Neisseria meningitidis is a commensal member of the human oropharyngeal microbiota that can cause devastating invasive meningococcal disease. This genetically and antigenically diverse 'accidental pathogen' has been a paradigm for the study of bacterial population biology. The meningococcus was the first organism for which a seven-locus multi-locus sequence typing (MLST) scheme was developed. With the addition of sequence-based characterisation of antigen genes, molecular typing has been widely employed to inform surveillance and public health interventions. Following the advent and widespread adoption of whole genome sequencing (WGS), precise delineation of variants is possible. Here, a WGS-based Life Identification Number (LIN) code typing scheme is described, providing a multi-resolution nomenclature for understanding meningococcal population diversity and molecular epidemiology. The LIN codes were developed using a set of 6,131 N. meningitidis genomes, comprising up to 200 isolates from each clonal complex (cc) previously described using MLST. Based on cluster-stability analysis and concordance with ccs, thirteen LIN 'thresholds' described the meningococcal population at different levels of resolution. LIN codes and human-readable 'nicknames' consistent with existent nomenclatures were assigned to the N. meningitidis genomes hosted in the PubMLST. Published outbreaks validated the LIN thresholds, illustrating the potential of this genomic tool in public health management.

Horizontal gene transfer events were crucial in the emergence of multicellular life. A striking example is the acquisition of Teneurins, putative surface-exposed toxins in bacteria that function as cell adhesion receptors in metazoan neuronal development. Here, we demonstrate the evolutionary relationships between metazoan and bacterial Teneurins. We use cryogenic electron microscopy and bioinformatic analysis to show that bacterial Teneurins harbour a toxic protein in a proteinaceous shell. They are rare but widely distributed across bacterial taxa and are predominantly seen in species with complex social behaviours, suggesting roles in cell-to-cell interaction. This work confirms that metazoan Teneurins are repurposed bacterial toxins that have evolved to be essential mediators of intercellular communication in all advanced nervous systems. Their acquisition was a key event in the evolution of metazoans.

The accurate identification of Streptococcus pneumoniae (pneumococcus) is crucial for diagnostics and surveillance but is complicated by the use of molecular assays that may also detect non-pneumococcal Streptococcus (NPS) species. Therefore, the aim of this study was to use a combination of in silico and in vitro analyses to evaluate PCR assays for the molecular detection and identification of pneumococci. A diverse dataset of over 9,300 pneumococcal and NPS genomes was investigated in silico to determine the sensitivity and specificity of assays for seven recommended gene targets: lytA , piaB , ply , psaA , Spn9802, SP2020 and Xisco. These in silico findings were used to design new diagnostic assays for two targets, Xisco and SP2020. The new assays were evaluated in vitro using three sets of isolates, one of which was selected based upon evidence for sequence diversity from a second in silico investigation of over 6,000 pneumococcal genomes sequenced by the United Kingdom Health Security Agency. Experimentally, the new Xisco and SP2020 assays were compared to published assays for lytA and piaB . The in vitro specificity was 100% (95% CI, 98.7–100%) across all assays. The in vitro sensitivity was 100% (95% CI, 98.5–100%) for lytA , SP2020_new and the Xisco assays and 99.6% (95% CI, 97.8–100%) for piaB . The new assays were found to be highly sensitive and specific and able to detect as few as two pneumococcal genome copies per quantitative PCR reaction. Overall, this study demonstrated the value of performing large-scale in silico genomic analyses of diagnostic targets, followed by in vitro testing that was specifically designed to account for global pneumococcal population-level diversity.

