 in Kathmandu). Resistance to first-line antimicrobials was common in Blantyre (98%) and Dhaka (32%) but not Kathmandu (1.4%). Quinolone-resistance mutations were common in Dhaka (99.8%) and Kathmandu (89%) but not Blantyre (2.1%). AcrB azithromycin-resistance mutations were rare (Dhaka only; n=5, 1.1%). Phylogenetic analyses showed that (a) most cases derived from pre-existing, locally- established pathogen variants; (b) nearly all (98%) drug-resistant infections resulted from local circulation of AMR variants, not imported variants or recent de novo emergence; (c) pathogen variants circulated across age groups. Most cases (67%) clustered with others that were indistinguishable by point mutations; individual clusters included multiple age groups and persisted for up to 2.3 years, and AMR determinants were invariant within clusters. InterpretationEnteric fever was associated with locally-established pathogen variants that circulate across age groups. AMR infections resulted from local transmission of resistant strains. These results form a baseline against which to monitor the impacts of control measures. FundingWellcome Trust, Bill & Melinda Gates Foundation, European Unions Horizon 2020, NIHR. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCurrent knowledge of the enteric fever pathogen populations in Dhaka, Kathmandu, and Blantyre comes from retrospective analysis of isolates captured from routine diagnostics or treatment trials. Due to these study designs, most focus on either adult or paediatric cohorts, which complicates assessment of pathogen variant transmission across age groups. Many studies report prevalence of antimicrobial resistance (AMR) and associated mechanisms amongst enteric fever cases. Genomic studies at these sites and elsewhere have identified the spread of AMR clones, and a recent genomic study quantified the inter- and intra-continental spread of resistant S. Typhi between countries. However, PubMed search of "(typhoid OR (enteric fever)) AND (genom*)" identified no studies quantifying the relative proportion of resistant infections that is attributable to local transmission of resistant variants vs imported strains or de novo emergence of AMR. Added value of this studyWe estimate the vast majority (98%) of drug-resistant enteric fever cases identified in our study resulted from local circulation of resistant variants. Further, we show genetically indistinguishable pathogen variants (either resistant or susceptible) persisting for up to 2.3 years and causing infections across all age groups (under 5 years; 5-15 years; [≥]15 years). Implications of all the available evidenceWhile inter-country transfer of resistant enteric fever pathogens does occur and is concerning, the burden of drug-resistant enteric fever at the study sites is currently caused mainly by transmission of locally-established variants, and transmits across age groups. These data confirm assumptions made in models of vaccine impact regarding heterogeneity of pathogen variants and AMR across age groups, and support that childhood immunisation programmes can be expected to reduce the overall burden of resistant infections in endemic settings.

BackgroundExtended spectrum beta-lactamases-producing (ESBL-P) and/or carbapenem-resistant (CR) Klebsiella pneumoniae have severely restricted available treatment options in healthcare settings in Vietnam. Understanding the diversity and transmission mechanisms of ESBL- and carbapenemase-encoding K. pneumoniae is important in both hospital and community settings for patient management. MethodsWe conducted a 6-month prospective cohort study of 69 Intensive care unit (ICU) patients from two hospitals in Hanoi, Vietnam. Longitudinally collected samples from patients and the ICU environment were cultured on selective media, and 357 K. pneumoniae colonies were whole genome sequenced. We performed phylogenetic analyses, and correlated phenotypic antimicrobial susceptibility testing with genotypic features of K. pneumoniae isolates. We constructed transmission networks of patient samples, relating ICU admission times and locations with genetic similarity of infecting K. pneumoniae. FindingsDespite being geographically and clinically separated, the two hospitals shared closely related strains carrying the same array of antimicrobial resistance genes. Many patients carried the same resistant K. pneumoniae clone from admission to discharge. 45.9% of total isolates carried both ESBL- and carbapenemase-encoding genes, with high minimum inhibitory concentrations. We found a novel co-occurrence of blaKPC-2 and blaNDM-1 in 46. 6% of samples from the globally successful ST15 lineage. InterpretationThese results highlight the high prevalence of ESBL-positive carbapenem-resistant K. pneumoniae in Vietnamese ICUs. Through studying K. pneumoniae ST15 in detail, we illustrated how important resistance genes are coalescing in stains carried broadly by patients entering the two hospitals directly or through referral. FundingThis study was supported by the Medical Research Council Newton Fund, United Kingdom (grant MR/N029399/1); the Ministry of Science and Technology, Vietnam (grant HNQT/SPThP/04.16); This research was funded in whole by the Wellcome Trust (grant 206194). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

