Biology subjects

Holt, K. E.

Publications and source records attributed to Holt, K. E..

At least 73 records · Page 4Linked to original sources

bioRxiv2019

Population structure and antimicrobial resistance patterns of Salmonella Typhi isolates in Bangladesh from 2004 to 2016

BackgroundMulti-drug resistant typhoid fever remains an enormous public health threat in low and middle-income countries. However, we still lack a detailed understanding of the epidemiology and genomics of S. Typhi in many regions. Here we have undertaken a detailed genomic analysis of typhoid in Bangladesh to unravel the population structure and antimicrobial resistance patterns in S. Typhi isolated in between 2004-2016.\n\nPrincipal findingsWhole genome sequencing of 202 S. Typhi isolates obtained from three study locations in urban Dhaka revealed a diverse range of S. Typhi genotypes and AMR profiles. The bacterial population within Dhaka were relatively homogenous with little stratification between different healthcare facilities or age groups. We also observed evidence of transmission of Bangladeshi genotypes with neighboring South Asian countries (India, Pakistan and Nepal) suggesting these are circulating throughout the region. This analysis revealed a decline in H58 (genotype 4.3.1) isolates from 2011 onwards, coinciding with a rise in a diverse range of non-H58 genotypes and a simultaneous rise in isolates with reduced susceptibility to fluoroquinolones, potentially reflecting a change in treatment practices. We identified a novel S. Typhi genotype, subclade 3.3.2 (previously defined only to clade level, 3.3), which formed two localised clusters (3.3.2.Bd1 and 3.3.2.Bd2) associated with different mutations in the Quinolone Resistance Determining Region (QRDR) of gene gyrA.\n\nSignificanceOur analysis of S. Typhi isolates from Bangladesh isolated over a twelve year period identified a diverse range of AMR profiles and genotypes. The observed increase in non-H58 genotypes associated with reduced fluoroquinolone susceptibility may reflect a change in treatment practice in this region and highlights the importance of continued molecular surveillance to monitor the ongoing evolution of AMR in Bangladesh. We have defined new genotypes and lineages of Bangladeshi S. Typhi which will facilitate identification of these emerging AMR clones in future surveillance efforts.\n\nAuthor SummaryTyphoid fever, caused by Salmonella enterica serovar Typhi, is an acute and often life-threatening febrile illness in developing countries. Until recently, there have been limited studies focusing on the epidemiology and disease burden of typhoid in poor resource settings including Bangladesh. This study highlights the urgent need for sustained genomics based surveillance studies to monitor the population structure and ongoing evolution of AMR. Our data revealed a diverse range of S. Typhi genotypes and AMR patterns among 202 isolates collected from three urban areas in Dhaka. Moreover, we defined a new genotype, subclade 3.3.2 (previously typed only to clade level, 3.3) with two Bangladesh-localiased clades 3.3.2.Bd1 and 3.3.2.Bd2 showing reduced susceptibility to fluoroquinolones. Our data shows a significant increase in non-H58 genotypes carrying QRDR mutations from 2012 onwards, replacing MDR H58 genotypes. Our data suggest that a shift in treatment practice towards third generation cephalosporins to control typhoid might be beneficial, in addition to the introduction of vaccination programs and improvements in water sanitation and hygiene (WASH) in Bangladesh.

genomics↗

bioRxiv2019

Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from south and southeast Asia.