Moraxella catarrhalis is an important cause of infectious exacerbations of chronic obstructive pulmonary disease and otitis media. Previously, seroresistant (SR) and serosensitive (SS) lineages that differed in virulence potential were described, which raised questions about their evolutionary relationship and species classification. To investigate the population structure of M. catarrhalis, we developed a core-genome multilocus sequence typing (cgMLST) scheme using 1,319 core genes, and a life identification number (LIN) barcode classification system. Whole-genome analyses of nearly 2,000 genomes confirmed two divergent SR and SS M. catarrhalis lineages with distinct evolutionary trajectories. SR genomes were more conserved, while SS genomes exhibited greater genetic variability. Virulence gene analyses revealed lineage-specific variations in ubiquitous surface proteins (UspA1 and UspA2) and lipooligosaccharide (LOS) types, and SR genomes had more diverse LOS variants. The bro β-lactamase gene, and the mcb bacteriocin cluster, were more common in SR, which suggested different selective pressures and ecological adaptation. This cgMLST scheme and LIN code system provides a robust method for characterising M. catarrhalis, distinguishes between SR and SS lineages, and offers a unified framework for population structure analyses. Implemented within PubMLST, this open-access resource facilitates high-resolution genomic studies and supports the scientific community in understanding the evolutionary complexity of M. catarrhalis.

In double-membraned bacteria, non-equilibrium processes that occur at the outer membrane are typically coupled to the chemiosmotically energized inner membrane. TolA and TonB are homologous proteins which energetically couple inner membrane motor proteins to the essential processes of outer membrane stabilization and substrate import, respectively. The evolutionary trajectories of these proteins have been difficult to elucidate due to low-sequence conservation, yet they are thought to transduce force similarly. Here, this problem was addressed using structural prediction approaches to identify and annotate force transduction operons to trace their distribution and evolutionary origins. In the process, we identify a novel outer membrane-tethering system and a previously unknown family of monomeric force transducers. This approach revealed putative tolA genes, and thus the core organizational principles of the tol-pal operon throughout diverse bacterial taxa. We discovered that the α-helical structure of the periplasm-spanning domain II of TolA previously thought its hallmark, is anomalous amongst most Tol-Pal systems. This structure is mainly prevalent in γ-proteobacteria, likely in adaptation to their lifestyle. Comparison of Tol-Pal and Ton system distribution suggests that TolA emerged from a TonB paralogue and co-emerged with Pal, the outer membrane-tethering lipoprotein that functionalizes the Tol-Pal system. We also determined that TolB, the Pal-mobilizing protein, likely emerged from a family of outer membrane proteins; and CpoB, a periplasmic factor that coordinates peptidoglycan remodeling with cell division, was originally a lipoprotein present in the ancestral Tol-Pal system. The extensive conservation of the Tol-Pal system throughout Gracilicutes highlights its significance in bacterial cell biology.

ContextDiagnosing meningitis remains challenging with etiological agents frequently unidentified. Using both in silico and in vitro approaches, this study evaluated published and novel genetic targets for the detection of common bacterial species known to cause meningitis: Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, and Haemophilus influenzae.MethodsA total of 29 genetic targets were investigated for the detection of N. meningitidis, S. agalactiae, S. pneumoniae, and H. influenzae, using the Gene Presence tool and whole genome sequence data (WGS) found in the genomics platform, PubMLST. These targets were further tested in silico by screening WGS using the PCR tool hosted on PubMLST allowing the sensitivity, specificity, Negative Predicted Values (NPV) and Positive Predictive Values (PPV) to be determined. Ten targets were then further evaluated in vitro by real-time PCR against a panel of 44 bacterial isolates representative of the genera evaluated.ResultsThe best performing in silico genetic determinants targeted: N. meningitidis, sodC (NEIS1339) (sensitivity 99.7%, specificity, 99.4%, PPV, 99.6% and NPV, 99.6%); S. pneumoniae, SP2020 (99.5%, 99.9%, 99.9%, and 81.5%) and H. influenzae, dmsA (HAEM1183) (98%, 100%, 99.6%, and 77.4%). All three of these targets also had the best in vitro sensitivity (100%), specificity [91.7% sodC (NEIS1339), 100% SP2020 and 97.3% dmsA (HAEM1183), PPV (72.7% sodC (NEIS1339), 100% SP2020 and 87.5% dmsA (HAEM1183)] and NPV (100% for all targets). The gene sip (SAG0032) encoding the surface immunogenic protein (sip) exhibited the best sensitivity (99.6%) and NPV (96.9%) for S. agalactiae compared to 99.3% and 94.8% for cfb (SAG2043), respectively in silico. However, in vitro, cfb showed the best sensitivity (100% vs. 85.7%) and NPV (100% vs. 97.4%) when compared to sip.ConclusionSodC, cfb, SP2020, and dmsA have the potential to enhance the accuracy of molecular diagnostics for the four most common bacterial species causing meningitis. Moreover, a combined in silico and in vitro approach that leverages WGS deposited in databases such as PubMLST, offers an efficient and cost-effective means for the preliminary evaluation of diagnostic targets.

