A population phylogenomic analysis of the origin and spread of Escherichia coli sequence type 131 (ST131)

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally due to their increasing resistance to standard antibiotics. This results in the use of broader-spectrum drugs, prolonged patient ill-health and more nosocomial infections. E. coli sequence type 131 (ST131) is the predominant ExPEC clone worldwide. The antimicrobial resistance (AMR) gene repertoire of ST131 is evolving rapidly due to the widespread use of β-lactam (bla) antibiotics. Here, we performed a genomic investigation of an ST131 outbreak in a long-term care facility (LTCF) to describe transmission, within-host clonal diversity, genetic diversity of antibiotic resistance and the evolution of ST131 in the LTCF over a seven-year period. We analyzed the population structure and inferred the genealogical history of the LTCF isolates in the context of local hospital and global collections of ST131 to elucidate the epidemiology of ST131. We confirmed our initial hypotheses by reconstructing the evolutionary history of a much larger population consisting of >4000 global ST131 genomes This provided a deeper resolution of their evolutionary trajectories and the adaptive mechanisms of AMR driven by their ESBL genes, particularly cefotaximase (blaCTX). We further investigated the intersection of the AMR genes (AMRGs) found in ST131 with that of the human microbiome to understand the extent of their loss, gain and spread across different bacterial species. Across all strains, a large number of ST131’s AMRGs were found in a total of 794 genes in the human microbiome. Various gene families were represented, including transporters, transcription factors, β-lactamases and cell wall biosynthesis enzymes. To establish the main culprit for the dynamic nature of the blaCTX-M genes, we performed long read sequencing using a GridION X5 instrument. Analysis of long read-only assemblies revealed a clear and robust result on the genetic flanking context of blaCTX-M genes in both plasmid and chromosomes. Overall, our findings underpin the tremendous potential power for improving our current treatment of bacterial infections using high-throughput analysis of whole genome sequence data

An Escherichia coli ST131 pangenome atlas reveals population structure and evolution across 4,071 isolates

This work was funded by a DCU O’Hare Ph.D. fellowship and a DCU Enhancing Performance grant.Escherichia coli ST131 is a major cause of infection with extensive antimicrobial resistance (AMR) facilitated by widespread beta-lactam antibiotic use. This drug pressure has driven extended-spectrum beta-lactamase (ESBL) gene acquisition and evolution in pathogens, so a clearer resolution of ST131’s origin, adaptation and spread is essential. E. coli ST131’s ESBL genes are typically embedded in mobile genetic elements (MGEs) that aid transfer to new plasmid or chromosomal locations, which are mobilised further by plasmid conjugation and recombination, resulting in a flexible ESBL, MGE and plasmid composition with a conserved core genome. We used population genomics to trace the evolution of AMR in ST131 more precisely by extracting all available high-quality Illumina HiSeq read libraries to investigate 4,071 globally-sourced genomes, the largest ST131 collection examined so far. We applied rigorous quality-control, genome de novo assembly and ESBL gene screening to resolve ST131’s population structure across three genetically distinct Clades (A, B, C) and abundant subclades from the dominant Clade C. We reconstructed their evolutionary relationships across the core and accessory genomes using published reference genomes, long read assemblies and k-mer-based methods to contextualise pangenome diversity. The three main C subclades have co-circulated globally at relatively stable frequencies over time, suggesting attaining an equilibrium after their origin and initial rapid spread. This contrasted with their ESBL genes, which had stronger patterns across time, geography and subclade, and were located at distinct locations across the chromosomes and plasmids between isolates. Within the three C subclades, the core and accessory genome diversity levels were not correlated due to plasmid and MGE activity, unlike patterns between the three main clades, A, B and C. This population genomic study highlights the dynamic nature of the accessory genomes in ST131, suggesting that surveillance should anticipate genetically variable outbreaks with broader antibiotic resistance levels. Our findings emphasise the potential of evolutionary pangenomics to improve our understanding of AMR gene transfer, adaptation and transmission to discover accessory genome changes linked to novel subtypes.Publisher PDFPeer reviewe

Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts.

