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Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
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Published in final edited form as:

Curr Opin Infect Dis. 2020 October ; 33(5): 372–380. doi:10.1097/QCO.0000000000000665.

Updates on defining and detecting diarrheagenic Escherichia

coli pathotypes
Kelsey J Jesser1, Karen Levy1
1Department of Environmental and Occupational Health Sciences, University of Washington,
Seattle, Washington, USA
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Abstract
Purpose of review—Several types of Escherichia coli cause acute diarrhea in humans and are
responsible for a large burden of disease globally. The purpose of this review is to summarize
diarrheagenic Escherichia coli (DEC) pathotype definitions and discuss existing and emerging
molecular, genomic, and gut microbiome methods to detect, define, and study DEC pathotypes.

Recent findings—DEC pathotypes are currently diagnosed by molecular detection of unique

virulence genes. However, some pathotypes have defied coherent molecular definitions because of
imperfect gene targets, and pathotype categories are complicated by hybrid strains and isolation of
pathotypes from asymptomatic individuals. Recent progress toward more efficient, sensitive, and
multiplex DEC pathotype detection has been made using emerging PCR-based technologies.
Genomics and gut microbiome detection methods continue to advance rapidly and are contributing
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to a better understanding of DEC pathotype diversity and functional potential.

Summary—DEC pathotype categorizations and detection methods are useful but imperfect. The
implementation of molecular and sequence-based methods and well-designed epidemiological
studies will continue to advance understanding of DEC pathotypes. Additional emphasis is needed
on sequencing DEC genomes from regions of the world where they cause the most disease and
from the pathotypes that cause the greatest burden of disease globally.

Keywords
Escherichia coli; diarrhea; pathotypes; genomics; molecular detection; gut microbiome

INTRODUCTION
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Escherichia coli is one of the most important and widely studied etiologic agents of diarrhea
worldwide [1-4]. Though usually a benign member of the commensal gut microbiota [5],
some E. coli strains have horizontally acquired virulence characteristics that enable them to
cause diarrheagenic and extraintestinal illness in humans and other animals [6]. Human
diarrheagenic E. coli (DEC) with specific combinations of virulence traits are grouped into
pathotypes, each with unique host preferences, global prevalence, disease burdens, and

Corresponding author Karen Levy, 4225 Roosevelt Way NE, Seattle, WA, 98105, 206-543-6991.
CONFLICTS OF INTEREST
None.
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modes of transmission [7]. DEC pathotypes include the enterotoxigenic E. coli (ETEC),
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enteropathogenic E. coli (EPEC), Shiga toxin producing E. coli (STEC), enteroinvasive E.

coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC).

Precise definitions for each pathotype are important to effectively diagnose and treat DEC
infections, design vaccines, and understand pathotype-specific disease burdens. However, E.
coli strains within each pathotype are genetically heterogenous, and some have defied
coherent molecular definitions of pathogenicity [8-10]. Pathotype definitions are
complicated by the frequent isolation of pathogenic E. coli from individuals who are
asymptomatic for acute diarrhea [11], and the existence of hybrid strains [12]. It is critical
that DEC detection methods are as sensitive and specific as possible, which requires
accurate and comprehensive characterization of each pathotype. Several excellent reviews
have summarized research efforts for DEC pathotypes [e.g. 2,7,12,13]. Here we provide a
brief overview of DEC pathotype definitions and provide updates on current and emerging
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technologies used to detect and define DEC.

DEC PATHOTYPES
In this section we provide a brief overview of the gene targets most commonly used to detect
the six DEC pathotypes and discuss hybrid pathotypes. Additional pathotype characteristics
are summarized in Table 1.

ETEC
ETEC causes loose, watery stools in children in low- and middle-income countries (LMICs)
and travelers to endemic regions. Diagnosis relies on the presence of colonization factors or
enterotoxin genes, usually the heat-labile (lt) and heat-stable (sta) enterotoxins [14]. The
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presence of either or both the lt and sta toxin genes defines ETEC strains [14,15], and the
contribution of both hemolysins to ETEC pathogenicity has been demonstrated in
epidemiological and volunteer studies [e.g. 16-18].

