Julian Parkhill

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the …

Background DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. This is exemplified by the success in predicting antibiotic resistance (ABR) from the genomes of bacteria such as Mycobacterium tuberculosis, Staphylococcus aureus or Streptococcus pneumoniae. Here, we aimed to assess and improve the accuracy of ABR determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. Methods We conducted a literature search to compile a catalogue of genes and mutations predictive of antibiotic resistance in E. faecium. We evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different antibiotics using a large and diverse population of 4,382 E. faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. We kept isolates tested with broth microdilution, Vitek2 and disk diffusion; and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations (MICs) and measured inhibition diameters; and harmonised using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A bioinformatics pipeline was developed to process raw sequencing reads, identify ABR genetic determinants and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus and LRE-Finder, to assess the accuracy of genotypic predictions against …

Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce …

BackgroundThe effect of antibiotic usage on the success of multidrug-resistant (MDR) clones in a population remains unclear. With this genomics-based molecular epidemiology study, we aimed to investigate the contribution of antibiotic use to Escherichia coli clone success, relative to intra-strain competition for colonisation and infection.MethodsWe sequenced all the available E coli bloodstream infection isolates provided by the British Society for Antimicrobial Chemotherapy (BSAC) from 2012 to 2017 (n=718) and combined these with published data from the UK (2001–11; n=1090) and Norway (2002–17; n=3254). Defined daily dose (DDD) data from the European Centre for Disease Prevention and Control (retrieved on Sept 21, 2021) for major antibiotic classes (β-lactam, tetracycline, macrolide, sulfonamide, quinolone, and non-penicillin β-lactam) were used together with sequence typing, resistance profiling …

BackgroundDNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes.MethodsIn this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from …

Objective Selective decontamination of the digestive tract (SDD) is a well-studied but hotly contested medical intervention of enhanced infection control. Here, we aim to characterise the changes to the microbiome and antimicrobial resistance (AMR) gene profiles in critically ill children treated with SDD-enhanced infection control compared with conventional infection control.Design We conducted shotgun metagenomic microbiome and resistome analysis on serial oropharyngeal and faecal samples collected from critically ill, mechanically ventilated patients in a pilot multicentre cluster randomised trial of SDD. The microbiome and AMR profiles were compared for longitudinal and intergroup changes. Of consented patients, faecal microbiome baseline samples were obtained in 89 critically ill children. Additionally, samples collected during and after critical illness were collected in 17 children treated with SDD …

The declining cost of performing bacterial whole-genome sequencing (WGS) coupled with the availability of large libraries of sequence data for well-characterized isolates have enabled the application of machine-learning (ML) methods to the development of nonlinear sequence-based predictive models. We tested the ML-based model developed by Next Gen Diagnostics for prediction of cefepime phenotypic susceptibility results in Escherichia coli. A cohort of 100 isolates of E. coli recovered from urine (n = 77) and blood (n = 23) cultures were used. The cefepime MIC was determined in triplicate by reference broth microdilution and classified as susceptible (MIC of ≤2 μg/mL) or not susceptible (MIC of ≥4 μg/mL) using the 2022 Clinical and Laboratory Standards Institute breakpoints. Five isolates generated both susceptible and not susceptible MIC results, yielding categorical agreement of 95% for the …

Exposure to different mutagens leaves distinct mutational patterns that can allow inference of pathogen replication niches. We therefore investigated whether SARS-CoV-2 mutational spectra might show lineage-specific differences, dependent on the dominant site(s) of replication and onwards transmission, and could therefore rapidly infer virulence of emergent variants of concern (VOCs). Through mutational spectrum analysis, we found a significant reduction in G>T mutations in the Omicron variant, which replicates in the upper respiratory tract (URT), compared to other lineages, which replicate in both the URT and lower respiratory tract (LRT). Mutational analysis of other viruses and bacteria indicates a robust, generalizable association of high G>T mutations with replication within the LRT. Monitoring G>T mutation rates over time, we found early separation of Omicron from Beta, Gamma and Delta, while …

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner …

Following publication of the original article [1], the authors identified a typographical error in Table 2 related to the accession numbers listed for genomes PN1 and PN2. The correct accession numbers are PN1, ERR163219 and PN2, ERR163220. The genomic analyses as described in the article were performed on the correct genomes, and the assembled genomes were correctly made public in PubMLST: PN1, PubMLST id 25638 and PN2, PubMLST id 37770. The authors apologise for any confusion caused by the typographical errors in Table 2. The corrected Table 2 is supplied in this correction article with the corrections marked in bold typeface.

Staphylococcus aureus is an important human pathogen but is primarily a commensal of the human nose and skin. Survival during colonisation is likely one of the major drivers of S. aureus evolution. Here we use a genome-wide mutation enrichment approach to analyse a genomic dataset of 3,060 S. aureus isolates from 791 individuals to show that despite limited within-host genetic diversity, an excess of protein-altering mutations can be found in genes encoding key metabolic pathways, in regulators of quorum-sensing and in known antibiotic targets. Nitrogen metabolism and riboflavin synthesis are the metabolic processes with strongest evidence of adaptation. Further evidence of adaptation to nitrogen availability was revealed by enrichment of mutations in the assimilatory nitrite reductase and urease, including mutations that enhance growth with urea as the sole nitrogen source. Inclusion of an additional 4,090 genomes from 802 individuals revealed eight additional genes including sasA/sraP, pstA, and rsbU with signals adaptive variation that warrant further characterisation. Our study provides the most comprehensive picture to date of the heterogeneity of adaptive changes that occur in the genomes of S. aureus during colonisation, revealing the likely importance of nitrogen metabolism, loss of quorum sensing and antibiotic resistance for successful human colonisation.

Summary 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone. Background Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017. Methods We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales …

Objectives: Abnormal cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with gut dysbiosis in people with cystic fibrosis. The recently introduced elexacaftor/tezacaftor/ivacaftor (ETI) therapy has proved highly effective at improving clinical status, reducing pulmonary exacerbations and increasing weight. There is presently little data on the impact of ETI on CF-related gut dysbiosis. In this study, we explored compositional changes of the gut microbiota following ETI therapy.Methods: Stool samples were prospectively collected (OMNIgene®. GUT) at two time points in pancreatic insufficient adults with CF as part of the Igloo-CF study. Whole genomic sequencing was conducted and changes in the gut microbiota pre and post commencing ETI therapy was analysed.Results: Data for 57 participants was analysed, age 33 years (IQR 29, 39.5) with 56% of participant being male. Median …

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within–host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time …