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays, and higher health care costs. E. coli sequence type 131 (ST131) is a major ExPEC clonal group worldwide, with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes blaCTX-M-14, blaCTX-M-15, and blaCTX-M-27, which are often rearranged, amplified, and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding blaCTX-M genes to be fully determined. Here, we performed long-read sequencing to decipher the genome structures of six E. coli ST131 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to more fragmented assemblies created with short reads alone. The long-read assemblies highlighted diverse accessory genomes with blaCTX-M-15, blaCTX-M-14, and blaCTX-M-27 genes identified in three, one, and one isolates, respectively. One sample had no blaCTX-M gene. Two samples had chromosomal blaCTX-M-14 and blaCTX-M-15 genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: ISEcp1, IS903B, and Tn2 This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts, even between closely related isolates within a clonal group such as E. coli ST131.IMPORTANCE Drug-resistant bacteria are a major cause of illness worldwide, and a specific subtype called Escherichia coli ST131 causes a significant number of these infections. ST131 bacteria become resistant to treatments by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to that of an existing approach. A combination of methods shows that drug resistance genes on plasmids are highly mobile because they can jump into ST131's chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes

Molecular characterizations of the coagulase-negative staphylococci species causing urinary tract infection in Tanzania : a laboratory-based cross-sectional study

Funding: This study is part of the Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) project funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care, Award (MR/S004785/1).Background: There is a growing body of evidence on the potential involvement of coagulase-negative Staphylococci (CoNS) in causing urinary tract infections (UTIs). The aim of this study was to delineate virulence potential, antimicrobial resistance genes, and sequence types of CoNS isolated from patients with UTI symptoms and pyuria in Tanzania. Methods: CoNS from patients with UTI symptoms and more than 125 leucocytes/μL were retrieved, subcultured, and whole-genome sequenced. Results: Out of 65 CoNS isolates, 8 species of CoNS were identified; Staphylococcus haemolyticus, n = 27 (41.5%), and Staphylococcus epidermidis, n = 24 (36.9%), were predominant. The majority of S. haemolyticus were sequence type (ST) 30, with 8 new ST138-145 reported, while the majority of S. epidermidis were typed as ST490 with 7 new ST1184-1190 reported. Sixty isolates (92.3%) had either one or multiple antimicrobial resistance genes. The most frequently detected resistance genes were 53 (21%) dfrG, 32 (12.9%) blaZ, and 26 (10.5%) mecA genes conferring resistance to trimethoprim, penicillin, and methicillin, respectively. Out of 65 isolates, 59 (90.8%) had virulence genes associated with UTI, with a predominance of the icaC 47 (46.5%) and icaA 14 (13.9%) genes. Conclusion: S. haemolyticus and S. epidermidis harboring icaC, dfrG, blaZ, and mecA genes were the predominant CoNS causing UTI in Tanzania. Laboratories should carefully interpret the significant bacteriuria due to CoNS in relation to UTI symptoms and pyuria before labeling them as contaminants. Follow-up studies to document the outcome of the treated patients is needed to add more evidence that CoNS are UTI pathogens.Publisher PDFPeer reviewe

A population phylogenomic analysis of the origin and spread of Escherichia coli sequence type 131 (ST131)

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally due to their increasing resistance to standard antibiotics. This results in the use of broader-spectrum drugs, prolonged patient ill-health and more nosocomial infections. E. coli sequence type 131 (ST131) is the predominant ExPEC clone worldwide. The antimicrobial resistance (AMR) gene repertoire of ST131 is evolving rapidly due to the widespread use of β-lactam (bla) antibiotics. Here, we performed a genomic investigation of an ST131 outbreak in a long-term care facility (LTCF) to describe transmission, within-host clonal diversity, genetic diversity of antibiotic resistance and the evolution of ST131 in the LTCF over a seven-year period. We analyzed the population structure and inferred the genealogical history of the LTCF isolates in the context of local hospital and global collections of ST131 to elucidate the epidemiology of ST131. We confirmed our initial hypotheses by reconstructing the evolutionary history of a much larger population consisting of >4000 global ST131 genomes This provided a deeper resolution of their evolutionary trajectories and the adaptive mechanisms of AMR driven by their ESBL genes, particularly cefotaximase (blaCTX). We further investigated the intersection of the AMR genes (AMRGs) found in ST131 with that of the human microbiome to understand the extent of their loss, gain and spread across different bacterial species. Across all strains, a large number of ST131’s AMRGs were found in a total of 794 genes in the human microbiome. Various gene families were represented, including transporters, transcription factors, β-lactamases and cell wall biosynthesis enzymes. To establish the main culprit for the dynamic nature of the blaCTX-M genes, we performed long read sequencing using a GridION X5 instrument. Analysis of long read-only assemblies revealed a clear and robust result on the genetic flanking context of blaCTX-M genes in both plasmid and chromosomes. Overall, our findings underpin the tremendous potential power for improving our current treatment of bacterial infections using high-throughput analysis of whole genome sequence data