BackgroundK. pneumoniae is a leading cause of blood stream infection (BSI). Strains producing extended spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial resistant (AMR) K. pneumoniae, and also for the characteristically antimicrobial sensitive, community-acquired hypervirulent strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. MethodsWe conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate. FindingsK. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common amongst South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes amongst K. pneumoniae. InterpretationSouth and Southeast Asia are high-risk regions for the emergence of AMR and convergent AMR-hypervirulent K. pneumoniae. Enhanced surveillance efforts, reporting STs, AMR and virulence information are urgently required to monitor this public health threat. FundingThis work was supported by the Wellcome Trust (grant #206194 to Wellcome Sanger Institute) and the Bill and Melinda Gates Foundation, Seattle (grant OPP1175797 to KEH). KEH is supported by a Senior Medical Research Fellowship from the Viertel Foundation of Australia. DAB and PNN are supported by the Wellcome Trust.

microbiology↗

bioRxiv2019

hicap: in silico serotyping of the Haemophilus influenzae capsule locus

Haemophilus influenzae exclusively colonises the human nasopharynx and can cause a variety of respiratory infections as well as invasive diseases including meningitis and sepsis. A key virulence determinant of H. influenzae is the polysaccharide capsule of which six serotypes are known, each encoded by a distinct variation of the capsule biosynthesis locus (cap-a to cap-f). H. influenzae type b (Hib) was historically responsible for the majority of invasive H. influenzae disease and prevalence has been markedly reduced in countries that have implemented vaccination programs targeting this serotype. In the postvaccine era, non-typeable H. influenzae emerged as the most dominant group causing disease but in recent years a resurgence of encapsulated H. influenzae strains has also been observed, most notably serotype a. Given the increasing incidence of encapsulated strains and the high frequency of Hib in countries without vaccination programs, there is growing interest in genomic epidemiology of H. influenzae. Here we present hicap, a software tool for rapid in silico serotype prediction from H. influenzae genome sequences. hicap is written using Python3 and is freely available at github.com/scwatts/hicap under a GPLv3 license. To demonstrate the utility of hicap, we used it to investigate the cap locus diversity and distribution in 691 high-quality H. influenzae genomes from GenBank. These analyses identified cap loci in 95 genomes and confirmed the general association of each serotype with a unique clonal lineage and also identified occasional recombination between lineages giving rise to hybrid cap loci (2% of encapsulated strains).

bioinformatics↗

bioRxiv2019

Performance of neural network basecalling tools for Oxford Nanopore sequencing

Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rules consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly via additional signal-level analysis with Nanopolish. Training basecallers on taxon-specific data resulted in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network was able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences ( polishing) with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy, so basecaller choice is still relevant even when Nanopolish is used.

bioinformatics↗

bioRxiv2019

Global phylogenomics of multidrug resistant Salmonella enterica serotype Kentucky ST198

Salmonella enterica serotype Kentucky (S. Kentucky) can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S. Kentucky ST198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug resistant (MDR) S. Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island 1 (variant SGI1-K) conferring resistance ampicillin, streptomycin, gentamicin, sulfamethoxazole, and tetracycline. Phylogeographic analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone resistance determining regions (QRDR) of DNA gyrase (gyrA) and DNA topoisomerase (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems, and/or azithromycin. These results indicate that most MDR S. Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials.

microbiology↗

bioRxiv2019

Informal genomic surveillance of regional distribution of Salmonella Typhi genotypes and antimicrobial resistance via returning travellers