Background and AimsCystic Fibrosis (CF) is associated with gut dysbiosis, local and systemic inflammation, and impaired immune function. Gut microbiota dysbiosis results from changes in the complex gut milieu in response to CF transmembrane conductance regulator (CFTR) dysfunction, pancreatic malabsorption, diet, medications, and environmental influences. In several diseases, alteration of the gut microbiota influences local and systemic inflammation and disease outcomes. We conducted a systematic review of the gut microbiota in CF and explored factors influencing dysbiosis.MethodsAn electronic search of three databases was conducted in January 2019, and re-run in June 2021. Human, animal, and in vitro studies were included. The primary outcome was differences in the gut microbiota between people with CF (pwCF) and healthy controls. Secondary outcomes included the relationship between the …

Concerns exist that widespread use of antiseptic or disinfectant biocides could contribute to the emergence and spread of multidrug-resistant bacteria. To investigate this, we performed transposon-directed insertion-site sequencing (TraDIS) on the multidrug-resistant pathogen, Acinetobacter baumannii, exposed to a panel of ten structurally diverse and clinically relevant biocides. Multiple gene targets encoding cell envelope or cytoplasmic proteins involved in processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, were identified to have effects on bacterial fitness upon biocide exposure, suggesting that these compounds may have intracellular targets in addition to their known effects on the cell envelope. As cell respiration genes are required for A. baumannii fitness in biocides, we confirmed that sub-inhibitory concentrations of the biocides that …

Functional genomics analysis of Mycobacterium abscessus clinical isolates from chronically infected patients to identify genes under strong evolutionary pressure during lung adaptation identified phoR as one of the most frequently mutated. phoR encodes the histidine kinase (HK) of the two-component regulatory system (TCS) PhoPR. While PhoPR has been extensively studied in Mycobacterium tuberculosis for its role in virulence, little is known about the function of this TCS and the signals governing its activation in M. abscessus. We here show that acidic pH leads to the upregulation of phoP in M. abscessus and that clinically relevant non-synonymous mutations identified in PhoR exacerbate this response. PhoR modulates the ability of its cognate response regulator, PhoP, to autoregulate itself by controlling its dephosphorylation. At low pH, the phosphatase activity of PhoR is reduced and the build-up of …

Background Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight‐component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. Objective To define the diversity of the eight Strangvac antigens across a diverse S equi population. Study design Genomic description. Methods Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 …

Aggregation of children in schools has been established to be a key driver of transmission of infectious diseases. Mathematical models of transmission used to predict the impact of control measures, such as vaccination and testing, commonly depend on self-reported contact data. However, the link between self-reported social contacts and pathogen transmission has not been well described. To address this, we used Staphylococcus aureus as a model organism to track transmission within two secondary schools in England and test for associations between self-reported social contacts, test positivity and the bacterial strain collected from the same students. Students filled out a social contact survey and their S. aureus colonization status was ascertained through self-administered swabs from which isolates were sequenced. Isolates from the local community were also sequenced to assess the representativeness of …

Infectious diseases have had devastating impacts on human populations throughout history. Still, the origins and past dynamics of human pathogens remain poorly understood (1). To create the first spatiotemporal map of diverse ancient human microorganisms and parasites, we screened shotgun sequencing data from 1,313 ancient human remains covering 35,000 years of Eurasian history for ancient DNA deriving from bacteria, viruses, and parasites. We demonstrate the widespread presence of ancient microbial DNA in human remains, identifying over 2,400 individual species hits in 896 samples. We report a wide range of pathogens detected for the first time in ancient human remains, including the food-borne pathogens Yersinia enterocolitica and Shigella spp., the animal-borne Leptospira interrogans, and the malaria-causing parasite Plasmodium vivax. Our findings extend the spatiotemporal range of previously described ancient pathogens such as Yersinia pestis, the causative agent of plague, Hepatitis B virus, and Borrelia recurrentis, the cause of louse-borne relapsing fever (LBRF). For LRBF we increase the known distribution from a single medieval genome to 31 cases across Eurasia covering 5,000 years. Grouping the ancient microbial species according to their type of transmission (zoonotic, anthroponotic, sapronotic, opportunistic, and other), we find that most categories are identified throughout the entire sample period, while zoonotic pathogens, which are transmitted from living animals to humans or which have made a host jump into humans from animals in the timeframe of this study, are only detected from ~6,500 years ago …

Background Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. Methods Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. Results In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized …

Horizontal gene transfer (HGT) and the resulting patterns of gene gain and loss are a fundamental part of bacterial evolution. Investigating these patterns can help us to understand the role of selection in the evolution of bacterial pangenomes and how bacteria adapt to a new niche. Predicting the presence or absence of genes can be a highly error-prone process that can confound efforts to understand the dynamics of horizontal gene transfer. This review discusses both the challenges in accurately constructing a pangenome and the potential consequences errors can have on downstream analyses. We hope that by summarizing these issues researchers will be able to avoid potential pitfalls, leading to improved bacterial pangenome analyses.

While symbiotic relationships between invertebrates and bacteria have been extensively described, studies of microbial communities inhabiting parasitic worms remain scarce. Exploring the microbiota associated with helminths responsible for major infectious diseases will inform on parasite biology, host-pathogen interactions, and disease pathophysiology. We investigated the presence of microorganisms inhabiting tissues of the human parasite Schistosoma mansoni. In situ hybridization using a pan-bacterial 16S rRNA gene probe revealed bacteria colonizing key developmental stages that were successfully removed after antibiotic treatment of live parasites. Understanding the composition and function of the S. mansoni-associated microbiota may lead to the development of novel microbiome-targeting control strategies

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in …

BackgroundPatients with prolonged hospitalisation have a significant risk of carriage of and subsequent infection with extended spectrum β-lactamase (ESBL)-producing and carbapenemase-producing Klebsiella pneumoniae. However, the distinctive roles of the community and hospital environments in the transmission of ESBL-producing or carbapenemase-producing K pneumoniae remain elusive. We aimed to investigate the prevalence and transmission of K pneumoniae within and between the two tertiary hospitals in Hanoi, Viet Nam, using whole-genome sequencing.MethodsWe did a prospective cohort study of 69 patients in intensive care units (ICUs) from two hospitals in Hanoi, Viet Nam. Patients were included if they were aged 18 years or older, admitted for longer than the mean length of stay in their ICU, and cultured K pneumoniae from their clinical samples. Longitudinally collected samples from …

BackgroundMelioidosis is a frequently fatal disease caused by an environmental bacterium Burkholderia pseudomallei. The disease is prevalent in northeast Thailand, particularly among rice field farmers who are at risk of bacterial exposure through contact with contaminated soil and water. However, not all exposure results in disease, and infection can manifest diverse outcomes. We postulate that genetic factors, whether from the bacterium, the host or the combination of both, may influence disease outcomes. To address this hypothesis, we aim to collect, sequence, and analyse genetic data from melioidosis patients and controls, along with isolates of B. pseudomallei obtained from patients. Additionally, we will study the metagenomics of the household water supply for both patients and controls, including the presence of B. pseudomallei.MethodsBurkHostGEN is an ongoing observational study being conducted …