An <i>Escherichia coli </i>ST131 pangenome atlas reveals population structure and evolution across 4,071 isolates

Escherichia coli ST131 is a major cause of infection with extensive antimicrobial resistance (AMR) facilitated by widespread beta-lactam antibiotic use. This drug pressure has driven extended-spectrum beta-lactamase (ESBL) gene acquisition and evolution in pathogens, so a clearer resolution of ST131’s origin, adaptation and spread is essential. E. coli ST131’s ESBL genes are typically embedded in mobile genetic elements (MGEs) that aid transfer to new plasmid or chromosomal locations, which are mobilised further by plasmid conjugation and recombination, resulting in a flexible ESBL, MGE and plasmid composition with a conserved core genome. We used population genomics to trace the evolution of AMR in ST131 more precisely by extracting all available high-quality Illumina HiSeq read libraries to investigate 4,071 globally-sourced genomes, the largest ST131 collection examined so far. We applied rigorous quality-control, genome de novo assembly and ESBL gene screening to resolve ST131’s population structure across three genetically distinct Clades (A, B, C) and abundant subclades from the dominant Clade C. We reconstructed their evolutionary relationships across the core and accessory genomes using published reference genomes, long read assemblies and k-mer-based methods to contextualise pangenome diversity. The three main C subclades have co-circulated globally at relatively stable frequencies over time, suggesting attaining an equilibrium after their origin and initial rapid spread. This contrasted with their ESBL genes, which had stronger patterns across time, geography and subclade, and were located at distinct locations across the chromosomes and plasmids between isolates. Within the three C subclades, the core and accessory genome diversity levels were not correlated due to plasmid and MGE activity, unlike patterns between the three main clades, A, B and C. This population genomic study highlights the dynamic nature of the accessory genomes in ST131, suggesting that surveillance should anticipate genetically variable outbreaks with broader antibiotic resistance levels. Our findings emphasise the potential of evolutionary pangenomics to improve our understanding of AMR gene transfer, adaptation and transmission to discover accessory genome changes linked to novel subtypes

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones.

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum β-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short- and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that Clade C was the most prevalent (686 out of 794 isolates, 86 %) of the three major ST131 clades circulating worldwide (A with fimH41, B with fimH22, C with fimH30), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in c. 1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1-mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10), highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments

Molecular characterizations of the coagulase-negative <i>staphylococci </i>species causing urinary tract infection in Tanzania:a laboratory-based cross-sectional study

Background: There is a growing body of evidence on the potential involvement of coagulase-negative Staphylococci (CoNS) in causing urinary tract infections (UTIs). The aim of this study was to delineate virulence potential, antimicrobial resistance genes, and sequence types of CoNS isolated from patients with UTI symptoms and pyuria in Tanzania. Methods: CoNS from patients with UTI symptoms and more than 125 leucocytes/μL were retrieved, subcultured, and whole-genome sequenced. Results: Out of 65 CoNS isolates, 8 species of CoNS were identified; Staphylococcus haemolyticus, n = 27 (41.5%), and Staphylococcus epidermidis, n = 24 (36.9%), were predominant. The majority of S. haemolyticus were sequence type (ST) 30, with 8 new ST138-145 reported, while the majority of S. epidermidis were typed as ST490 with 7 new ST1184-1190 reported. Sixty isolates (92.3%) had either one or multiple antimicrobial resistance genes. The most frequently detected resistance genes were 53 (21%) dfrG, 32 (12.9%) blaZ, and 26 (10.5%) mecA genes conferring resistance to trimethoprim, penicillin, and methicillin, respectively. Out of 65 isolates, 59 (90.8%) had virulence genes associated with UTI, with a predominance of the icaC 47 (46.5%) and icaA 14 (13.9%) genes. Conclusion: S. haemolyticus and S. epidermidis harboring icaC, dfrG, blaZ, and mecA genes were the predominant CoNS causing UTI in Tanzania. Laboratories should carefully interpret the significant bacteriuria due to CoNS in relation to UTI symptoms and pyuria before labeling them as contaminants. Follow-up studies to document the outcome of the treated patients is needed to add more evidence that CoNS are UTI pathogens