EPEC
Like ETEC and most other DEC pathotypes, EPEC is often associated with watery diarrhea
in children in LMICs. EPEC virulence genes are encoded on the chromosomal locus of
enterocyte effacement (LEE) pathogenicity island [19], and detection centers on the LEE-
encoded intimin gene eae [20]. Some EPEC strains also carry the EPEC adherence factor
plasmid (pEAF), which encodes the bundle-forming pilus gene bfp [13]. Strains that are bfp
+ are termed typical EPEC (tEPEC), and strains that are bfp− are defined as atypical EPEC
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(aEPEC) [21].

STEC/EHEC
STEC cause mild or bloody diarrhea, often accompanied by fever and vomiting. Infections
are usually self-limiting, but the STEC subtype enterohemorrhagic E. coli (EHEC) is
frequently linked to life-threatening foodborne disease outbreaks. STEC strains are defined
by the presence of the phage-encoded Shiga toxin, and EHEC strains have additional

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virulence factors whose expression results in hemorrhagic colitis (bloody diarrhea) and, in
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some cases, life-threatening hemolytic uremic syndrome (HUS). All EHEC are STEC but
not all STEC are EHEC. STEC diagnosis relies on the molecular detection of Shiga toxin
variants (stx genes) and accessory virulence genes, including markers encoded by the LEE
pathogenicity island also present in EPEC. Diagnostic methods for EHEC strains often
target plasmid-encoded hemolysin genes [22,23]. Serotyping is used for the identification of
E. coli O157:H7 and other EHEC strains that often cause food-related outbreaks [24,25].”

EIEC
Diarrhea caused by EIEC occurs worldwide but is especially common in children in LMICs
[22-24]. The clinical presentation and virulence mechanisms of EIEC are indistinguishable
from those initiated by closely-related Shigella spp. and both EIEC and Shigella carry the
pINV F-type plasmid which encodes the genes necessary for enteroinvasive pathogenesis
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[22,25]. Molecular detection of the pINV-encoded gene ipaH, a type-III effector protein, is
used to differentiate Shigella and EIEC from other pathotypes [26,27]. EIEC isolates are
distinguished from Shigella using biochemical characteristics [27] or molecular assays,
many of which detect the E. coli-specific lacY gene [22,28,29].

EAEC
EAEC is an emerging diarrheagenic and extraintestinal pathogen that affects all age groups
and is prevalent in industrialized and LMIC settings. The pathogenicity and clinical
relevance of EAEC are questionable because asymptomatic carriage is common and
volunteer studies have inconsistently linked ingestion to diarrhea [30,31]. EAEC cells adhere
to each other and host intestinal epithelial cells during infection, forming a characteristic
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stacked-brick or honeycomb formation when cultured on HEp-2 cells [9,32]. The

microscopic HEp-2 cell assay is considered the gold standard for EAEC diagnosis [13].
Several molecular targets have also been used for detection, but there is significant diversity
within the EAEC pathotype and a coherent molecular definition of EAEC does not yet exist
[33]. Frequently used marker genes for EAEC include aatA, a plasmid-encoded gene
important for biofilm formation, aggR, a plasmid-encoded transcriptional activator, and
aaiC, a gene located on a genomic island with a type-VI secretion system [34-36]. To date
the best definition of EAEC may be the presence of the aggregative pattern in the HEp-2
assay and the lack of markers associated with other pathotypes [13,37]. Whole genome
studies of EAEC isolates are needed in order to further define gene targets and the molecular
epidemiology of EAEC.
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DAEC
There is epidemiological evidence linking DAEC to diarrhea in children in LMICs, but, like
EAEC, its status as a diarrheal disease agent is uncertain due to inconclusive challenge
studies and high rates of asymptomatic carriage [38-40]. Methodologies for DAEC detection
are not well-defined because strains are heterogenous and DAEC-specific molecular
characteristics have not been established. DAEC were first defined by their distinctive
pattern of diffuse adherence to cells in culture [32,41]. However, this pattern of cell adhesion

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is not suitable for diagnosis because some aEPEC strains also have this phenotype [42].
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DAEC strains have also been defined based on the presence of adhesin genes encoded by the
afa, dra, or daa operons, which are structurally and functionally similar to one another
[8,43]. Molecular assays to detect daaC, daaE, afaB, afaC, and other genes in the afa, dra,
and daa operons have been developed, but are cross-reactive with well-characterized EAEC
genes [8]. As with EAEC, whole-genome sequencing studies coupled with epidemiological
data are needed to better characterize DAEC.