BackgroundMost studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. MethodsPlasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FindingsStrong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months. Nasal and plasma anti-S IgG remained elevated for at least 12 months with high plasma neutralising titres against all variants. Of 180 with complete data, 160 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal. Samples 12 months after admission showed no association between nasal IgA and plasma IgG responses, indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. InterpretationThe decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. Research in contextO_ST_ABSEvidence before the studyC_ST_ABSWhile systemic immunity to SARS-CoV-2 is important in preventing severe disease, mucosal immunity prevents viral replication at the point of entry and reduces onward transmission. We searched PubMed with search terms "mucosal", "nasal", "antibody", "IgA", "COVID-19", "SARS-CoV-2", "convalescent" and "vaccination" for studies published in English before 20th July 2022, identifying three previous studies examining the durability of nasal responses that generally show nasal antibody to persist for 3 to 9 months. However, these studies were small or included individuals with mild COVID-19. One study of 107 care-home residents demonstrated increased salivary IgG (but not IgA) after two doses of mRNA vaccine, and another examined nasal antibody responses after infection and subsequent vaccination in 20 cases, demonstrating rises in both nasal IgA and IgG 7 to 10 days after vaccination. Added value of this studyStudying 446 people hospitalised for COVID-19, we show durable nasal and plasma IgG responses to ancestral (B.1 lineage) SARS-CoV-2, Delta and Omicron (BA.1) variants up to 12 months after infection. Nasal antibody induced by infection with pre-Omicron variants, bind Omicron virus in vitro better than plasma antibody. Although nasal and plasma IgG responses were enhanced by vaccination, Omicron binding responses did not reach levels equivalent to responses for ancestral SARS-CoV-2. Using paired plasma and nasal samples collected approximately 12 months after infection, we show that nasal IgA declines and shows a minimal response to vaccination whilst plasma antibody responses to S antigen are well maintained and boosted by vaccination. Implications of all the available evidenceAfter COVID-19 and subsequent vaccination, Omicron binding plasma and nasal antibody responses are only moderately enhanced, supporting the need for booster vaccinations to maintain immunity against SARS-CoV-2 variants. Notably, there is distinct compartmentalisation between nasal IgA and plasma IgA and IgG responses after vaccination. These findings highlight the need for vaccines that induce robust and durable mucosal immunity.

BackgroundBecause M. tuberculosis evolves slowly, transmission clusters often contain multiple individuals with identical consensus genomes, making it difficult to reconstruct transmission chains. Finding additional sources of shared M. tuberculosis variation could help overcome this problem. Previous studies have reported M. tuberculosis diversity within infected individuals; however, whether within-host variation improves transmission inferences remains unclear. MethodsTo evaluate the transmission information present in within-host M. tuberculosis variation, we re-analyzed publicly available sequence data from three household transmission studies, using household membership as a proxy for transmission linkage between donor-recipient pairs. FindingsWe found moderate levels of minority variation present in M. tuberculosis sequence data from cultured isolates that varied significantly across studies (mean: 6, 7, and 170 minority variants above a 1% minor allele frequency threshold, outside of PE/PPE genes). Isolates from household members shared more minority variants than did isolates from unlinked individuals in the three studies (mean 98 shared minority variants vs. 10; 0.8 vs. 0.2, and 0.7 vs. 0.2, respectively). Shared within-host variation was significantly associated with household membership (OR: 1.51 [1.30,1.71], for one standard deviation increase in shared minority variants). Models that included shared within-host variation improved the accuracy of predicting household membership in all three studies as compared to models without within-host variation (AUC: 0.95 versus 0.92, 0.99 versus 0.95, and 0.93 versus 0.91). InterpretationWithin-host M. tuberculosis variation persists through culture and could enhance the resolution of transmission inferences. The substantial differences in minority variation recovered across studies highlights the need to optimize approaches to recover and incorporate within-host variation into automated phylogenetic and transmission inference. FundingNIAID: 5K01AI173385