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a systemic human infection with a burden exceeding 20 million cases each year that occur disproportionately among children in low and middle income countries. Antimicrobial therapy is the mainstay for treatment, but resistance to multiple agents is common. Here we report genotypes and antimicrobial resistance (AMR) determinants detected from routine whole-genome sequencing (WGS) of 533 S. Typhi isolates referred to Public Health England between April 2014 and April 2017, 488 (92%) of which had accompanying patient travel information obtained via an enhanced surveillance questionnaire. The majority of cases involved S. Typhi 4.3.1 (H58) linked with travel to South Asia (58%). Travel to East and West Africa were associated with genotypes 4.3.1 and 3.3.1, respectively. Point mutations in the quinolone resistance determining region (QRDR), associated with reduced susceptibility to fluoroquinolones, were very common (85% of all cases) but the frequency varied significantly by region of travel: 95% in South Asia, 43% in East Africa, 27% in West Africa. QRDR triple mutants, resistant to ciprofloxacin, were restricted to 4.3.1 lineage II and associated with travel to India, accounting for 23% of cases reporting travel to the country. Overall 24% of isolates were MDR, however the frequency varied significantly by region and country of travel: 27% in West Africa, 52% in East Africa, 55% in Pakistan, 24% in Bangladesh, 3% in India. MDR determinants were plasmid-borne (IncHI1 PST2 plasmids) in S. Typhi 3.1.1 linked to West Africa, but in all other regions MDR was chromosomally integrated in 4.3.1 lineage I. We propose that routine WGS data from travel-associated cases in industrialised countries could serve as informal sentinel AMR genomic surveillance data for countries where WGS is not available or routinely performed.\n\nAuthor SummaryOur data demonstrate how routine WGS data produced by Public Health England can be further mined for informal passive surveillance of Salmonella Typhi circulating in different geographical regions where typhoid is endemic. We have shown the public health utility of a simplified approach to WGS reporting based on the GenoTyphi genotyping framework and nomenclature, which doesnt require the generation of a phylogenetic tree or other phylogenetic analysis. These approaches yielded results consistent with previously reported antimicrobial resistance (AMR) patterns of S. Typhi, including prevalence of multi-drug resistant (MDR) and fluoroquinolone resistance in different regions in association with different pathogen variants. These data provide a rationale and framework for the extraction and reporting of geographically stratified genotype and AMR data from public health labs in non-endemic countries. Prospective analysis and reporting of such data could potentially detect shifts in regional S. Typhi populations, such as replacement or spread of different subclades and the emergence and dissemination of MDR, fluoroquinolone resistant and/or extensively drug resistant S. Typhi, providing valuable data to inform typhoid control measures in low and middle income countries that are still building their genomics capacity.

microbiology↗

bioRxiv2018

Convergence of virulence and multidrug resistance in a single plasmid vector in multidrug-resistant Klebsiella pneumoniae ST15

SYNOPSISO_ST_ABSBackgroundC_ST_ABSMultidrug resistance (MDR) and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options.\n\nObjectivesTo characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15.\n\nMethodsThe complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing.\n\nResultsBoth isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and eight other AMR genes (blaTEM, aac3-IIa, aph3-Ia, dfrA1, satA2, blaSHV, sul1, aadA1). The other carried remnants of these elements encoding blaTEM and aac3-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania.\n\nConclusionsThe presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.

genomics↗

bioRxiv2018

Yersiniabactin, Colibactin and Wider Resistome Contribute to Enhanced Virulence and Persistence of KPC-2-Producing Klebsiella pneumoniae CG258 in South America

The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.

microbiology↗

bioRxiv2018

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) has emerged as an important cause of two distinct public health threats: multidrug resistant (MDR) healthcare-associated infections1 and community-acquired invasive infections, particularly pyogenic liver abscess2. The majority of MDR hospital outbreaks are caused by a subset of Kp clones with a high prevalence of acquired antimicrobial resistance (AMR) genes, while the majority of community-acquired invasive infections are caused by hypervirulent clones that rarely harbour acquired AMR genes but have high prevalence of key virulence loci3-5. Worryingly, the last few years have seen increasing reports of convergence of MDR and the key virulence genes within individual Kp strains6, but it is not yet clear whether these represent a transient phenomenon or a significant ongoing threat. Here we perform comparative genomic analyses for 28 distinct Kp clones, including 6 hypervirulent and 8 MDR, to better understand their evolutionary histories and the risks of convergence. We show that MDR clones are highly diverse with frequent chromosomal recombination and gene content variability that far exceeds that of the hypervirulent clones. Consequently, we predict a much greater risk of virulence gene acquisition by MDR Kp clones than of resistance gene acquisition by hypervirulent clones.