Moraxella catarrhalis is an important cause of infectious exacerbations of chronic obstructive pulmonary disease and otitis media. To investigate the population structure of M. catarrhalis, we developed a core-genome multilocus sequence typing (cgMLST) scheme using 1319 core genes, and a life identification number (LIN) barcode classification system. Whole-genome analyses of nearly 2000 genomes confirmed divergent seroresistant (SR) and serosensitive (SS) M. catarrhalis lineages with distinct evolutionary trajectories. SR genomes are more conserved, while SS genomes exhibited greater genetic variability. Virulence gene analyses revealed lineage-specific variations in ubiquitous surface proteins (UspA1 and UspA2) and lipooligosaccharide (LOS) types. The bro β-lactamase, and mcb bacteriocin cluster, are more common in SR lineages, which suggested different selective pressures and adaptation. Here, we show that this cgMLST scheme and LIN code system provide a robust method for characterising M. catarrhalis, distinguish between SR and SS lineages, and offer a unified framework for population structure analyses.

Staphylococcus aureus infects both humans and animals, and antimicrobial resistance, including multidrug resistance, complicates the treatment of S. aureus infections. Understanding the population structure and distribution of genetic lineages of S. aureus is central to understanding the biology, epidemiology and pathogenesis of this organism. This study exploited a large, publicly available dataset of nearly 27,000 S. aureus genomes to (i) develop a core genome multilocus sequence typing (cgMLST) scheme, (ii) stratify hierarchical clusters based on allelic similarity thresholds, and (iii) define the clusters with a life identification number (LIN) code classification system. The cgMLST scheme characterised allelic variation at 1,716 core gene loci, and 13 classification thresholds were defined, which discriminated S. aureus variants across a range of genetic similarity thresholds. LIN code 'lineages' and clonal complexes defined by seven- locus multilocus sequence typing were highly concordant, but the LIN codes permitted a wider range of genetic discrimination among S. aureus genomes. This S. aureus cgMLST scheme and LIN code system is a high- resolution, stable genotyping tool that enables detailed genomic analyses of S. aureus.

MOTIVATION:Target enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high-throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present an analysis pipeline, Castanet, for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. Also included are an optional graphical interface and installer script. RESULTS:In addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in $<$2 h. AVAILABILITY AND IMPLEMENTATION:Source code freely available under GPL-3 license at https://github.com/MultipathogenGenomics/castanet, implemented in Python 3.10 and supported in Ubuntu Linux 22.04. The data underlying this article are available in Europe Nucleotide Archives, at https://www.ebi.ac.uk/ena/browser/view/PRJEB77004.

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants <12 months in Spain, the UK, and the Netherlands during 2017–20. We show, using targeted metagenomic sequencing of >100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03–9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity.

Investigating the genomic epidemiology of major bacterial pathogens is integral to understanding transmission, evolution, colonization, disease, antimicrobial resistance and vaccine impact. Furthermore, the recent accumulation of large numbers of whole genome sequences for many bacterial species enhances the development of robust genome-wide typing schemes to define the overall bacterial population structure and lineages within it. Using the previously published data, we developed the Pneumococcal Genome Library (PGL), a curated dataset of 30 976 genomes and contextual data for carriage and disease pneumococci recovered between 1916 and 2018 in 82 countries. We leveraged the size and diversity of the PGL to develop a core genome multilocus sequence typing (cgMLST) scheme comprised of 1222 loci. Finally, using multilevel single-linkage clustering, we stratified pneumococci into hierarchical clusters based on allelic similarity thresholds and defined these with a taxonomic life identification number (LIN) barcoding system. The PGL, cgMLST scheme and LIN barcodes represent a high-quality genomic resource and fine-scale clustering approaches for the analysis of pneumococcal populations, which support the genomic epidemiology and surveillance of this leading global pathogen.