Hybrid strains
Several studies have identified hybrid strains that carry genes associated with multiple DEC
pathotypes. These include Shiga toxin-producing EAEC strains, which have caused disease
outbreaks in Europe [44,45], as well as Shiga toxin-producing ETEC in livestock [46,47],
EPEC strains that carry the ETEC lt hemolysin gene [48], and aEPEC strains that encode
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genes typically found in extraintestinal pathogenic E. coli (ExPEC) [49]. Hybrid strains are
relatively rare, which is perhaps surprising given the mobile nature of DEC virulence genes
[50], but are important examples of the limitations of DEC pathotype designations.

RECENT ADVANCES IN DIAGNOSTIC METHODS

We focus here on research efforts to detect and define DEC pathotypes, citing recent studies
that have used established and novel applications of PCR- and sequence-based assays.

Molecular methods
PCR-based molecular methods are widely used to detect and study DEC because they are
sensitive, specific, and relatively rapid and easy to use. Both conventional and real-time
quantitative PCR (qPCR) are widely used for molecular detection of DEC virulence factors.
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In contrast to conventional PCR, in which PCR products are visualized using gel
electrophoresis, qPCR amplification is measured via fluorescent reporter molecules and
targets are quantified relative to a standard curve. Recent qPCR-based DEC research efforts
include the development of multiplex qPCR assays for DEC and other enteropathogens
[51,52] and new assays to detect all known subtypes of Shiga toxin genes [53].

Newer tools are also being used for DEC pathotype detection. These include Luminex and
BioFire panels designed to detect and diagnose an array of gastrointestinal pathogens. Both
the Luminex and BioFire platforms take advantage of known DEC pathotype targets to
deliver fast diagnostic results in clinical settings [54]. In addition to other common
diarrheagenic pathogens, the Luminex panel detects ETEC, STEC, EHEC O157:H7, and
Shigella/EIEC. The BioFire panel also detects ETEC, STEC, EHEC O157:H7, and Shigella/
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EIEC, and has additional gene targets for EAEC and EPEC [55]. Other emerging methods
such as digital droplet PCR (ddPCR) and Taqman Array Card technologies have contributed
to improved amplification, detection, multiplexing, and automation capabilities for DEC
pathotypes. In ddPCR, qPCR reactions are partitioned into thousands of oil droplets,
enabling absolute target quantification and reducing sample inhibition. A comparison of
ddPCR to conventional and qPCR methods for detecting the EIEC/Shigella gene target ipaH
found that ddPCR shortened detection time and was 10X and 100X more sensitive than

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conventional PCR and qPCR, respectively [56]. TAC relies on a microfluidic card to run
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qPCR assays for multiple gene targets simultaneously, effectively eliminating variability
between methods, requiring less labor and time, and, most importantly, contributing to a
better and more complete understanding of the prevalence and clinical relevance of diarrheal
disease agents. TAC has most recently been used to identify DEC and other enteropathogens
in travelers [57], U.S. military personnel [58], and children in LMICs [59,60], and studies
have confirmed that TAC methods have good sensitivity and specificity for DEC gene targets
[61-63]. The utility of TAC to study childhood diarrhea in LMIC settings has been
demonstrated by its use in the GEMS (Global Enteric Multicenter Study) and MAL-ED
(Malnutrition and Enteric Disease) studies [62-66], as well as its recent implementation in
CHAMPS (Child Health and Mortality Prevention Surveillance) study sites in Asia and
Africa [67].

