Long-Read-Sequenced Reference Genomes of the Seven Major Lineages of Enterotoxigenic Escherichia Coli (ETEC) Circulating in Modern Time

Abstract Abstract Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 fatalities per year, with the highest rates of burden, ca 75 million cases per year, amongst children under five years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades and that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

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Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time

Scientific Reports ◽

10.1038/s41598-021-88316-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Astrid von Mentzer ◽

Grace A. Blackwell ◽

Derek Pickard ◽

Christine J. Boinett ◽

Enrique Joffré ◽

...

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Bacterial Species ◽

Enterotoxigenic Escherichia Coli ◽

Poor Countries ◽

Depth Analysis ◽

Sequencing Studies ◽

Long Read ◽

To Come ◽

Reference Genomes

AbstractEnterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

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Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time

10.1101/2020.07.16.203430 ◽

2020 ◽

Author(s):

Astrid von Mentzer ◽

Grace Blackwell ◽

Derek Pickard ◽

Christine J. Boinett ◽

Enrique Joffré ◽

...

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Bacterial Species ◽

Enterotoxigenic Escherichia Coli ◽

Poor Countries ◽

Depth Analysis ◽

Sequencing Studies ◽

Long Read ◽

To Come ◽

Reference Genomes

AbstractBackgroundEnterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 fatalities per year, with the highest burden, ca 75 million cases per year, amongst children under five years of age in resource poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation world-wide.MethodsHere we have used PacBio long read sequencing in combination with Illumina sequencing to create high quality complete reference genomes for each of these lineages with manually curated chromosomes and plasmids. The plasmids carrying ETEC virulence genes were compared to other available long-read sequenced ETEC strains using blastn.ResultsThe ETEC reference strains harbour between two and five plasmids, including virulence, antibiotic resistance and phage-plasmids. The virulence plasmids carrying the colonization factors are highly conserved as shown by comparison with plasmids with other ETEC strains and confirm that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and superiority as pathogens.ConclusionWe confirm that the major ETEC lineages all harbor conserved plasmids that have been associated to their respective background genomes for decades. The in-depth analysis of gene content and order and correct annotations of plasmids will help to elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for rapid and accurate comparison between different ETEC strains and these data will form the foundation of ETEC genomics research for years to come.

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Comparison of different technologies for the decipherment of the whole genome sequence of Campylobacter jejuni BfR-CA-14430

Gut Pathogens ◽

10.1186/s13099-019-0340-7 ◽

2019 ◽

Vol 11 (1) ◽

Author(s):

Lennard Epping ◽

Julia C. Golz ◽

Marie-Theres Knüver ◽

Charlotte Huber ◽

Andrea Thürmer ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Genome Sequence ◽

Bacterial Species ◽

Illumina Miseq ◽

Chicken Meat ◽

Whole Genome ◽

Short Read ◽

Plasmid Sequence ◽

Depth Analysis ◽

Long Read

Abstract Background Campylobacter jejuni is a zoonotic pathogen that infects the human gut through the food chain mainly by consumption of undercooked chicken meat, raw chicken cross-contaminated ready-to-eat food or by raw milk. In the last decades, C. jejuni has increasingly become the most common bacterial cause for food-born infections in high income countries, costing public health systems billions of euros each year. Currently, different whole genome sequencing techniques such as short-read bridge amplification and long-read single molecule real-time sequencing techniques are applied for in-depth analysis of bacterial species, in particular, Illumina MiSeq, PacBio and MinION. Results In this study, we analyzed a recently isolated C. jejuni strain from chicken meat by short- and long-read data from Illumina, PacBio and MinION sequencing technologies. For comparability, this strain is used in the German PAC-CAMPY research consortium in several studies, including phenotypic analysis of biofilm formation, natural transformation and in vivo colonization models. The complete assembled genome sequence most likely consists of a chromosome of 1,645,980 bp covering 1665 coding sequences as well as a plasmid sequence with 41,772 bp that encodes for 46 genes. Multilocus sequence typing revealed that the strain belongs to the clonal complex CC-21 (ST-44) which is known to be involved in C. jejuni human infections, including outbreaks. Furthermore, we discovered resistance determinants and a point mutation in the DNA gyrase (gyrA) that render the bacterium resistant against ampicillin, tetracycline and (fluoro-)quinolones. Conclusion The comparison of Illumina MiSeq, PacBio and MinION sequencing and analyses with different assembly tools enabled us to reconstruct a complete chromosome as well as a circular plasmid sequence of the C. jejuni strain BfR-CA-14430. Illumina short-read sequencing in combination with either PacBio or MinION can substantially improve the quality of the complete chromosome and epichromosomal elements on the level of mismatches and insertions/deletions, depending on the assembly program used.