Enterococcus faecium is a member of the human gastrointestinal (GI) tract microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity that ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least two years, and that invasive isolates likely emerged from a large E. faecium population in the patients GI tract. The addition of bacteriophage (phage) therapy to the patients antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. Eventual recurrence of E. faecium BSI was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage neutralizing antibody response occurred simultaneously with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. ImportancePhage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patients quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patients stool. Eventually an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

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 3GC-resistant (3GCR) 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 3GC-resistant 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.

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.

BackgroundAs mpox virus (MPXV) has continued to expand geographically, critical knowledge gaps in environmental persistence in resource-limited healthcare settings remain. Despite evidence of fomite-mediated transmission, no empirical data exist on surface contamination in West African hospitals during active mpox outbreaks. MethodsIn this cross-sectional study, we conducted a systematic environmental surveillance study at two major hospitals in Sierra Leone (Connaught Hospital, Freetown; Bo Government Hospital, Bo) during peak transmission (June 2025). A total of 89 high-contact surfaces were sampled across clinical and non-clinical zones using standardized protocols. MPXV DNA was extracted via robotic MagMax protocols and detected through quantitative real-time PCR targeting the B6R gene. ResultsOverall, 13.5% (12/89) of surfaces tested positive by PCR for MPXV, with geographic variation: Freetown (14.0%, 7/50) vs. Bo (12.8%, 5/39). Cycle threshold values (32.34-39.86) indicated low-to-moderate viral genomes. Critical contamination hotspots were identified, with doors representing 42% (5/12) of positive samples; predominantly ward entrances, staff offices, and bathrooms. Patient beds and clinical instruments constituted secondary risk zones (8.3% each). ConclusionsThis first-in-region study demonstrates quantifiable MPXV genomes from surfaces in Sierra Leonean healthcare facilities, providing support for patients with mpox, highlighting areas for infection prevention and control (IPC) considerations. The predominance of doors as high-risk fomites underscores the need for targeted disinfection protocols. Our findings establish environmental surveillance as a vital component of mpox control in clinical settings and provide evidence for IPC resource prioritization, including enhanced disinfection of high-touch surfaces and integration of IPC monitoring into national outbreak response frameworks.

BackgroundThe rise of antimicrobial resistance (AMR) is a growing concern globally and a deeper understanding of AMR gene carriage vs usage is vital for future responses to reduce the spread of AMR. Identification of AMR phenotype by laboratory-based assays are often hindered by difficulties in establishing cultures. This issue could be resolved by rapid computational assessment of an organisms genome, however, AMR gene finder tools are not intended to infer AMR phenotype which is likely to be a product of multiple gene interactions. MethodsTo understand the importance of multi-gene interactions to the relationship between AMR genotype and AMR phenotype, we applied machine learning approaches to 16,950 genomes from microbial isolates representing 28 different genera with 1.2 million corresponding laboratory-determined MICs for 23 different antibiotics. We then elucidated the genomic paths to phenotypic antimicrobial resistance with the aim of allowing for the development of rapid determination of AMR phenotype from genomes or even whole microbiomes. ResultsThe application of machine learning models resulted in a >1.5-fold increase in average prediction accuracy of AMR phenotype across the 23 antibiotic models. Interpretation of these models revealed 528 distinct genomic pathways to phenotypic resistance, many of which were species-specific and involved genes which have not previously been associated with AMR phenotype. This is the first study to demonstrate the utility of machine learning models in the prediction of AMR phenotype for a wide range of clinically relevant organisms and antibiotics. This could be applied as a rapid and affordable alternative to culture-based techniques, estimating taxonomy in addition to AMR phenotype, and providing real-time monitoring of multi-drug resistant pathogens. Availability and implementationO_ST_ABSContactC_ST_ABSldillon05@qub.ac.uk View supplementary information at this linkhttps://osf.io/cj4bq/?view_only=c0ee87b7609543b688953089be4c376f See Code Availability for scripts used.