BACKGROUND Respiratory syncytial virus (RSV) is the leading cause of hospitalisation associated with acute respiratory infection in infants and young children, with substantial disease burden globally. The impact of additional respiratory pathogens on RSV disease severity is not completely understood. OBJECTIVES The objective of this study was to explore the associations between RSV disease severity and the presence of other respiratory pathogens. METHODS Nasopharyngeal swabs were prospectively collected from two infant cohorts: a prospective longitudinal birth cohort study and an infant cross-sectional study recruiting infants <1 year of age with RSV infection in Spain, the UK, and the Netherlands during 2017–20 [part of the REspiratory Syncytial virus Consortium in EUrope (RESCEU) project]. The samples were sequenced using targeted metagenomic sequencing with a probe set optimised for high-resolution capture of sequences of over 100 pathogens, including all common respiratory viruses and bacteria. Viral genomes and bacterial genetic sequences were reconstructed. Associations between clinical severity and presence of other pathogens were evaluated after adjusting for potential confounders, including age, gestational age, RSV viral load, and presence of comorbidities. RESULTS RSV was detected in 433 infants. Nearly one in four of the infants (24%) harboured at least one additional non-RSV respiratory virus, with human rhinovirus being the most frequently detected (15% of the infants), followed by seasonal coronaviruses (4%). In this cohort, RSV-infected infants harbouring any other virus tended to be older (median age: 4.3 vs. 3.7 months) and were more likely to require intensive care and mechanical ventilation than those who did not. Moraxella, Streptococcus , and Haemophilus species were the most frequently identified target bacteria, together found in 392 (91%) of the 433 infants ( S. pneumoniae in 51% of the infants and H. influenzae in 38%). The strongest contributors to severity of presentation were younger age and the co-detection of Haemophilus species alongside RSV. Across all age groups in both cohorts, detection of Haemophilus species was associated with higher overall severity, as captured by ReSVinet scores, and specifically with increased rates of hospitalisation and respiratory distress. In contrast, presence of Moraxella species was associated with lower ReSVinet scores and reduced need for intensive care and mechanical ventilation. Infants with and without Streptococcus species (or S. pneumoniae in particular) had similar clinical outcomes. No specific RSV strain was associated with co-detection of other pathogens. CONCLUSION Our findings provide strong evidence for associations between RSV disease severity and the presence of additional respiratory viruses and bacteria. The associations, while not indicating causation, are of potential clinical relevance. Awareness of coexisting microorganisms could inform therapeutic and preventive measures to improve the management and outcome of RSV-infected infants. ### Competing Interest Statement G.-L. L. is currently an employee of Mundipharma Research Ltd., a position he commenced subsequent to the completion of this study. S. B. D. has been an investigator for clinical trials of vaccines and antimicrobials for pharmaceutical companies, including AstraZeneca, Merck, Pfizer, Valneva, Iliad, Sanofi, and Janssen, and previously sat on RSV advisory boards for Sanofi and Merck. M. D. S. has been an investigator on behalf of the University of Oxford for studies funded or supported by vaccine manufacturers, including Pfizer, GSK, Novavax, MCM vaccines and Janssen. M. D. S. is currently an employee of Moderna Biotech UK, a position he commenced subsequent to the completion of this study. M. A. A. is supported by a Sir Henry Dale Fellowship, jointly funded by the Royal