Introduction Maternal immunization against Group B Streptococcus (GBS) has the potential to significantly reduce the burden of neonatal GBS infections. Population genetics of GBS from maternal carriage can offer key insights into vaccine target distribution. Methods In this study we characterized the population structure of GBS isolates from maternal carriage (n = 535) in an ethnically diverse community in London, using whole genome sequencing. Results The isolates clustered into nine clonal complexes (CCs) but the majority (95%) belonged to five lineages: CC1 (26%), CC19 (26%), CC23 (20%), CC17 (13%) and CC8/10 (10%). Nine serotypes were identified, the most common were serotypes III (26%), V (21%), II (19%) and Ia (19%). Other serotypes (Ib, IV, VI, VII, IX) represented less than 10% of all isolates each. Intra-lineage serotype diversity was observed in all major CCs but was highest in CC1, which revealed nine serotypes. Nearly all isolates (99%) carried at least one of the four alpha family protein genes (alpha, alp1, alp23, and rib). All isolates were susceptible to penicillin. We found 21% and 13% of isolates to be resistant to clarithromycin and clindamycin, respectively. Prevalence of macrolide-lincosamide-streptogramin B (MLSB) resistance genes was 22% and they were most common in CC19 (37%) and CC1 (28%), and isolates with serotypes V (38%) and IV (32%). We identified some associations between maternal ethnicity and GBS population structure. Serotype Ib was significantly less common among the South Asian compared to Black women (S. Asian: 3/142, Black: 15/135, p = 0.03). There was also a significantly …

The genetics underlying tuberculosis (TB) pathophysiology are poorly understood. Human genome-wide association studies have failed so far to reveal reproducible susceptibility loci, attributed in part to the influence of the underlying Mycobacterium tuberculosis (Mtb) bacterial genotype on the outcome of the infection. Several studies have found associations of human genetic polymorphisms with Mtb phylo-lineages, but studies analysing genome-genome interactions are needed. By implementing a phylogenetic tree-based Mtb-to-human analysis for 714 TB patients from Thailand, we identify eight putative genetic interaction points (P < 5 × 10−8) including human loci DAP and RIMS3, both linked to the IFNγ cytokine and host immune system, as well as FSTL5, previously associated with susceptibility to TB. Many of the corresponding Mtb markers are lineage specific. The genome-to-genome analysis reveals …

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing …

Streptococcus agalactiae (Group B Streptococcus; GBS) is a common cause of sepsis in neonates. Previous work detected GBS DNA in the placenta in ~5% of women before the onset of labour, but the clinical significance of this finding is unknown. Here we re-analysed this dataset as a case control study of neonatal unit (NNU) admission. Of 436 infants born at term (≥37 weeks of gestation), 7/30 with placental GBS and 34/406 without placental GBS were admitted to the NNU (odds ratio (OR) 3.3, 95% confidence interval (CI) 1.3–7.8). We then performed a validation study using non-overlapping subjects from the same cohort. This included a further 239 cases of term NNU admission and 686 term controls: 16/36 with placental GBS and 223/889 without GBS were admitted to the NNU (OR 2.4, 95% CI 1.2–4.6). Of the 36 infants with placental GBS, 10 were admitted to the NNU with evidence of probable but culture …

Mutational spectra describe the pattern of mutations acquired during evolution and are driven by factors including mutagens, repair processes and selection. Calculating mutational spectra of pathogen genomic datasets may enable analysis of factors that influence these mutational processes, including replication niches, transmission routes and pathogen biology. Here, we introduce MutTui, which can leverage multiple types of sequence data to calculate and compare mutational spectra of DNA and RNA pathogens. MutTui is available at https://github.com/chrisruis/MutTui.

BackgroundNon-tuberculous mycobacteria (NTM) are environmental microorganisms and opportunistic pathogens in individuals with pre-existing lung conditions such as cystic fibrosis (CF) and non-CF bronchiectasis. While recent studies of Mycobacterium abscessus have identified transmission within single CF centres as well as nationally and globally, transmission of other NTM species is less well studied.MethodsTo investigate the potential for transmission of the Mycobacterium avium complex (MAC) we sequenced 996 isolates from 354 CF and non-CF patients at the Royal Brompton Hospital (London, UK; collected 2013–2016) and analysed them in a global context. Epidemiological links were identified from patient records. Previously published genomes were used to characterise global population structures.ResultsWe identified putative transmission clusters in three MAC species, although few …

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free …

The National Collection of Type Cultures (NCTC) was founded on 1 January 1920 in order to fulfil a recognized need for a centralized repository for bacterial and fungal strains within the UK. It is among the longest-established collections of its kind anywhere in the world and today holds approximately 6000 type and reference bacterial strains – many of medical, scientific and veterinary importance – available to academic, health, food and veterinary institutions worldwide. Recently, a collaboration between NCTC, Pacific Biosciences and the Wellcome Sanger Institute established the NCTC3000 project to long-read sequence and assemble the genomes of up to 3000 NCTC strains. Here, at the beginning of the collection’s second century, we introduce the resulting NCTC3000 sequence read datasets, genome assemblies and annotations as a unique, historically and scientifically relevant resource for the benefit of …

We report the complete genome sequence of the Ornithobacterium hominis type strain MSHR-COH1 (ATCC TSD-185/NCTC 14317), a bacterial species isolated from the human nasopharynx. Long-read sequencing reveals that the genome is 2,036,909 bp in length, with a GC content of 35.72%.

As observed in cancers, individual mutagens and defects in DNA repair create distinctive mutational signatures that combine to form context-specific spectra within cells. We reasoned that similar processes must occur in bacterial lineages, potentially allowing decomposition analysis to detect both disruption of DNA repair processes and exposure to niche-specific mutagens. Here we reconstruct mutational spectra for 84 clades from 31 diverse bacterial species and find distinct mutational patterns. We extract signatures driven by specific DNA repair defects using hypermutator lineages, and further deconvolute the spectra into multiple signatures operating within different clades. We show that these signatures are explained by both bacterial phylogeny and replication niche. By comparing mutational spectra of clades from different environmental and biological locations, we identify niche-associated mutational …