BackgroundLower respiratory tract infections (LRTIs) remain a major cause of morbidity and mortality among hospitalized patients.1 However, isolating organisms from respiratory samples often leads to diagnostic uncertainty due to the coexistence of colonizers, commensals, and contaminants.2 To address this challenge, this study employed a structured, stepwise exploratory model to differentiate true pathogens from non-pathogens in aerobic respiratory cultures and Multiplex PCR (Biofire(R) FilmArray) results. MethodsThis prospective, longitudinal time-bound study was conducted over three months (August- October 2024) at a tertiary care center in northern India. Adult patients ([≥] 18 years) with positive lower respiratory tract samples (aerobic culture or Multiplex PCR (Biofire(R) FilmArray) were enrolled. Each isolate was independently classified by the treating clinician, microbiologist, and study investigator using a six-step clinical-pathological algorithm that incorporated clinical signs, Sequential Organ Failure Assessment (SOFA) score trends, alternative infection sources, host factors, and outcome data. The final classification was determined by the investigator. Outcomes, including treatment response and mortality at 28 days, were compared across pathogen and non-pathogen groups. FindingsOf the 145 included cases, 131 (90{middle dot}3%) were classified as pathogens and 14 (9{middle dot}7%) as non-pathogens. Cohens Kappa between investigator and microbiologist classifications was 0{middle dot}28, indicating fair agreement. Among pathogen cases, 68 (51{middle dot}9%) responded to treatment, while 63 (48{middle dot}1%) did not respond to treatment in the pathogenic group. In contrast, 12 of 14 non-pathogen cases (85{middle dot}7%) were not treated, with favourable outcomes in most, and only one unrelated death (7{middle dot}1%). InterpretationThe structured clinico -microbiological model strongly correlates with treatment outcomes, making it useful for differentiating infection from colonization. Crucially, microbiological detection alone doesnt determine pathogenicity. Integrating clinical, laboratory, and outcome data is essential for rational antibiotic use and effective antimicrobial stewardship. FundingNone

BackgroundA substantial and rapid increase, followed by a sharp decline in vanB-type vancomycin-resistant E. faecium (VRE), occurred in Germany in the late 2010s. This unusual epidemiological trend prompted detailed genomic investigations to explore the underlying dynamics at a German university hospital. MethodsWe retrospectively analyzed 344 E. faecium bloodstream infection (BSI) isolates collected between 2017 and 2022. Isolates were classified as vanA-positive, vanB-positive, or van-negative, while VRE was defined as vanA- or vanB-positive. Molecular typing included multilocus sequence typing (MLST), core genome MLST (cgMLST), and split k-mer analysis (SKA). Clonal relationships and potential patient-to-patient transmission events were assessed by integrating genomic data with machine-aided analysis of available longitudinal patient movement data. ResultsHigh-resolution genomic analysis of BSI isolates revealed an oligoclonal scenario involving multiple epidemic lineages (e.g., sequence type (ST)117/complex type (CT)71, ST117/CT929, ST117/CT2505, ST80, and ST262) with distinct van genotypes and dynamic changes over time. Overall, genomic overlap between vanA-positive, vanB-positive, and van-negative populations was minimal. The vanB surge, peaking in late 2018-early 2019, was mainly driven by ST117/CT71, ST117/CT36, and ST117/CT1917. SKA provided enhanced discriminatory power over cgMLST. The inferred transmission events linked bloodstream infections separated by long intervals (median, 106 days). ConclusionsAccurate characterization of the transient vanB-type VRE peak required an integrated genomic approach combining cgMLST, high-resolution SKA, and epidemiological data. These comprehensive methods enable infection prevention and control (IPC) teams to distinguish true outbreaks from apparent epidemiologic clusters of polyclonal isolates. This allows for accurate interpretation and precisely targeted interventions where evidence supports transmission. ImportanceVancomycin-resistant Enterococcus faecium is an increasingly important nosocomial pathogen worldwide. Understanding its epidemiology and transmission dynamics is critical to effectively control its spread. This study documents shifting E. faecium populations within a high-risk hospital environment, including both vancomycin-resistant and vancomycin-susceptible bloodstream isolates. Virulence gene profiling demonstrated that hospital-associated variants predominated across all major lineages, independent of vancomycin resistance, indicating that hospital adaptation is a common feature of both resistant and susceptible populations. Detailed genomic analyses, combining core genome multilocus sequence typing with high-resolution split k-mer analysis, integrated with comprehensive epidemiological tracking, were critical to accurately depict complex epidemiological dynamics. This combined approach allows precise differentiation between monoclonal outbreaks and oligoclonal transmission, enabling more targeted infection prevention and control strategies.