microbiology↗

bioRxiv2018

Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulent K. pneumoniae clones that cause severe community-associated infections such as liver abscess and pneumonia. Concerningly iuc has also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in the K. pneumoniae population.\n\nMethodsHere we examined the genetic diversity, distribution and mobilisation of iuc and iro loci among 2503 K. pneumoniae genomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance.\n\nResultsIro and iuc were detected at low prevalence (<10%). Considerable genetic diversity was observed, resolving into five iro and six iuc lineages that show distinct patterns of mobilisation and dissemination in the K. pneumoniae population. The major burden of iuc and iro amongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% and iuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBK virulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids of K. pneumoniae. The other iuc and iro lineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported from E. coli; the exceptions were iro3(mobilised by ICEKp1), and iuc4 (fixed in the chromosome of K. pneumoniae subspecies rhinoscleromatis). Iro/iuc MGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258.\n\nConclusionsIuc and iro are mobilised in K. pneumoniae via a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K. pneumoniae strains.

genomics↗

bioRxiv2018

Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks

Multiplexing, the simultaneous sequencing of multiple barcoded DNA samples on a single flow cell, has made Oxford Nanopore sequencing cost-effective for small genomes. However, it depends on the ability to sort the resulting sequencing reads by barcode, and current demultiplexing tools fail to classify many reads. Here we present Deepbinner, a tool for Oxford Nanopore demultiplexing that uses a deep neural network to classify reads based on the raw electrical read signal. This signal-space approach allows for greater accuracy than existing base-space tools (Albacore and Porechop) for which signals must first be converted to DNA base calls, itself a complex problem that can introduce noise into the barcode sequence. To assess Deepbinner and existing tools, we performed multiplex sequencing on 12 amplicons chosen for their distinguishability. This allowed us to establish a ground truth classification for each read based on internal sequence alone. Deepbinner had the lowest rate of unclassified reads (7.8%) and the highest demultiplexing precision (98.5% of classified reads were correctly assigned). It can be used alone (to maximise the number of classified reads) or in conjunction with other demultiplexers (to maximise precision and minimise false positive classifications). We also found cross-sample chimeric reads (0.3%) and evidence of barcode switching (0.3%) in our dataset, which likely arise during library preparation and may be detrimental for quantitative studies that use multiplexing. Deepbinner is open source (GPLv3) and available at https://github.com/rrwick/Deepbinner.

bioinformatics↗

bioRxiv2018

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307

Recent reports indicate the emergence of a new carbapenemase producing Klebsiella pneumoniae clone, ST307. Here we show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 extended-spectrum beta-lactamase (ESBL) gene plus other antimicrobial resistance determinants. Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance is now emerging.

microbiology↗

bioRxiv2018

FastSpar: Rapid and scalable correlation estimation for compositional data

A common goal of microbiome studies is the elucidation of community composition and member interactions using counts of taxonomic units extracted from sequence data. Inference of interaction networks from sparse and compositional data requires specialised statistical approaches. A popular solution is SparCC, however its performance limits the calculation of interaction networks for very high-dimensional datasets. Here we introduce FastSpar, an efficient and parallelisable implementation of the SparCC algorithm which rapidly infers correlation networks and calculates p-values using an unbiased estimator. We further demonstrate that FastSpar reduces network inference wall time by 2-3 orders of magnitude compared to SparCC. FastSpar source code, precompiled binaries, and platform packages are freely available on GitHub: github.com/scwatts/FastSpar

bioinformatics↗

bioRxiv2018

Kaptive Web: user-friendly capsule and lipopolysaccharide serotype prediction for Klebsiella genomes