Protocol for an interdisciplinary cross-sectional study investigating the social, biological and community-level drivers of antimicrobial resistance (AMR) : Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA)

The Holistic Approach to Unravel Antibacterial Resistance in East Africa is a 3-year Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care. The award is also part of the EDCTP2 programme supported by the European Union. This work is supported in part by the Makerere University-Uganda Virus Research Institute Centre of Excellence for Infection and Immunity Research and Training (MUII). MUII is supported through the DELTAS Africa Initiative (grant number 107743). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences and Alliance for Accelerating Excellence in Science in Africa, and is supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust (grant number 107743) and the UK Government. This paper was funded in part by a grant from the National Institutes of Health (grant number U01CA207167).Introduction Antimicrobial resistance (AMR) is a global health threat that requires urgent research using a multidisciplinary approach. The biological drivers of AMR are well understood, but factors related to treatment seeking and the social contexts of antibiotic (AB) use behaviours are less understood. Here we describe the Holistic Approach to Unravel Antibacterial Resistance in East Africa, a multicentre consortium that investigates the diverse drivers of drug resistance in urinary tract infections (UTIs) in East Africa. Methods and analysis This study will take place in Uganda, Kenya and Tanzania. We will conduct geospatial mapping of AB sellers, and conduct mystery client studies and in-depth interviews (IDIs) with drug sellers to investigate AB provision practices. In parallel, we will conduct IDIs with doctors, alongside community focus groups. Clinically diagnosed patients with UTI will be recruited from healthcare centres, provide urine samples and complete a questionnaire capturing retrospective treatment pathways, sociodemographic characteristics, attitudes and knowledge. Bacterial isolates from urine and stool samples will be subject to culture and antibiotic sensitivity testing. Genomic DNA from bacterial isolates will be extracted with a subset being sequenced. A follow-up household interview will be conducted with 1800 UTI-positive patients, where further environmental samples will be collected. A subsample of patients will be interviewed using qualitative tools. Questionnaire data, microbiological analysis and qualitative data will be linked at the individual level. Quantitative data will be analysed using statistical modelling, including Bayesian network analysis, and all forms of qualitative data analysed through iterative thematic content analysis.Publisher PDFPeer reviewe

Unravelling patient pathways in the context of antibacterial resistance in East Africa

BACKGROUND A key factor driving the development and maintenance of antibacterial resistance (ABR) is individuals' use of antibiotics (ABs) to treat illness. To better understand motivations and context for antibiotic use we use the concept of a patient treatment-seeking pathway: a treatment journey encompassing where patients go when they are unwell, what motivates their choices, and how they obtain antibiotics. This paper investigates patterns and determinants of patient treatment-seeking pathways, and how they intersect with AB use in East Africa, a region where ABR-attributable deaths are exceptionally high.METHODS The Holistic Approach to Unravelling Antibacterial Resistance (HATUA) Consortium collected quantitative data from 6,827 adult outpatients presenting with urinary tract infection (UTI) symptoms in Kenya, Tanzania, and Uganda between February 2019- September 2020, and conducted qualitative in-depth patient interviews with a subset (n = 116). We described patterns of treatment-seeking visually using Sankey plots and explored explanations and motivations using mixed-methods. Using Bayesian hierarchical regression modelling, we investigated the associations between socio-demographic, economic, healthcare, and attitudinal factors and three factors related to ABR: self-treatment as a first step, having a multi-step treatment pathway, and consuming ABs.RESULTSAlthough most patients (86%) sought help from medical facilities in the first instance, many (56%) described multi-step, repetitive treatment-seeking pathways, which further increased the likelihood of consuming ABs. Higher socio-economic status patients were more likely to consume ABs and have multi-step pathways. Reasons for choosing providers (e.g., cost, location, time) were conditioned by wider structural factors such as hybrid healthcare systems and AB availability.CONCLUSION There is likely to be a reinforcing cycle between complex, repetitive treatment pathways, AB consumption and ABR. A focus on individual antibiotic use as the key intervention point in this cycle ignores the contextual challenges patients face when treatment seeking, which include inadequate access to diagnostics, perceived inefficient public healthcare and ease of purchasing antibiotics without prescription. Pluralistic healthcare landscapes may promote more complex treatment seeking and therefore inappropriate AB use. We recommend further attention to healthcare system factors, focussing on medical facilities (e.g., accessible diagnostics, patient-doctor interactions, information flows), and community AB access points (e.g., drug sellers).</p