Ciprofloxacin is the first-line drug for treating typhoid fever in many high burden countries in Africa, but the emergence of non-susceptibility poses a grave challenge to public health programmes. Through enhanced surveillance as part of vaccine evaluation, we set out to investigate the occurrence and determinants of ciprofloxacin non-susceptibility in Blantyre, Malawi. We performed systematic typhoid fever and antibiotic prescription surveillance in two health centres in Blantyre, Malawi between 01/10/2016 and 31/10/2019, as part of the STRATAA and TyVAC studies. Blood culture isolates from study participants underwent i) pefloxacin screening and ciprofloxacin E-tests to identify ciprofloxcain non-susceptibility and ii) whole genome sequencing (WGS) to identify drug resistance mutations and phylogenetic relationships between non-susceptible and sensitive isolates. We constructed generalised linear regression models to investigate associations between ciprofloxacin prescription rates and S. Typhi isolates with Quinolone Resistance Determining Region (QRDR) mutations. We carried out 11295 blood cultures and microbiologically confirmed 239 cases of typhoid fever, with isolates from 193 participants sequenced (mean age of participants with sequenced genomes 12.8 years, 47% male). Between October 2016 and August 2019 2% (n=4/175) of WGS-confirmed typhoid fever cases were caused by S. Typhi with QRDR mutations, compared with 33% (n=6/18) in September and October 2019. Nine of the ten S. Typhi with QRDR mutations had a decreased ciprofloxacin susceptibility phenotype. Every additional prescription of ciprofloxacin given to study participants in the preceding month was associated with a 4.2% increase in the relative risk of isolating S. Typhi with a QRDR mutation (95% CI, 1.8-7.0%, p=0.0008). Phylogenetic analysis showed that S. Typhi isolates with QRDR mutations in September/October 2019 belonged to two distinct sub-clades encoding two different QRDR mutations, and were closely related (0-6 SNPs) to susceptible S. Typhi endemic to Blantyre. We have shown a close temporal association between empiric antimicrobial usage with an increase of fluoroquinolone non-susceptibility in S. Typhi, with two sub-clades responsible for the increase. Decreasing ciprofloxacin usage by improving typhoid diagnostics could help to limit the emergence of resistance.

The diarrhoeal disease, shigellosis, can be sustained as a sexually transmissible enteric illness among gay, bisexual, and other men who have sex with men (GBMSM). Multiple extensively drug-resistant strains of Shigella have been detected through genomic surveillance, which have typically been associated with plasmids carrying the gene variant blaCTX-M-27. We report an increase in likely sexually transmissible cases of Shigella carrying blaCTX-M-15, which was previously associated with travel. In 2023, there were 117 cases belonging to the single 10-SNP single linkage cluster, t10.1814. While this cluster had been present in England since August 2019, genetic analyses revealed that blaCTX-M-15 entered the lineage on a novel resistance plasmid coincident with the first case of the outbreak. This highlights the shifting antimicrobial resistance landscape of sexually transmissible Shigella and the parallel emergence of resistance determinants against third generation cephalosporins in sexual transmission networks suggests high levels of antimicrobial selection pressure among GBMSM.