Background: The interplay between antibiotic usage levels, and other factors influencing the success of multi-drug resistant (MDR) clones in a population, remains unclear. Our study aimed to investigate the contribution of antibiotic use to clone success, relative to genetic determinants of intra-strain competition for colonisation and infection.Methods: We sequenced 718 E. coli bloodstream infection (BSI) isolates provided by the British Society for Antimicrobial Chemotherapy (BSAC) from 2012–17 and combined these with published data from the UK (2001–11; n= 1090) and Norway (2002–17; n= 3254). Defined daily doses (DDD) for major antibiotic classes were obtained from the European Centre for Disease Prevention and Control database. Sequence-typing, resistance profiling and simulation-based modelling were used to enable systematic comparison of resistance levels, clone success and antibiotic usage between the UK and Norway.Findings: New BSAC data showed that the extended-spectrum β-lactamase (ESBL)-carrying, globally disseminated MDR clone ST131–C2 continues to display higher success in the UK (7 [6· 3%] of 112 isolates) compared to Norway (7 [2· 0%] of 355 isolates; p= 0· 049) in 2017. DDD data indicated generally similar trends between the two countries, except notably for the class of non-penicillin β-lactams, which were used 2· 7–5· 1 times more in the UK per annum (ratio mean= 3· 74; SD= 0· 79). This difference was associated with the higher success of the MDR clone ST131–C2. A population genetics non-neutral Wright–Fisher model replicated the observed growth of non-MDR and MDR sequence types …

The dynamics of pathogen genetic diversity, including the emergence of lineages with increased fitness, is a foundational concept of disease ecology with key public health implications. However, the identification of distinct lineages and estimation of associated fitness remain challenging, and are rarely done outside densely sampled systems. Here, we present a scalable framework that summarizes changes in population composition in phylogenies, allowing for the automatic detection of lineages based on shared fitness and evolutionary relationships. We apply our approach to a broad set of viruses and bacteria (SARS-CoV-2, H3N2 influenza, Bordetella pertussis and Mycobacterium tuberculosis) and identify previously undiscovered lineages, as well as specific amino acid changes linked to fitness changes, the findings of which are robust to uneven and limited observation. This widely-applicable framework provides an avenue to monitor evolution in real-time to support public health action and explore fundamental drivers of pathogen fitness.

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial …

Objectives Escherichia coli bloodstream infections have shown a sustained increase in England, for reasons that are unknown. Furthermore, the contribution of MDR lineages such as ST131 to overall E. coli disease burden and outcome is undetermined. Methods We genome-sequenced E. coli blood isolates from all patients with E. coli bacteraemia in north-west London from July 2015 to August 2016 and assigned MLST genotypes, virulence factors and AMR genes to all isolates. Isolate STs were then linked to phenotypic antimicrobial susceptibility, patient demographics and clinical outcome data to explore relationships between the E. coli STs, patient factors and outcomes. Results A total of 551 E. coli genomes were analysed. Four STs (ST131, 21.2%; ST73, 14.5%; ST69, 9.3%; and ST95, 8.2%) accounted for over half of cases. E. coli genotype …

The medical and scientific response to emerging and established pathogens is often severely hampered by ignorance of the genetic determinants of virulence, drug resistance and clinical outcomes that could be used to identify therapeutic drug targets and forecast patient trajectories. Taking the newly emergent multidrug-resistant bacteria Mycobacterium abscessus as an example, we show that combining high-dimensional phenotyping with whole-genome sequencing in a phenogenomic analysis can rapidly reveal actionable systems-level insights into bacterial pathobiology. Through phenotyping of 331 clinical isolates, we discovered three distinct clusters of isolates, each with different virulence traits and associated with a different clinical outcome. We combined genome-wide association studies with proteome-wide computational structural modelling to define likely causal variants, and employed direct coupling …

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organism Escherichia coli, as well as several major human pathogens including multiple serovars of Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the E. coli Keio single-gene deletion library often regarded as a gold standard for gene essentiality studies. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, surprisingly we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss …

jats: pCampylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuniresponsible for 80% of these infections. There is no current vaccine and antibiotic resistance is emerging. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which impacts studies into the function of essential and conditionally essential genes. My project aims to address this by applying a CRISPR-based interference system known as CRISPRi in C. jejuni as a means to control gene expression and thereby investigate gene function. To validate the CRISPRi system in C. jejuni, I have paired the dCas9 and sgRNA backbone from the Streptococcus pyogenesCRISPRi system with several C. jejuni-derived promoters to develop a series of CRISPRi constructs targeting several genes. Through rigorous sgRNA target design I have successfully targeted and repressed expression of the endogenous arylsulphatase (AstA) enzyme, as well as achieving partial repression of expression of the regulatory flagellar protein FlgR in two clinically relevant C. jejuni strains. This is the first report of a CRISPRi system for Campylobacter.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus—a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest …

Humanity is in the grip of another pandemic beyond coronavirus disease (COVID). Although it is not spreading as rapidly as the viral pandemic, it is insidious and probably presents a greater threat in the long run. Through the large-scale use of antibiotics, we have driven the emergence of antimicrobial resistance (AMR) in bacteria, and these resistant bacteria are increasing in number and prevalence, threatening our ability to treat common infections and reducing our opportunities to use other life-saving treatments such as chemotherapy and surgery. A recent study showed that bacterial AMR was associated with nearly 5 million deaths in 2019, with about 1.3 million being directly attributable to resistance [1]. These numbers are likely to increase. Antibiotics are used in human medicine reactively, to treat infection, and prophylactically, to prevent infection. They are also used in agriculture, both reactively and …

Recent years have seen a remarkable increase in the practicality of sequencing whole genomes from large numbers of bacterial isolates. The availability of this data has huge potential to deliver new insights into the evolution and epidemiology of bacterial pathogens, but the scalability of the analytical methodology has been lagging behind that of the sequencing technology. Here we present a step-by-step approach for such large-scale genomic epidemiology analyses, from bacterial genomes to epidemiological interpretations. A central component of this approach is the dated phylogeny, which is a phylogenetic tree with branch lengths measured in units of time. The construction of dated phylogenies from bacterial genomic data needs to account for the disruptive effect of recombination on phylogenetic relationships, and we describe how this can be achieved. Dated phylogenies can then be used to perform fine …

Acute respiratory distress syndrome (ARDS) is an inflammatory disorder of the lungs, with sepsis as the predominant aetiology. Despite advances in ventilation strategies, mortality for moderate to severe ARDS remains at 40-46%(1). ARDS is associated with neutrophil influx into alveoli. Persistently high neutrophil and low alveolar macrophage (AM) numbers in broncho-alveolar lavage (BAL) fluid are associated with greater mortality (2). While the inflammatory alveolar environment of early ARDS initially delays apoptosis, these neutrophils ultimately undergo apoptosis within alveoli (3). Efficient efferocytosis of apoptotic neutrophils by AMs is critical for resolution of inflammation (3). Apoptotic neutrophils may accumulate in ARDS due to defective AM efferocytosis and/or overwhelmed efferocytosis capacity, then undergo secondary necrosis, releasing inflammatory mediators into the alveolar space (4). This may …

Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found …

As observed in cancers, individual mutagens and defects in DNA repair create distinctive mutational signatures that combine to form context-specific spectra within cells. We reasoned that similar processes must occur in bacterial lineages, potentially allowing decomposition analysis to identify disrupted DNA repair processes and niche-specific mutagen exposure. Here we reconstructed mutational spectra for 84 clades from 31 diverse bacterial species, assigned signatures to specific DNA repair pathways using hypermutator lineages, and, by comparing mutational spectra of clades from different environmental and biological locations, extracted reproducible niche-associated mutational signatures. We show that mutational spectra can predict general and specific bacterial niches and therefore reveal the site of infection and types of transmission routes for established and emergent human bacterial pathogens.One sentence summaryVariable mutagen exposure and DNA repair drive differential mutational spectra between bacteria and enable niche inference

BackgroundCampylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive (“dead”) cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C …

Genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) could transform outbreak investigations, but its clinical introduction is hampered by the lack of automated data analysis tools to rapidly and accurately define transmission based on sequence relatedness. We aimed to evaluate a fully automated bioinformatics system for MRSA genome analysis versus a bespoke researcher-led manual informatics pipeline. We analyzed 781 MRSA genomes from 777 consecutive patients identified over a 9-month period in a clinical microbiology laboratory in the United Kingdom. Outputs were bacterial species identification, detection of mec genes, assignment to sequence types (STs), identification of pairwise relatedness using a definition of ≤25 single nucleotide polymorphisms (SNPs) apart, and use of genetic relatedness to identify clusters. There was full concordance between the two analysis …

Distinct bacterial trophic networks exist in the gut microbiota of individuals in industrialized and non-industrialized countries. In particular, non-industrialized gut microbiomes tend to be enriched with Prevotella species. To study the development of these Prevotella-rich compositions, we investigated the gut microbiota of children aged between 7 and 37 months living in rural Gambia (616 children, 1,389 stool samples, stratified by 3-month age groups). These infants, who typically eat a high-fibre, low-protein diet, were part of a double-blind, randomized iron intervention trial (NCT02941081) and here we report the secondary outcome. We found that child age was the largest discriminating factor between samples and that anthropometric indices (collection time points, season, geographic collection site, and iron supplementation) did not significantly influence the gut microbiome. Prevotella copri, Faecalibacterium …

As with other pathogens, competitive interactions between Bordetella pertussis strains drive infection risk. Vaccines are thought to perturb strain diversity through shifts in immune pressures; however, this has rarely been measured because of inadequate data and analytical tools. We used 3344 sequences from 23 countries to show that, on average, there are 28.1 transmission chains circulating within a subnational region, with the number of chains strongly associated with host population size. It took 5 to 10 years for B. pertussis to be homogeneously distributed throughout Europe, with the same time frame required for the United States. Increased fitness of pertactin-deficient strains after implementation of acellular vaccines, but reduced fitness otherwise, can explain long-term genotype dynamics. These findings highlight the role of vaccine policy in shifting local diversity of a pathogen that is responsible for 160,000 …

Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. In this study, we performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand the genomic diversity. A retrospective collection of 498 M. tuberculosis isolates submitted to the National Institute for Research in Tuberculosis for phenotypic susceptibility testing between 2014 to 2016 were sequenced. Genotypic resistance prediction was performed using known resistance-conferring determinants. Genotypic and phenotypic results for 12 antituberculosis drugs were compared, and sequence data were explored to characterize lineages and their association with drug resistance. Four lineages were identified although lineage …

Exposure to different mutagens leaves distinct mutational patterns that can allow prediction of pathogen replication niches . We therefore hypothesised that analysis of SARS-CoV-2 mutational spectra might show lineage-specific differences, dependant on the dominant site(s) of replication and onwards transmission, and could therefore rapidly infer virulence of emergent variants of concern (VOC; ). Through mutational spectrum analysis, we found a significant reduction in G>T mutations in Omicron, which replicates in the upper respiratory tract (URT), compared to other lineages, which replicate in both upper and lower respiratory tracts (LRT). Mutational analysis of other viruses and bacteria indicates a robust, generalisable association of high G>T mutations with replication within the LRT. Monitoring G>T mutation rates over time, we found early separation of Omicron from Beta, Gamma and Delta, while the mutational burden in Alpha varied consistent with changes in transmission source as social restrictions were lifted. This supports the use of mutational spectra to infer niches of established and emergent pathogens.

Emergence of the colistin resistance gene, mcr-1, has attracted worldwide attention. Despite the prevalence of mcr-1-positive Escherichia coli (MCRPEC) strains in human carriage showing a significant decrease between 2016 and 2019, genetic differences in MCRPEC strains remain largely unknown. We therefore conducted a comparative genomic study on MCRPEC strains from fecal samples of healthy human subjects in 2016 and 2019. We identified three major differences in MCRPEC strains between these two time points. First, the insertion sequence ISApl1 was often deleted and the percentage of mcr-1-carrying IncI2 plasmids was increased in MCRPEC strains in 2019. Second, the antibiotic resistance genes (ARGs), aac(3)-IVa and blaCTX-M-1, emerged and coexisted with mcr-1 in 2019. Third, MCRPEC strains in 2019 contained more virulence genes, resulting in an increased proportion of …

Genome-wide association studies (GWAS) are increasingly being applied to investigate the genetic basis of bacterial traits. However, approaches to perform power calculations for bacterial GWAS are limited. Here we implemented two alternative approaches to conduct power calculations using existing collections of bacterial genomes. First, a sub-sampling approach was undertaken to reduce the allele frequency and effect size of a known and detectable genotype-phenotype relationship by modifying phenotype labels. Second, a phenotype-simulation approach was conducted to simulate phenotypes from existing genetic variants. We implemented both approaches into a computational pipeline (PowerBacGWAS) that supports power calculations for burden testing, pan-genome and variant GWAS; and applied it to collections of Enterococcus faecium, Klebsiella pneumoniae and Mycobacterium tuberculosis. We …

Broad-spectrum antimicrobial use during the treatment of critical illness influences gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary to secondary bile acids. We previously observed reduced fermentation capacity in the faecal microbiota of critically ill children upon hospital admission. Here, we further explore the timecourse of the relationship between the microbiome and bile acid profile in faecal samples collected from critically ill children. The microbiome was assayed by sequencing of the 16S rRNA gene, and faecal water bile acids were measured by liquid chromatography mass spectrometry. In comparison to admission faecal samples, members of the Lachnospiraceae recovered during the late-acute phase (days 8–10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than controls …

The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of “co-selection”—that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress …

Pit latrines are used by billions of people globally, often in developing countries where they provide a low-tech and low-cost sanitation method. However, health and social problems can arise from a lack of emptying or maintenance of these facilities. A better understanding of the biological and environmental parameters within pit latrines could inform attempts to enhance material decomposition rates, and therefore slow fill-up rate. In this study, we have performed a spatial analysis of 35 Tanzanian pit latrines to identify bacteria and environmental factors that are associated with faster or slower pit latrine fill-up rates. Using ordination of microbial community data, we observed a linear gradient in terms of beta diversity with increasing pit latrine sample depth, corresponding to a shift in microbial community structure from gut-associated families in the top layer to environmental- and wastewater-associated taxa at greater depths. We also investigated the bacteria and environmental parameters associated with fill-up rates, and identified pH, volatile solids, and volatile fatty acids as features strongly positively correlated with pit latrine fill-up rates, whereas phosphate was strongly negatively correlated with fill-up rate. A number of pit latrine microbiota taxa were also correlated with fill-up rates. Using a multivariate regression, we identified the Lactobacillaceae and Incertae_Sedis_XIII taxa as particularly strongly positively and negatively correlated with fill-up rate, respectively. This study therefore increases knowledge of the microbiota within pit latrines, and identifies potentially important bacteria and environmental variables associated with fill-up rates …

BackgroundViet Nam has high rates of antimicrobial resistance (AMR) but little capacity for genomic surveillance. This study used whole genome sequencing to examine the prevalence and transmission of three key AMR pathogens in two intensive care units (ICUs) in Hanoi, Viet Nam.MethodsA prospective surveillance study of all adults admitted to ICUs at the National Hospital for Tropical Diseases and Bach Mai Hospital was done between June 19, 2017, and Jan 16, 2018. Clinical and environmental samples were cultured on selective media, characterised with MALDI TOF mass spectrometry, and sequenced with Illumina. Phylogenies based on the de-novo assemblies (SPAdes) were constructed with MAFFT (PARsnp), Gubbins, and RAxML. Resistance genes were detected with Abricate against the US National Center for Biotechnology Information database.Findings3153 Escherichia coli, Klebsiella …

Biocides, such as antiseptics and disinfectants, are used ubiquitously for hygiene in households and for life-saving infection control in hospitals. An increasing concern is that the widespread use of biocides may contribute to the emergence and spread of multidrug-resistant bacteria. We performed transposon directed insertion site sequencing (TraDIS) to identify genes and key cellular pathways of the multidrug resistant nosocomial pathogen Acinetobacter baumannii, that affect host fitness during exposure to a panel of ten structurally-diverse and clinically-relevant biocides: silver nitrate, benzalkonium, cetyltrimethylammonium bromide (CTAB), chlorhexidine, triclosan, chloroxylenol, polyvidone iodine, bleach, glutaraldehyde and ethanol. Multiple genes encoding proteins localised either in the cell envelope or in the cytoplasm were shown to affect biocide susceptibility. These proteins are involved in multiple processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, suggesting that these biocides may have intracellular targets in addition to their known effects on the cell envelope. Based on the importance of cell respiration genes for A. baumannii fitness on biocides, we proposed and confirmed that apart from triclosan, the other 9 biocides at sub-inhibitory concentration can dissipate the membrane potential and lead to A. baumannii tolerance to antibiotics that have intracellular targets. Our results support the concern that residual biocides in clinical or community environments can promote the development of antibiotic resistance in pathogenic bacteria.

We take advantage of a historic collection of 133 Staphylococcus aureus strains accessioned between 1924 and 2016, whose genomes have been long-read sequenced as part of a major National Collection of Type Cultures (NCTC) initiative, to conduct a gene family-wide computational analysis of enterotoxin genes. We identify two novel staphylococcal enterotoxin (pseudo)genes (sel29p and sel30), the former of which has not been observed in any contemporary strain to date. We provide further information on five additional enterotoxin genes or gene variants that either have recently entered the literature or for which the nomenclature or description is currently unclear (selz, sel26, sel27, sel28, and ses-2p). An examination of over 11,000 RefSeq genomes in search of wider support for these seven (pseudo)genes led to the identification of an additional three novel enterotoxin gene family members (sel31, sel32 …

Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 M. bovis isolates sourced from naturally infected cattle in the State of Rio Grande do Sul (RS), southern Brazil, was used to evaluate the population structure of M. bovis in the region, identify potential transmission events and date the introduction of clonal complex (CC) European 2 (Eu2). In silico spoligotyping identified 11 distinct patterns including four new profiles and two CCs, European 1 (Eu1) and Eu2. The analyses revealed a high level of genetic diversity in the majority of herds and identified putative transmission clusters that suggested that within …

The medical and scientific response to emerging pathogens is often severely hampered by ignorance of the genetic determinants of virulence, drug resistance, and clinical outcomes that could be used to identify therapeutic drug targets and forecast patient trajectories –. Taking the newly emergent multidrug-resistant bacteria Mycobacterium abscessus as an example , we show that combining high dimensional phenotyping with whole genome sequencing in a phenogenomic analysis can rapidly reveal actionable systems-level insights into bacterial pathobiology. Using in vitro and in vivo phenotyping, we discovered three distinct clusters of isolates, each associated with a different clinical outcome. We combined genome-wide association studies (GWAS) with proteome-wide computational structural modelling to define likely causal variants, and employed direct coupling analysis (DCA) to identify co-evolving, and therefore potentially epistatic, gene networks. We then used in vivo CRISPR-based silencing to validate our findings, defining a novel secretion system controlling virulence in M. abscessus, and illustrating how phenogenomics can reveal critical pathways within emerging pathogenic bacteria.

BackgroundEscherichia coli is a leading cause of bloodstream infections. Developing interventions to reduce E coli infections requires an understanding of the frequency of nosocomial transmission, but the available evidence is scarce. We aimed to detect and characterise transmission of E coli and associated plasmids in a hospital setting.MethodsIn this prospective observational cohort study, patients were admitted to two adult haematology wards at the Cambridge University Hospitals NHS Foundation Trust in England. Patients aged 16 years and older who were treated for haematological malignancies were included. Stool samples were collected from study participants on admission, once per week, and at discharge. We sequenced multiple E coli isolates (both extended spectrum β-lactamase [ESBL]-producing and non-ESBL-producing) from each stool sample. A genetic threshold to infer E coli transmission …

BackgroundSepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis.MethodsIn BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood …

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli …

Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a …

Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies …

Chronic pulmonary infections caused by non-tuberculous mycobacteria of the Mycobacterium abscessus complex (MABSC) are emerging as a global health problem and pose a threat to susceptible individuals with structural lung disease such as cystic fibrosis. The molecular mechanisms underlying the pathogenicity and intrinsic resistance of MABSC to antibiotics remain largely unknown. The involvement of Msp-type porins in the virulence and biocide resistance of some rapidly growing non-tuberculous mycobacteria and the finding of deletions and rearrangements in the porin genes of serially collected MABSC isolates from cystic fibrosis patients prompted us to investigate the contribution of these major surface proteins to MABSC infection. Inactivation by allelic replacement of the each of the two Msp-type porin genes of M. abscessus subsp. massiliense CIP108297, mmpA and mmpB, led to a marked increase in the virulence and pathogenicity of both mutants in murine macrophages and infected mice. Neither of the mutants were found to be significantly more resistant to antibiotics. These results suggest that adaptation to the host environment rather than antibiotic pressure is the key driver of the emergence of porin mutants during infection.