Unfortunately, molecular diagnostics do not work well for all DEC pathotypes, in particular
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due to issues defining molecular targets for EAEC and DAEC. In addition, high rates of
asymptomatic carriage in epidemiological studies indicate inconsistent relationships
between marker gene presence and diarrhea symptoms [4,65]. qPCR and ddPCR are
extremely sensitive, allowing for the detection of very small amounts of pathogen, which
may or may not represent biologically or clinically relevant infections [11,65]. Also, while
molecular methods have been increasingly implemented in LMICs, access to appropriate
training, supplies, and equipment may be limited. Nonetheless, both established and
emerging PCR-based methods are critical for detecting and diagnosing DEC pathotypes in
both clinical and research settings.

Genomics
In contrast to molecular approaches that detect specific gene targets, genomic methods
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sequence all of the genetic material encoded in the bacterial genome. Genomics can be used
to understand the structure, function, and relatedness of DEC pathotype strains, and genome-
based analyses can reliably predict DEC pathotypes, serotype, multilocus sequence type, and
other characteristics [68,69]. Comparative genome analyses have been used to find new
pathotype-specific and diarrhea-associated genes, investigate antibiotic resistance, and
examine transmission pathways [70-75]. Results of whole-genome sequencing can be
entered into disease surveillance database resources such as the CDC’s PulseNet [76] and
FDA’s GenomeTrakr [77] to track disease outbreaks. Recent research efforts have identified
unique plasmids, diverse colonization factors, pathotype and strain-specific gene
duplications [17,78-80]. Comparative genomics studies are also used to investigate the
virulence potential and origins of hybrid DEC pathotypes [49,81,82].
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Microbial genomics continues to advance rapidly, but important limitations remain. Most
genomic sequencing approaches require isolation of the microorganism of interest on culture
media before sequencing. This culture-based step requires additional effort and time-to-
result. However, exceptions to the culture-based step of strain isolation have been
demonstrated by studies in which high-quality bacterial genomes have been recovered
directly from shotgun metagenomes [83,84] or using single cell sequencing [85]. Also,
despite the ongoing rapid development and implementation of user-friendly sequencing

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technologies and analysis pipelines, genome sequencing usually necessitates complex

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library preparation methods and data processing requires bioinformatics skills.

Perhaps the largest limitation of genomics is that taxonomic and functional annotation of
sequence-based datasets relies on large public databases that have high levels of
misannotation and uneven coverage of taxonomic groups [86]. While E. coli is the most
widely studied organism in the world, DEC pathotypes are not evenly represented in
sequencing databases. We demonstrated this by performing a blastn analysis of DEC
pathotype genes against all complete and draft E. coli genomes in the NCBI Genome
Database (Table 2). This exploration of DEC pathotype marker gene presence in published
E. coli genomes indicates that some pathotypes (especially STEC/EHEC) may have higher
representation in NCBI than others. STEC/EHEC prevalence in the NCBI Genome Database
is perhaps unsurprising given their importance as foodborne pathogens in industrialized
nations. However, EPEC and ETEC are the DEC pathotypes with the highest global burden
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of disease, and are closely linked to childhood mortality in LMICs [3,87], and additional
sequencing efforts are needed for these pathotypes. Importantly, the global prevalence of
DAEC and EAEC has been difficult to determine because the gene targets used to detect
these pathotypes are not specific. Additional genome sequencing efforts are warranted in
order to better define molecular targets for DAEC and EAEC.

DEC and the gut microbiome

Sequence-based gut microbiome studies of DEC infection can characterize pathogen-
induced shifts in the composition of the complex microbial communities of the gut. The two
most widely used approaches for analyzing the gut microbiome are marker-based amplicon
sequencing and whole-metagenome shotgun sequencing. Amplicon sequencing studies
usually target the 16S ribosomal RNA (rRNA gene) and are used to characterize the
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taxonomic composition and diversity of microbial communities in the gut [88]. 16S rRNA
sequencing has been used to investigate shifts in the gut microbiome during ETEC infection
[89], but 16S gene studies generally do not have sufficient resolution to identify bacteria to
the species level.