BackgroundDespite advances in molecular diagnostics, only 48% of newly diagnosed tuberculosis (TB) cases were confirmed using nucleic acid amplification tests (NAATs) in 2023. The BD MAXTM MDR-TB (MAX MDR-TB) assay, a moderate complexity NAAT, detects Mycobacterium tuberculosis complex (MTBC) and resistance to rifampicin (RIF) and isoniazid (INH), but data on clinical performance is limited. This study assessed assay performance on raw sputum, NALC/NaOH decontaminated sputum, and tongue swab (TS) specimens. MethodsThis evaluation assessed the MAX MDR-TB assay for MTBC detection and RIF and INH resistance profiling on sputum, using liquid culture as the reference standard. Additionally, diagnostic accuracy for MTBC detection in TS specimens was evaluated under different transport and processing conditions. ResultsAssay sensitivity was similar for sputum pellet (87%) and raw sputum (89%), with one additional case detected using raw sputum. Two false RIF-resistant results were observed. INH resistance was missed in two cases. Although specimen numbers were small, TS demonstrated better diagnostic accuracy when using a diluted (66%) STR buffer. A total of 15/55 (27%) were classified as "MTB Low POS." ConclusionThese findings suggest that MAX MDR-TB assay performance is comparable between sputum pellet and raw sputum. While TS showed promise, further validation in larger studies is warranted. The high rate of "MTB Low POS" results across specimen types underscores the importance of assay optimisation to reduce the burden of repeat testing and improve diagnostic reliability. Future research should enhance sensitivity and integration into diagnostic algorithms to improve patient outcomes.

BackgroundNew Delhi metallo-{beta}-lactamase (NDM) represents an emergent mechanism of carbapenem resistance associated with high mortality and limited antimicrobial treatment options. Because the blaNDM resistance gene is often carried on plasmids, traditional infection prevention and control (IP&C) surveillance methods like speciation, antimicrobial resistance testing, and reactive whole genome sequencing (WGS) may not detect plasmid transfer in multispecies outbreaks. MethodsInitial outbreak detection of NDM-producing Enterobacterales identified at an acute care hospital occurred via traditional IP&C methods and was supplemented by real-time WGS surveillance, which was performed weekly using the Illumina platform. To resolve NDM-encoding plasmids, we performed long-read Oxford Nanopore sequencing and constructed hybrid assemblies using Illumina and Nanopore sequencing data. Reports of relatedness between NDM-producing organisms and reactive WGS for suspected outbreaks were shared with the IP&C team for assessment and intervention. FindingsWe observed a multispecies outbreak of NDM-5-producing Enterobacterales isolated from 15 patients between February 2021 and February 2023. The 19 clinical and surveillance isolates sequenced included seven bacterial species and each encoded the same NDM-5 plasmid, which showed high homology to NDM plasmids previously observed in Asia. WGS surveillance and epidemiologic investigation characterized ten horizontal plasmid transfer events and six bacterial transmission events between patients housed in varying hospital units. Transmission prevention focused on enhanced observation and adherence to basic infection prevention measures. InterpretationOur investigation revealed a complex, multispecies outbreak of NDM that involved multiple plasmid transfer and bacterial transmission events, increasing the complexity of outbreak identification and transmission prevention. Our investigation highlights the utility of combining traditional IP&C and prospective genomic methods in identifying and containing plasmid-associated outbreaks. FundingThis work was funded in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) (R01AI127472) (R21AI1783691). 5. SummaryWe investigated a multispecies outbreak of Enterobacterales harboring the same New-Delhi metallo-{beta}-lactamase-encoding plasmid using both traditional infection prevention and genomic approaches. Our investigation revealed a complex outbreak involving 7 bacterial species, including both bacterial transmission and plasmid transfer events.