As whole genome sequencing becomes an established component of the microbiologists toolbox, it is imperative that researchers, clinical microbiologists and public health professionals have access to genomic analysis tools for rapid extraction of epidemiologically and clinically relevant information. For the gram-negative hospital pathogens such as Klebsiella pneumoniae, initial efforts have focused on detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting Klebsiella surface polysaccharides, the ability to extract information about these antigens is increasingly important.\n\nHere we present Kaptive Web, an online tool for rapid typing of Klebsiella K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyse genome assemblies in a web browser. Results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise.\n\nWe demonstrate Kaptive Webs utility by analysis of >500 K. pneumoniae genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase- associated clonal group 258 (25 K and six O loci). Characterisation of a further 309 genomes indicates that such diversity is common among the multi-drug resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable and accessible typing methods such as Kaptive Web.\n\nKaptive Web is available for use at kaptive.holtlab.net and source code is available at github.com/kelwyres/Kaptive-Web.

microbiology↗

bioRxiv2017

Non-parametric mixture models identify trajectories of childhood immune development relevant to asthma and allergy

Events in early life contribute to subsequent risk of asthma; however, the causes and trajectories of childhood wheeze are heterogeneous and do not always result in asthma. Similarly, not all atopic individuals develop wheeze, and vice versa. The reasons for these differences are unclear. Using unsupervised model-based cluster analysis, we identified latent clusters within a prospective birth cohort with deep immunological and respiratory phenotyping. We characterised each cluster in terms of immunological profile and disease risk, and replicated our results in external cohorts from the UK and USA. We discovered three distinct trajectories, one of which is a high-risk \"atopic\" cluster with increased propensity for allergic diseases throughout childhood. Atopy contributes varyingly to later wheeze depending on cluster membership. Our findings demonstrate the utility of unsupervised analysis in elucidating heterogeneity in asthma pathogenesis and provide a foundation for improving management and prevention of childhood asthma.

systems biology↗

bioRxiv2017

Drivers of antimicrobial resistance amongst intestinal Escherichia coli isolated from children in South Asia and sub-Saharan Africa

Antimicrobial resistance (AMR) dynamics are poorly understood in developing countries, where data on the prevalence of AMR in enteric bacteria are sparse, particularly among children and in the community setting. Here we use a combination of phenotyping, genomics and antimicrobial usage data to investigate patterns of AMR amongst atypical enteropathogenic E. coli (aEPEC) strains isolated from children <5 years old in seven countries (four in sub-Saharan Africa and three in South Asia) over a three-year period. We detected very high rates of AMR, with 65% of isolates displaying resistance to [≥]3 drug classes; the rates of AMR were the same amongst strains associated with diarrhea and strains that were carried asymptomatically. Whole genome sequencing identified a diversity of genetic mechanisms for AMR, which could explain >95% of observed phenotypic resistance. Analysis of AMR gene co-occurrence revealed clusters of acquired AMR genes that were frequently co-located on small plasmids and transposons, providing opportunities for acquisition of multidrug resistance in a single step. We used discriminant analysis to investigate potential drivers of AMR within the bacterial population, and found that genetic determinants of AMR were associated with geographical location of isolation but not with phylogenetic lineage of the E. coli strain or disease status of the human host. Comparison with antimicrobial usage data showed that the prevalence of resistance to newer drugs (fluoroquinolones and third-generation cephalosporins) was correlated with usage, which was generally higher in South Asia than Africa. In particular, fluoroquinolone resistance-associated mutations in gyrA were significantly associated with use of these drugs for treatment of diarrheic children. Notably resistance to older drugs such as trimethoprim, chloramphenicol and ampicillin, which are conferred by acquired AMR genes that were frequently clustered together in mobile genetic elements, were common in all locations despite differences in usage; this suggests that reversion to sensitivity is unlikely to occur even if these drugs are removed from circulation. This study provides much-needed insights into the frequencies of AMR in intestinal E. coli in community-based children in developing countries and to antimicrobial usage for diarrhea where the burden of infections is greatest.

microbiology↗

bioRxiv2017

Population genomics of hypervirulent Klebsiella pneumoniae clonal group 23 reveals early emergence and rapid global dissemination