Determining the composition of bacterial communities beyond the level of a genus or species is challenging because of the considerable overlap between genomes representing close relatives. Here, we present the mSWEEP pipeline for identifying and estimating the relative sequence abundances of bacterial lineages from plate sweeps of enrichment cultures. mSWEEP leverages biologically grouped sequence assembly databases, applying probabilistic modelling, and provides controls for false positive results. Using sequencing data from major pathogens, we demonstrate significant improvements in lineage quantification and detection accuracy. Our pipeline facilitates investigating cultures comprising mixtures of bacteria, and opens up a new field of plate sweep metagenomics.

Background Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. The use of antimicrobials in livestock production can lead to emergence of AMR, which can have direct effects on humans through spread of zoonotic disease. Pigs pose a particular risk as they are a source of zoonotic diseases and receive more antimicrobials than most other livestock. Here we use a large-scale genomic approach to characterise AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans. Results We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into ‘resistant’ and …

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral …

Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the “modern hospital” era, suggesting selection in another niche …

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis , which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole-genome sequencing to examine the population structure of M. bovis in Ethiopia. A total of 134 M . bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis , based on …

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered to be monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate strains of the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum) strains, from each other. However, this genome variability and gene content, especially of L5 strains, has not been fully explored and may be important for pathobiology and current approaches for genomic analysis of MTBC strains, including transmission studies. By comparing the genomes of 355 L5 clinical strains (including 3 complete genomes and 352 Illumina whole-genome sequenced isolates) to each other and to H37Rv, we identified multiple genes that were differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found …

Understanding how multidrug-resistant Enterobacterales (MDRE) are transmitted in low- and middle-income countries (LMICs) is critical for implementing robust policies to curb the increasing burden of antimicrobial resistance (AMR). Here, we analysed samples from surgical site infections (SSIs), hospital surfaces (HSs) and arthropods (summer and winter 2016) to investigate the incidence and transmission of MDRE in a public hospital in Pakistan. We investigated Enterobacterales containing resistance genes (blaCTX-M-15, blaNDM and blaOXA-48-like) for identification, antimicrobial susceptibility testing and whole-genome sequencing. Genotypes, phylogenetic relationships and transmission events for isolates from different sources were investigated using single-nucleotide polymorphism (SNP) analysis with a cut-off of ≤20 SNPs. Escherichia coli (14.3%), Klebsiella pneumoniae (10.9%) and Enterobacter …

A recent study by Rackaityte et al. reported evidence for a low level of bacterial colonization, specifically of Micrococcus luteus, in the intestine of second trimester human fetuses. We have re-analyzed their sequence data and identified a batch effect which violates the underlying assumptions of the bioinformatic method used for contamination removal. This batch effect resulted in Micrococcus not being identified as a contaminant in the original work and being falsely assigned to the fetal samples. We further provide evidence that the micrographs presented by Rackaityte et al. are unlikely to show Micrococci or other bacteria as the size of the particles shown exceeds that of related bacterial cells. Finally, phylogenetic analysis showed that the microbes cultured from the fetal samples differed significantly from those detected by sequencing. Overall, our findings show that the presence of Micrococcus in the …

48 The lateral transfer of genes among bacterial strains and species has opposing effects, 49 conferring potentially beneficial adaptations whilst introducing disharmony in coadapted 50 genomes. The prevailing outcome will depend upon the fitness cost of disrupting established 51 epistatic interactions between genes. It is challenging to understand this in nature because it 52 requires population-scale analysis of recombination and genomic coadaptation, and 53 laboratory confirmation of the functional significance of genotype variation. By assigning the 54 ancestry of DNA in the genomes of two species of the world’s most common enteric bacterial 55 pathogen, we show that up to 28% of the Campylobacter coli genome has been recently 56 introgressed from Campylobacter jejuni. Then, by quantifying covariation across the genome 57 we show that> 83% of putative epistatic links are between introgressed C. jejuni genes in 58 divergent genomic positions (> 20kb apart), consistent with independent acquisition. Much of 59 this covariation is between 16 genes, with just 5 genes accounting for 99% of epistatic SNP 60 pairs. Laboratory mutagenesis and complementation cloning assays demonstrated functional 61 links between these genes, specifically related to formate dehydrogenase (FDH) activity. 62 These findings suggest that the genetic confederations that define genomic species may be 63 transient. Even for complex traits such as central metabolism in the bacterial cell, conditions 64 can arise where epistatic genes can be decoupled, transferred and reinstated in a new genetic 65 background. 66

Distinctive bacterial trophic networks exist in the gut microbiota of individuals in industrialized and non-industrialized countries. To study the development of these networks, we investigated the gut microbiota of 7-37 month old children living in rural Gambia (616 children, 1407 stool samples, stratified by 3-month age groups). We found that child age was the largest discriminating factor between samples, and that anthropometric indices WAZ, HAZ, and WHZ, collection timepoints, and iron supplementation did not significantly influence the gut microbiome in this data set. Prevotella copri, Faecalibacterium prausnitzii, and Prevotella stercorea were the most abundant species (35%, 11%, and 7%, respectively). Distinct bacterial trophic network clusters were identified, centered around Prevotella stercorea and Faecalibacterium prausnitzii, which were found to develop steadily as the gut microbiome matured. The Prevotella stercorea trophic network cluster is distinct from those found in individuals in industrialized countries and therefore this dataset, set within a critical developmental timeframe, provides an excellent opportunity to understand the influence of a high fiber, low-protein diet on the development of a Prevotella-enriched gut microbiome.

INTRODUCTION Nearly all mycobacterial species are free-living environmental saprophytes. A few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection and eventually to become obligate human pathogens. The recent emergence and global spread of virulent clones of the environmental nontuberculous mycobacterium M. abscessus has provided a unique opportunity to examine the pathogenic evolution of mycobacteria. RATIONALE M. abscessus, a multidrug-resistant species of nontuberculous mycobacteria, has recently emerged as a major threat to individuals with cystic fibrosis (CF) and other chronic lung conditions. Infection rates within the CF community are increasing globally, driven in part by indirect person-to-person transmission of M. abscessus. Currently more than 70% of infections in CF patients are caused by genetically clustered (and thus transmitted) isolates …