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A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization

Biochemical Journal ◽

10.1042/bj2010505 ◽

1982 ◽

Vol 201 (3) ◽

pp. 505-513 ◽

Cited By ~ 12

Author(s):

K Altmann ◽

N A Pyliotis ◽

T K S Mukkur

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Average Length ◽

Bacterial Species ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Enterotoxigenic Escherichia Coli ◽

Total Amino Acid ◽

Apparent Homogeneity

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN.

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Microbiome Profiling of Enterotoxigenic Escherichia Coli (ETEC) Carriers Highlights Signature Differences Between Symptomatic and Asymptomatic Individuals

10.21203/rs.3.rs-1030513/v1 ◽

2021 ◽

Author(s):

Ellen Elizabeth Higginson ◽

Md Abu Sayeed ◽

Joana Pereira Dias ◽

Vignesh Shetty ◽

Mamatha Ballal ◽

...

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Alpha Diversity ◽

Enterotoxigenic Escherichia Coli ◽

Healthy Children ◽

Middle Income ◽

E Coli ◽

Adults And Children ◽

Asymptomatic Individuals ◽

Pathogen Burden

Abstract BackgroundEscherichia coli (ETEC) are one of the top causes of diarrhoea in children in low- and middle-income countries (LMICs). However, large-scale pathogen burden studies in children have identified ETEC in the guts of symptomatic patients and controls. The factors that influence this balance between carriage and disease are poorly understood, but it is postulated that the gut microbiome may play a role in either resistance or progression to disease. In this study, we investigated the microbiome profiles, using shotgun DNA sequencing, of children and adults from Bangladesh who were asymptomatically or symptomatically infected with ETEC. ResultsSymptomatic patients had significantly higher numbers of sequenced reads mapping to both E. coli and the two ETEC toxins (LT and ST), suggesting higher bacterial burden. They were also significantly more likely to be co-infected with enteroaggregative E. coli (EAEC) and had higher proportions of other Gammaproteobacteria, including Klebsiella, Salmonella, and Haemophilus. Colonisation with ETEC (symptomatic or asymptomatic) was also associated with increased prevalence of antimicrobial resistance (AMR) genes, most notably those of the b-lactamase class. Taxonomic profiles were distinctly different between all groups in both species richness (alpha diversity) and composition (beta diversity), although the direction of these changes was different in adults and children. As seen in previous studies, children with high E. coli burdens also had higher proportions of Streptococcus spp., while healthy children were more heavily colonised by several Bifidobacterium spp. ConclusionsOur study provides insight into the microbiome changes that occur upon infection with ETEC in an endemic setting, and provides rationale for future studies investigating how the microbiome may protect or predispose individuals to symptomatic infections with gastrointestinal pathogens.

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Lineage-Specific Distribution of Insertion Sequence Excision Enhancer in Enterotoxigenic Escherichia coli Isolated from Swine

Applied and Environmental Microbiology ◽

10.1128/aem.03696-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1394-1402 ◽

Cited By ~ 7

Author(s):

Masahiro Kusumoto ◽

Dai Fukamizu ◽

Yoshitoshi Ogura ◽

Eiji Yoshida ◽

Fumiko Yamamoto ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Species ◽

Bacterial Genome ◽

Enterotoxigenic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Genomic Deletions ◽

Specific Distribution ◽

Multiple Copies ◽

Genomic Locations

ABSTRACTInsertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagicEscherichia coli(EHEC) O157. IEE promotes the excision of IS elements belonging to the IS3family, such as IS629, as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequencedE. colistrains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenicE. colistrains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenicE. coli(ETEC) strains of serotypes O139 or O149 isolated from swine. Theieegene is located within integrative elements that are similar to SpLE1 of EHEC O157. Alliee-positive ETEC lineages also contained multiple copies of IS629, a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS629is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE.

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What can we learn from over 100,000 Escherichia coli genomes?

10.1101/708131 ◽

2019 ◽

Cited By ~ 4

Author(s):

Kaleb Abram ◽

Zulema Udaondo ◽

Carissa Bleker ◽

Visanu Wanchai ◽

Trudy M. Wassenaar ◽

...