Since the mid-1980s there have been increasing reports of severe community-acquired pyogenic liver abscess, meningitis and bloodstream infections caused by hypervirulent Klebsiella pneumoniae, predominantly encompassing clonal group (CG) 23 serotype K1 strains. Common features of CG23 include a virulence plasmid associated with iron scavenging and hypermucoidy, and a chromosomal integrative and conjugative element (ICE) encoding the siderophore yersiniabactin and the genotoxin colibactin. Here we investigate the evolutionary history and genomic diversity of CG23 based on comparative analysis of 98 genomes. Contrary to previous reports with more limited samples, we show that CG23 comprises several deep branching sublineages dating back to the 1870s, many of which are associated with distinct chromosomal insertions of ICEs encoding yersiniabactin. We find that most liver abscess isolates (>80%) belong to a dominant sublineage, CG23-I, which emerged in the 1920s following acquisition of ICEKp10 (encoding colibactin in addition to yersiniabactin) and has undergone clonal expansion and global dissemination within the human population. The unique genomic feature of CG23-I is the production of colibactin, which has been reported previously as a promoter of gut colonisation and dissemination to the liver and brain in a mouse model of CG23 K. pneumoniae infection, and has been linked to colorectal cancer. We also identify an antibiotic-resistant subclade of CG23-I associated with sexually-transmitted infections in horses dating back to the 1980s. These data show that hypervirulent CG23 K. pneumoniae was circulating in humans for decades before the liver abscess epidemic was first recognised, and has the capacity to acquire and maintain AMR plasmids. These data provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To further support such studies we present an open access and completely sequenced human liver abscess isolate, SGH10, which is typical of the globally disseminated CG23-I sublineage.

genomics↗

bioRxiv2017

Evolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection

Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem resistant A. baumannii is considered a major public health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45-day period via Illumina and Oxford Nanopore sequencing. All isolates were multi-drug resistant, carrying two genomic islands that harboured several antibiotic resistance genes. Most isolates were genetically identical and represent a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to [≥]8 g/mL; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 g/mL. Structural modelling of AdeB and FtsI showed that these mutations affected their drug binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacteria has not yet been exposed.\n\nDATA SUMMARYO_LIAll raw genome sequences, including Illumina paired end short reads and Oxford Nanopore long reads, have been deposited in the SRA under project PRJNA396979. Individual accessions for each strain are provided in Table S1.\nC_LIO_LIThe annotated genome assembly for strain A2, the reference genome for the founder genotype, has been submitted to GenBank under accession CP024124 (chromosome) and CP024125 (plasmid).\nC_LIO_LIHybrid assemblies for strains A1, A3, A8, A13, A15, A17 and A20 are available in FigShare, doi: 10.4225/49/5987e14e9b530 (note they were not deposited in GenBank as they differ from A2 by only 1-4 SNPs as indicated).\nC_LI\n\nIMPACT STATEMENTSAcinetobacter baumannii is a highly drug resistant pathogen that is frequently found within intensive care units (ICUs) and especially impacts patients with severe burns. While several studies have examined the global population structure of A. baumannii, few have investigated within-host evolution of A. baumannii in direct response to antibiotic treatment in a single patient. Here, we analysed the genetic evolution of A. baumannii isolated from a patient with severe burns over the course of their stay in ICU. The A. baumannii population on this patient was highly drug resistant, carrying two distinct genomic islands encoding resistance to several antibiotics but not carbapenems. The bacterial population comprised four distinct subclades, two of which had evolved carbapenem resistance over the course of antibiotic treatment through novel mutations in genes associated with drug binding. One subclade was also transmitted to another patient on the ward. While carbapenem resistance is common in A. baumannii, this is generally attributed to horizontally transferred carbapenemase genes. These data provide evidence for carbapenem resistance arising in vivo via non-synonymous substitutions during a single infection episode, demonstrating carbapenem resistance can emerge in genetic isolation in response to exposure to carbapenems and other drugs.

genomics↗