Shotgun metagenomes are obtained by sequencing the genome content of all microbes
present in a sample and provide both taxonomic and functional gene information. In contrast
to 16S rRNA marker-based sequencing, strain-level taxonomic resolution can be inferred
using metagenomic data [90]. Recent studies demonstrate how metagenomic methods can be
used to understand the implications of DEC infections. For example, a study that relied on
shotgun metagenomes and PCR-pathotyped DEC isolates from an epidemiological study of
DEC-associated diarrhea in Ecuador assembled high-quality E. coli genomes from
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metagenome data and used these to directly measure the population diversity of E. coli in the
gut and the functional and virulence characteristics of DEC [84]. The potential and power of
shotgun metagenome-based DEC pathotype detection has also been demonstrated in food
and water samples [91,92].

As with genomics, annotation of gut microbiome data relies on database comparisons, and,
though E. coli are well represented in reference databases, it is important to consider the
implications of database biases and misannotations. Gut microbiome methods are not

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currently practical in many LMIC settings due to significant equipment and training
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requirements, and are usually more time- and computationally-intensive than PCR-based
detection methods. In addition, both marker-based and shotgun metagenome sequencing are
considered only semi-quantitative. Thus, pathogen detection and quantification usually rely
on quantitative PCR-based methods. However, emerging approaches for sequencing and
analysis are contributing to faster and more quantitative gut microbiome methods [93-95].
Gut microbiome methods are helping to untangle the complex relationships between
commensal and pathogenic bacteria in the host, and these efforts have important
implications for future development of diagnostics, therapeutics, and vaccines.

CONCLUSIONS
Grouping DEC into pathotypes based on shared characteristics is useful in order to
understand differences in the virulence mechanisms, prevalence, modes of transmission, and
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other characteristics associated with various subtypes. However, pathotype designations have
limited capacity to accommodate DEC strains that do not neatly fit pathotype definitions,
and genomics and metagenomics have and will continue to highlight the imperfections in
classic DEC pathotype definitions while concurrently providing new information to help
improve diagnostic methods and definitions. E. coli virulence determinants are horizontally
acquired, and it is likely that gene losses and gains will continue to result in emerging
pathogenic strains of DEC that do not align with current pathotype definitions. It is
important to acknowledge and understand exceptions to DEC pathotype definitions and to
continue to use molecular and sequence-based tools to understand the breadth of DEC
diversity at both the strain and population levels. Continuing to do this in combination with
well-designed epidemiological studies will help us to better understand DEC-associated
disease outcomes and design treatment and intervention strategies to improve health
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outcomes.

ACKNOWLEDGEMENTS
Our research is supported by the National Institute for Allergy and Infectious Diseases (Grant number
R01AI137679 to K.L.). At the time of writing K.J. was supported by a NIEHS T32 Postdoctoral Fellowship/
Traineeship at Emory University (5T32ES012870-15). K.J. is currently funded by an NIEHS T32 Postdoctoral
Fellowship/Traineeship at the University of Washington (T32ES007032-37). This content is solely the
responsibility of the authors and does not necessarily represent the official views of the National Institutes of
Health.

REFERENCES
1. Pearson JS, Giogha C, Wong T, Lung F, Hartland EL: The genetics of enteropathogenic Escherichia
coli virulence. Annu Rev Genet 2016, 50:493–513. [PubMed: 27893961]
Author Manuscript

2. Kaper JB, Nataro JP, Mobley HLT: Pathogenic Escherichia coli. Nat Rev Microbiol 2004, 2:123–
140. [PubMed: 15040260]
3. Troeger C, Forouzanfar M, Rao PC, Khalil I, Brown A, Reiner RC, Fullman N, Thompson RL,
Abajobir A, Ahmed M, et al.: Estimates of global, regional, and national morbidity, mortality, and
aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study
2015. Lancet Infect Dis 2017, 17:909–948. [PubMed: 28579426]
4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur
D, Breiman RF, et al.: Burden and aetiology of diarrhoeal disease in infants and young children in

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 8

developing countries (the Global Enteric Multicenter Study, GEMS): A prospective, case-control
study. Lancet 2013, 382:209–222. [PubMed: 23680352]
Author Manuscript