BackgroundMonkeypox virus (MPXV) clade Ib, first detected in the Democratic Republic of the Congo (DRC) in September 2023, spread internationally within months, prompting a WHO emergency declaration. Data on its incubation period, which both shapes outbreak dynamics and informs epidemic response strategies remain limited. ObjectiveTo estimate the incubation periods of mpox clade Ib examining evidence for differences by route of exposure and demographic factors. DesignBayesian analysis of clinical surveillance data collected between June and October 2024. SettingSouth Kivu, Democratic Republic of Congo, the epicenter of the current mpox clade Ib global outbreak. ParticipantsClinically attended persons with confirmed mpox clade Ib infection. MeasurementsDemographics, exposure history, symptom onset, and transmission route. ResultsAmong 37 PCR-confirmed cases with high viral load (Cycle Threshold [Ct] values less than 34), the median incubation period from exposure to rash was 13.6 days (95% CrI: 10.7-20.2). Five percent of cases are expected to develop a rash within 3.8 days (95% CrI; 1.7-6.6) and 95% within 33.4 days (95% CrI: 24.1-46.4). The incubation period appeared to differ by putative transmission route: sexual transmission had a shorter median (10.3 days, 95% CrI 3.1-20.3) than non-sexual transmission (13.5 days, 95% CrI: 9.5-19.1), though the confidence intervals overlapped. LimitationSurveillance data lacked detailed exposure histories and a lower bound for exposure periods, but models accounted for these uncertainties, yielding robust median estimates. ConclusionEvidence from this study suggests that clade Ib may have a longer incubation period than other MPXV clades, and this may vary by transmission route. The shorter incubation for sexual transmission mirrors patterns seen in the predominantly sexually transmitted clade IIb outbreak, highlighting the potential role of exposure route in disease progression. These findings have im-plications for global recommendation on post-exposure monitoring periods and prophylaxis. Primary Funding SourceThis work was supported by the Gates Foundation (INV-079976) and funds from the Geneva Centre for Emerging Viruses. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ceftazidime-avibactam (CZA) has revolutionized care for carbapenem-resistant Enterobacterales infections, yet increasing New Delhi metallo-{beta}-lactamase (NDM) prevalence has driven use of CZA plus aztreonam (CZA/ATM). We performed a comprehensive analysis of CZA-resistant Escherichia coli (CZA-R-Ec) at a tertiary cancer center (2017-2024) by integrating clinical data, comparative genomics, and CZA/ATM susceptibility testing. CZA-R-Ec were isolated from forty-eight unique patients of whom 28 (58%) had confirmed infection. Oxford Nanopore Technologies long-read sequencing performed on 34 isolates from unique patients showed a diverse population enriched for ST167 (35%). Most sequenced isolates carried blaNDM-5 (26/34, 76%); among blaNDM-5 strains, 88% (23/26) harbored PBP3 YRI(K/N) insertions. Eleven isolates (32%) carried blaCMY variants, predominantly blaCMY-42. BlaNDM-5 and blaCMY genes were largely plasmid-borne (IncF-type and IncI-{gamma}/K1) in distinct genomic contexts. Among 32 confirmed CZA-R and ATM-R index isolates, 21 (66%) were CZA/ATM synergy positive to CZA/ATM (MIC[≤]4 {micro}g/mL; SYN+). Compared to patients with SYN+ strains, patients with SYN-isolates (CZA/ATM MIC>4 {micro}g/mL) were significantly more likely to have had a prior E. coli infection (73% vs. 0%, P-value=1.6x10-5). SYN- isolates were strongly associated with blaCMY carriage (81% vs 9% in SYN+; adjusted P-value=4x10-4). Among 18 confirmed CZA-R-Ec bacteremias, 15 carried blaNDM; 14/15 (92%) received CZA/ATM and all responded clinically. In conclusion, CZA-R-Ec at our center are dominated by PBP3-insertion, blaNDM-5-positive lineages, for which CZA/ATM retains substantial in vitro activity and clinical efficacy. However, recent carbapenem exposure and prior E. coli infection identifies patients at risk for CZA/ATM non-synergy, frequently linked to blaCMY-42 variant positive isolates.