Society and Wellcome Trust (220171/Z/20/Z). J. E. B. and K. A. J. are funded on a Wellcome Trust Biomedical Resources Grant (218205/Z/19/Z, PubMLST: Disseminating and exploiting bacterial diversity data for public health benefit). J. A. is an employee of Janssen Pharmaceutica NV. P. J. M. O. has received honoraria from GSK, Pfizer Inc, Sanofi Pasteur, Seqirus, and Janssen for taking part in advisory boards and expert meetings and for acting as a speaker in congresses outside the scope of the submitted work. P. J. M. O. is also a National Institute for Health and Care Research (NIHR) Senior Investigator. F. M.-T. has received honoraria from GSK, Pfizer Inc, Sanofi Pasteur, MSD, Seqirus, and Janssen for taking part in advisory boards and expert meetings and for acting as a speaker in congresses outside the scope of the submitted work. F. M.-T. has also acted as principal investigator in randomised controlled trials of the above-mentioned companies as well as Ablynx, Regeneron, Roche, Abbott, Novavax, and MedImmune, with honoraria paid to his institution. F. M.-T. receives support for his research activities from the Instituto de Salud Carlos III (Proyecto de Investigación en Salud, Acción Estratégica en Salud): Fondo de Investigación Sanitaria (FIS;PI1601569/PI1901090/PI22/00406) del plan nacional de I+D+I and 'fondos FEDER'. H. N. received grants from Innovative Medicine Initiative, NIHR, Bill and Melinda Gates Foundation, WHO, and Pfizer. H. N. also received consultancy or honoraria and speaker fees from Sanofi, Merck, Novavax, ReViral and GSK (all paid to institution). A. J. P. is chair of the UK Department of Health and Social Care's Joint Committee on Vaccination and Immunisation. A. J. P. has also provided advice to Shionogi on COVID-19 vaccines and his institution receives research funding from NIHR, Bill & Melinda Gates Foundation, Wellcome Trust, Medical Research Council and AstraZeneca for vaccine research. All other authors report no potential conflicts. The views expressed in this article are those of the authors and may not be understood or quoted as being made on behalf of or reflecting the position of the organizations with which the authors are employed/affiliated. ### Funding Statement This work was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, the NIHR Thames Valley and South Midlands Clinical Research Network, the British Research Council, and the Respiratory Syncytial Virus Consortium in Europe (RESCEU) project. RESCEU has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (grant number 116019). This Joint Undertaking receives support from the European Union Horizon 2020 Research and Innovation Program and European Federation of Pharmaceutical Industries and Associations. ### 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: Comité de ética de la Investigación de Santiago-Lugo of Hospital Clínico Universitario de Santiago de Compostela, Spain, gave ethical approval for this work (no. 2017/395). Ethics committee of the University of Oxford, UK, gave ethical approval for this work. Health Research Authority, UK, gave ethical approval for this work (no. 231136). NHS National Research Ethics Service Oxfordshire Research Ethics Committee A, UK, gave ethical approval for this work (no. 15/SC/0335). NHS National Research Ethics Service South Central - Hampshire A, UK, gave ethical approval for this work (no. 17/SC/0522). Medical Ethical Committee of the University Medical Center Utrecht, the Netherlands, gave ethical approval for this work (no. 17/563). 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All raw sequencing reads of the samples included in this study are accessible at the European Nucleotide Archive under the study accession PRJEB34042 (). The RSV consensus sequences included in the phylogenetic analyses have been deposited in GenBank. The accession numbers can be found in Table S14.