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Phylogenetic Analyses ◽

Bacterial Species ◽

Single Copy ◽

Gene Gain ◽

E Coli ◽

Phylogenetic Profiles ◽

Genomic Studies ◽

Gain Loss

ABSTRACTThe explosion of microbial genome sequences in public databases allows for large-scale population genomic studies of bacterial species, such as Escherichia coli. In this study, we examine and classify more than one hundred thousand E. coli and Shigella genomes. After removing outliers, a semi-automated Mash-based analysis of 10,667 assembled genomes reveals 14 distinct phylogroups. A representative genome or medoid identified for each phylogroup serves as a proxy to classify more than 95,000 unassembled genomes. This analysis shows that most sequenced E. coli genomes belong to 4 phylogroups (A, C, B1 and E2(O157)). Authenticity of the 14 phylogroups described is supported by pangenomic and phylogenetic analyses, which show differences in gene preservation between phylogroups. A phylogenetic tree constructed with 2,613 single copy core genes along with a matrix of phylogenetic profiles is used to confirm that the 14 phylogroups change at different rates of gene gain/loss/duplication. The methodology used in this work is able to identify previously uncharacterized phylogroups in E. coli species. Some of these new phylogroups harbor clonal strains that have undergone a process of genomic adaptation to the acquisition of new genomic elements related to virulence or antibiotic resistance. This is, to our knowledge, the largest E. coli genome dataset analyzed to date and provides valuable insights into the population structure of the species.

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Escherichia coli HS and Enterotoxigenic Escherichia coli Hinder Stress Granule Assembly

Microorganisms ◽

10.3390/microorganisms9010017 ◽

2020 ◽

Vol 9 (1) ◽

pp. 17

Author(s):

Felipe Velásquez ◽

Josefina Marín-Rojas ◽

Ricardo Soto-Rifo ◽

Alexia Torres ◽

Felipe Del Canto ◽

...

Keyword(s):

Escherichia Coli ◽

Host Cell ◽

Stress Responses ◽

Bacterial Species ◽

Coli Strain ◽

Stress Granule ◽

Enterotoxigenic Escherichia Coli ◽

E Coli ◽

Eif2α Phosphorylation ◽

Successful Infection

Escherichia coli, one of the most abundant bacterial species in the human gut microbiota, has developed a mutualistic relationship with its host, regulating immunological responses. In contrast, enterotoxigenic E. coli (ETEC), one of the main etiologic agents of diarrheal morbidity and mortality in children under the age of five in developing countries, has developed mechanisms to reduce the immune-activator effect to carry out a successful infection. Following infection, the host cell initiates the shutting-off of protein synthesis and stress granule (SG) assembly. This is mostly mediated by the phosphorylation of translation initiator factor 2α (eIF2α). We therefore evaluated the ability of a non-pathogenic E. coli strain (E. coli HS) and an ETEC strain (ETEC 1766a) to induce stress granule assembly, even in response to exogenous stresses. In this work, we found that infection with E. coli HS or ETEC 1766a prevents SG assembly in Caco-2 cells treated with sodium arsenite (Ars) after infection. We also show that this effect occurs through an eIF2α phosphorylation (eIF2α-P)-dependent mechanism. Understanding how bacteria counters host stress responses will lay the groundwork for new therapeutic strategies to bolster host cell immune defenses against these pathogens.

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Socru: Typing of genome level order and orientation in bacteria

10.1101/543702 ◽

2019 ◽

Author(s):

Andrew J. Page ◽

Gemma C. Langridge

Keyword(s):

Large Scale ◽

Genome Rearrangement ◽

Bacterial Species ◽

Level Structure ◽

Typing Method ◽

Long Read ◽

Multiple Copies ◽

Ribosomal Operons ◽

Genome Level ◽

Species Specific

AbstractSummaryGenome rearrangements occur in bacteria between repeat sequences and impact growth and gene expression. Homologous recombination can occur between ribosomal operons, which are found in multiple copies in many bacteria. Inversion between indirect repeats and excision/translocation between direct repeats enable structural genome rearrangement. To identify what these rearrangements are by sequencing, reads of several thousand bases are required to span the ribosomal operons. With long read sequencing aiding the routine generation of complete bacterial assemblies, we have developed socru, a typing method for the order and orientation of genome fragments between ribosomal operons, defined against species-specific baselines. It allows for a single identifier to convey the order and orientation of genome level structure and 434 of the most common bacterial species are supported. Additionally, socru can be used to identify large scale misassemblies.Availability and implementationSocru is written in Python 3, runs on Linux and OSX systems and is available under the open source license GNU GPL 3 from https://github.com/quadram-institute-bioscience/[email protected]

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