Antimicrobial resistance (AMR) is designated as a global health crisis by the United Nations. In 2019, an estimated 4.95 million deaths were associated with AMR globally. The increasing global spread of resistant pathogens is a major threat to global health security (GHS). AMR has economic, social, and political ramifications worldwide with a disproportionate burden on resource-limited countries. Despite the growing concern over AMR globally, there are multiple challenges to bridge the gap between policies and practices. A strong interface among the human, animal, and environment through One Health approach is crucial to combat AMR effectively. There is a need to strengthen AMR surveillance, improve laboratory diagnostic capacity, initiate antimicrobial stewardship, reinforce infection prevention, and control programs in both humans and veterinary medicine. Effective implementation of national action plans …

Mycobacterium abscessus, a multidrug-resistant nontuberculous mycobacterium, has emerged as a major pathogen affecting people with cystic fibrosis (CF). Although originally thought to be acquired independently from the environment, most individuals are infected with one of several dominant circulating clones (DCCs), indicating the presence of global transmission networks of M. abscessus. How and when these clones emerged and spread globally is unclear. Here, we use evolutionary analyses of isolates from individuals both with and without CF to reconstruct the population history, spatiotemporal spread and recent transmission networks of the DCCs. We demonstrate synchronous expansion of six unrelated DCCs in the 1960s, a period associated with major changes in CF care and survival. Each of these clones has spread globally as a result of rare intercontinental transmission events. We show that the …

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

Nosocomial acquisition and transmission of vancomycin-resistant Enterococcus faecium (VREfm) is the driver for E. faecium carriage in hospitalized patients, which, in turn, is a risk factor for invasive infection in immunocompromised patients. In the present study, we provide a comprehensive picture of E. faecium transmission in an entire sampled patient population using a sequence-driven approach. We prospectively identified and followed 149 haematology patients admitted to a hospital in England for 6 months. Patient stools (n = 376) and environmental swabs (n = 922) were taken at intervals and cultured for E. faecium. We sequenced 1,560 isolates (1,001 stool, 559 environment) and focused our genomic analyses on 1,477 isolates (95%) in the hospital-adapted clade A1. Of 101 patients who provided two or more stool samples, 40 (40%) developed E. faecium carriage after admission based on culture …

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi , establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such ‘carrier’ animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing …

Bacterial coordination of stress resistance mechanisms in harsh environments is key to long-term survival and evolutionary success. In many Gram-negative pathogens, both general-and specific-stress response are controlled by alternative sigma factors such as RpoS. The critically important pathogen Acinetobacter baumannii is notoriously recalcitrant to external stressors, yet it lacks RpoS, so the molecular control of its resilience remains unclear. Here, we used transposon insertion sequencing to characterize the molecular responses of Acinetobacter baumannii to two biologically-important metals stressors, zinc and copper, and discovered that the transcriptional regulator DksA acts as a major regulatory stress-protection switch. We mapped the highly pleiotropic nature of DksA using transcriptomics and phenomics and found that it controls ribosomal protein expression, metabolism of gluconeogenic substrates and survival in stresses that cause oxidative damage. A. baumannii strains lacking DksA were no longer virulent in both murine and Galleria mellonella in vivo models. In vitro, DksA mutants exhibited increased sensitivity to human serum and antibiotics yet promoted biofilm and capsule formation. Our study provides detailed insight into the unique role that DksA plays in stress protection and virulence for A. baumannii and lays the groundwork for understanding of RpoS-independent regulatory general stress response.

Background: Critical illness frequently requires the use of broad-spectrum antimicrobials to treat life-threatening infection. The resulting impact on microbiome diversity is profound, influencing gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary bile acids to secondary bile acids. We previously observed reduced fermentation capacity in the gut microbiota of critically ill children upon hospital admission, but the functional recovery trajectory of the paediatric gut microbiome during critical illness has not been well defined. Here, we longitudinally studied the intestinal microbiome and faecal bile acid profile of critically ill children during hospitalisation in a paediatric intensive care unit (PICU). The composition of the microbiome was determined by sequencing of the 16s rRNA gene, and bile acids were measured from faecal water by liquid chromatography hyphenated to mass spectrometry.Results: In comparison to admission faecal samples, members of Clostridium cluster XIVa and Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than control microbiota and patients with admitting diagnoses. The proportion of Recovery Associated Bacteria (RAB) was observed to decline with the length of PICU admission. Additionally, the proportions of RAB were reduced in those with systemic infection, respiratory failure, and undergoing surgery. Notably, Clostridioides were positively associated with the secondary bile acid deoxycholic acid, which we hypothesised to …

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion–site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin–immunity pairs, including Tse8–Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the …

Competitive interactions between pathogen strains drive infection risk. Vaccines are thought to perturb strain diversity through shifts in immune pressures, however, this has rarely been measured due to inadequate data and analytical tools. Bordetella pertussis (B. pertussis), responsible for 160,000 deaths annually, provides a rare natural experiment as many countries have switched from whole cell vaccines to acellular vaccines, which have very different immunogenic properties,. Here we use 3,344 sequences from 23 countries and build phylogenetic models to reveal that B. pertussis has substantial diversity within communities, with the relative fitness of local genotypes changing in response to switches in vaccine policy. We demonstrate that the number of transmission chains circulating within subnational regions is strongly associated with host population size. It takes 5-10 years for individual lineages to be homogeneously distributed throughout Europe or the United States. Increased fitness of pertactin-deficient strains following implementation of acellular vaccines, but reduced fitness otherwise, can explain long-term genotype dynamics. These findings highlight the role of national vaccine policies in shifting local diversity of a pathogen that still poses a large burden on global public health.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin …

BackgroundVietnam has high rates of antimicrobial resistance (AMR) but limited genomic surveillance, impeding our ability to assess transmission dynamics. This study aimed to use whole genome ssequencing (WGS) to examine the transmission of key AMR pathogens in two intensive care units in Hanoi, Vietnam.MethodsA prospective surveillance study of all adults admitted to two intensive care units (ICUs) at the National Hospital for Tropical Diseases (NHTD) and Bach Mai Hospital (BMH) was conducted between June 2017 and January 2018. Clinical and environmental samples were cultured on selective media, characterised using MALDI TOF MS, and Illumina sequenced. Phylogenies based on the de novo assemblies (SPAdes) were constructed using Mafft (PARsnp), Gubbins and RAxML. Resistance genes were detected using Abricate against the NCBI database.Findings3,153 Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii isolates from 369 patients were analysed. Phylogenetic analysis revealed predominant lineages within A. baumannii (global clone [GC]2, sequence types [ST]2, ST571) and K. pneumoniae (ST15, ST16, ST656, ST11, ST147) isolates. Colonisation was most common with E. coli (88.9%) followed by K. pneumoniae (62.4%). 91% of E. coli carried a blaCTX-M variant, while 81% of K. pneumoniae isolates carried blaNDM (54%) and/or blaKPC (45%). Transmission analysis using single nucleotide polymorphisms (SNPs) identified 167 clusters involving 251 (68%) patients, in some cases involving patients from both ICUs. There were no significant differences between the lineages or AMR genes …