IntroductionNon-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies.MethodsPhylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment.ResultsThe Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species.ConclusionIn conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n=2), healthcare facility wastewater (n=2), wastewater treatment plant influent (n=1) and integrated constructed wetland influent (piggery farm waste) (n=1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, blaOXA-48 (n=2) and blaNDM-1 (n=1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

Bacterial genomics is making an increasing contribution to the fields of medicine and public health microbiology. Consequently, accurate species identification of bacterial genomes is an important task, particularly as the number of genomes stored in online databases increases rapidly and new species are frequently discovered. Existing database entries require regular re-evaluation to ensure that species annotations are consistent with the latest species definitions. We have developed an automated method for bacterial species identification that is an extension of ribosomal multilocus sequence typing (rMLST). The method calculates an ‘rMLST nucleotide identity’ (rMLST-NI) based on the nucleotides present in the protein-encoding ribosomal genes derived from bacterial genomes. rMLST-NI was used to validate the species annotations of 11839 publicly available Klebsiella and Raoultella genomes based on a comparison with a library of type strain genomes. rMLST-NI was compared with two whole-genome average nucleotide identity methods (OrthoANIu and FastANI) and the k-mer based Kleborate software. The results of the four methods agreed across a dataset of 11839 bacterial genomes and identified a small number of entries (n=89) with species annotations that required updating. The rMLST-NI method was 3.5 times faster than Kleborate, 4.5 times faster than FastANI and 1600 times faster than OrthoANIu. rMLST-NI represents a fast and generic method for species identification using type strains as a reference.

Bacterial meningitis is an infectious disease of the brain that occurs worldwide and is a major public health challenge. A leading cause of this often-fatal disease is the bacterium Neisseria meningitidis, also called meningococcus. Genomics is having a transformational impact on medicine. It enables advances in accurate diagnosis, analysis and prediction of anti-microbial resistance, development and assessment of vaccines etc. PubMLST.org, developed and run by the authors, is a major open-access genomics reference database. It is an integrated collection of databases consisting of phenotype metadata linked to nucleotide sequence data including genome assemblies, for molecular typing of many bacterial species. This paper discusses visual analytics and visualization work conducted to explore and analyse Neisseria meningitidis data from PubMLST.

Objective: Serogroup W and Y invasive meningococcal disease increased globally from 2000 onwards. Responding to a rapid increase in serogroup W clonal complex 11 (W:cc11) invasive meningococcal disease, the UK replaced an adolescent booster dose of meningococcal C conjugate vaccine with quadrivalent MenACWY conjugate vaccine in 2015. By 2018, the vaccine coverage in the eligible school cohorts aged 14 to 19 years was 84%. We assessed the impact of the MenACWY vaccination programme on meningococcal carriage. Methods: An observational study of culture-defined oropharyngeal meningococcal carriage prevalence before and after the start of the MenACWY vaccination programme in UK school students, aged 15 to 19 years, using two cross-sectional studies: 2014 to 2015 "UKMenCar4" and 2018 "Be on the TEAM" (ISRCTN75858406). Results: A total of 10 625 participants preimplementation and 13 438 postimplementation were included. Carriage of genogroups C, W, and Y (combined) decreased from 2.03 to 0.71% (OR 0.34 [95% CI 0.27e0.44], p < 0.001). Carriage of genogroup B meningococci did not change (1.26% vs 1.23% [95% CI 0.77 e1.22], p = 0.80) and genogroup C remained rare (n = 7/10 625 vs 17/13 438, p = 0.135). The proportion of serogroup positive isolates (i.e. those expressing capsule) decreased for genogroup Wby 53.8% (95% CI -5.0 e 79.8, p = 0.016) and for genogroup Y by 30.1% (95% CI 8.946.3, p = 0.0025). Discussion: The UK MenACWY vaccination programme reduced carriage acquisition of genogroup and serogroup Y and W meningococci and sustained low levels of genogroup C carriage. These data support the use of quadrivalent MenACWY conjugate vaccine for indirect ( herd) protection. Jeremy P. Carr, Clin Microbiol Infect 2022;28:1649.e1-e1649.e8

Background The incidence of invasive meningococcal disease in the UK decreased by approximately four times from 1999 to 2014, with reductions in serogroup C and serogroup B disease. Lower serogroup C invasive meningococcal disease incidence was attributable to implementation of the meningococcal serogroup C conjugate vaccine in 1999, through direct and indirect protection, but no vaccine was implemented against serogroup B disease. UK Meningococcal Carriage surveys 1-3 (UKMenCar1-3), conducted in 1999, 2000, and 2001, were essential for understanding the impact of vaccination. To investigate the decline in invasive meningococcal disease incidence, we did a large oropharyngeal carriage survey in 2014-15, immediately before the changes to meningococcal vaccines in the UK national immunisation schedule. Methods UKMenCar4 was a cross-sectional survey in adolescents aged 15-19 years who were enrolled from schools and colleges geographically local to one of 11 UK sampling centres between Sept 1, 2014, and March 30, 2015. Participants provided an oropharyngeal swab sample and completed a questionnaire on risk factors for carriage, including social behaviours. Samples were cultured for putative Neisseria spp, which were characterised with serogrouping and whole-genome sequencing. Data from this study were compared with the results from the UKMenCar1-3 surveys (1999-2001). Findings From the 19 641 participants (11 332 female, 8242 male, 67 not stated) in UKMenCar4 with culturable swabs and completed risk-factor questionnaires, 1420 meningococci were isolated, with a carriage prevalence of 7.23% (95% CI 6.88-7.60). Carriage prevalence was substantially lower in UKMenCar4 than in the previous surveys: carriage prevalence was 16.6% (95% CI 15.89-17.22; 2306/13901) in UKMenCar1 (1999), 17.6% (17.05-18.22; 2873/16 295) in UKMenCar2 (2000), and 18.7% (18.12-19.27; 3283/17569) in UKMenCar3 (2001). Carriage prevalence was lower for all serogroups in UKMenCar4 than in UKMenCar1-3, except for serogroup Y, which was unchanged. The prevalence of carriage-promoting social behaviours decreased from 1999 to 2014-15, with individuals reporting regular cigarette smoking decreasing from 2932 (21.5%) of 13 650 to 2202 (11.2%) of 19 641, kissing in the past week from 6127 (44.8%) of 13 679 to 7320 (37.3%) of 19 641, and attendance at pubs and nightclubs in the past week from 8436 (62.1%) of 13 594 to 7662 (39.0%) of 19 641 (all p<0.0001). Interpretation We show that meningococcal carriage prevalence in adolescents sampled nationally during a low incidence period (2014-15) was less than half of that in an equivalent population during a high incidence period (1999-2001). Disease and carriage caused by serogroup C was well controlled by ongoing vaccination. The prevalence of behaviours associated with carriage declined, suggesting that public health policies aimed at influencing behaviour might have further reduced disease. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd.

Expansion of quinolone-resistant Neisseria meningitidis clone ChinaCC4821-R1-C/B from sequence type (ST) 4821 clonal complex (CC4821) caused a serogroup shift from serogroup A to serogroup C invasive meningococcal disease (IMD) in China. To determine the relationship among globally distributed CC4821 meningococci, we analyzed whole-genome sequence data from 173 CC4821 meningococci isolated from 4 continents during 1972–2019. These meningococci clustered into 4 sublineages (1–4); sublineage 1 primarily comprised of IMD isolates (41/50, 82%). Most isolates from outside China (40/49, 81.6%) formed a distinct sublineage, the Europe–USA cluster, with the typical strain designation B:P1.17-6,23:F3-36:ST-3200(CC4821), harboring mutations in penicillin-binding protein 2. These data show that the quinolone-resistant clone ChinaCC4821-R1-C/B has expanded to other countries. The increasing distribution worldwide of serogroup B CC4821 raises the concern that CC4821 has the potential to cause a pandemic that would be challenging to control, despite indirect evidence that the Trumenba vaccine might afford some protection.

Benign cephalic histiocytosis is a rare non-Langerhans histiocytosis characterized by a self-healing eruption of papules and macules on the head and neck that occurs during infancy or childhood. Histologic and ultrastructural evaluations show a dermal proliferation of histiocytes with intracytoplasmic comma-shaped bodies, coated vesicles, and desmosome-like structures with an absence of Birbeck granules. We report a case of benign cephalic histiocytosis in a 9-month-old boy who presented with tan papules on his face that spread to his lower extremity and subsequently began to regress at 30 months of age. We review the features of this rare entity through a literature review and discuss the differential diagnosis.