5. Tenaillon O, Skurnik D, Picard B, Denamur E: The population genetics of commensal Escherichia

coli. Nat Rev Microbiol 2010, 8:207–217. [PubMed: 20157339]
6. Dobrindt U: (Patho-)genomics of Escherichia coli. Int J Med Microbiol 2005, 295:357–371.
[PubMed: 16238013]
7. Nataro JP, Kaper JB: Diarrheagenic Escherichia coli. 1998.
8. Servin AL: Pathogenesis of human diffusely adhering Escherichia coli expressing Afa/Dr adhesins
(Afa/Dr DAEC): Current insights and future challenges. Clin Microbiol Rev 2014, 27:823–869.
[PubMed: 25278576]
9. Kaur P, Asea A, Chakraborti A: Enteroaggregative Escherichia coli: An emerging enteric food borne
pathogen. Interdiscip Perspect Infect Dis 2010, 2010.
10. Rasko DA, Rosovitz MJ, Myers GSA, Mongodin EF, Fricke WF, Gajer P, Crabtree J, Sebaihia M,
Thomson NR, Chaudhuri R, et al.: The pangenome structure of Escherichia coli: Comparative
genomic analysis of E. coli commensal and pathogenic isolates. J Bacteriol 2008, 190:6881–6893.
[PubMed: 18676672]
Author Manuscript

11. Levine MM, Robins-Browne RM: Factors that explain excretion of enteric pathogens by persons
without diarrhea. Clin Infect Dis 2012, 55:S303–S311. [PubMed: 23169942]
12. Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB: Recent advances in
understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev 2013, 26:822–880.
[PubMed: 24092857]
13. Gomes TAT, Elias WP, Scaletsky ICA, Guth BEC, Rodrigues JF, Piazza RMF, Ferreira LCS,
Martinez MB: Diarrheagenic Escherichia coli. Brazilian J Microbiol 2016, 47:3–30.
14. Youmans BP, Ajami NJ, Jiang ZD, Petrosino JF, DuPont HL, Highlander SK: Development and
accuracy of quantitative real-time polymerase chain reaction assays for detection and
quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in
travelers’ diarrhea samples. Am J Trop Med Hyg 2014, 90:124–132. [PubMed: 24189361]
15. Qadri F, Svennerholm A-M, Faruque ASG, Sack RB: Enterotoxigenic Escherichia coli in
developing countries: epidemiology, microbiology, clinical features, treatment, and prevention.
Clin Microbiol Rev 2005, 18:465–483. [PubMed: 16020685]
Author Manuscript

16. Kim KH, Suh IS, Kim JM, Kim CW, Cho YJ: Etiology of childhood diarrhea in Korea. J Clin
Microbiol 1989, 27:1192. [PubMed: 2666437]
17 *. Vidal RM, Muhsen K, Tennant SM, Svennerholm A-M, Sow SO, Sur D, Zaidi AKM, Faruque
ASG, Saha D, Adegbola R, et al.: Colonization factors among enterotoxigenic Escherichia coli
isolates from children with moderate-to-severe diarrhea and from matched controls in the Global
Enteric Multicenter Study (GEMS). PLoS Negl Trop Dis 2019, 13:e0007037. [ [PubMed:
30608930] This comparative genomics study investigated the prevalence of ETEC colonization
factors in children <5 with and without diarrhea in Asia and Africa, and determined that
colonization factors that are vaccine candidates could potentially prevent up to 66% of pediatric
diarrhea associated with sta-encoding ETEC.]
18. Ifeanyi CIC, Ikeneche NF, Bassey BE, Al-Gallas N, Casmir AA, Nnennaya IR: Characterization of
toxins and colonization factors of enterotoxigenic escherichia coli isolates from children with
acute Diarrhea in Abuja, Nigeria. Jundishapur J Microbiol 2018, 11.
19. Schmidt MA: LEEways: tales of EPEC, ATEC and EHEC. Cell Microbiol 2010, 12:1544–1552.
[PubMed: 20716205]
Author Manuscript

20. Ochoa TJ, Contreras CA: Enteropathogenic Escherichia coli infection in children. Curr Opin Infect
Dis 2011, 24:478–483. [PubMed: 21857511]
21. Alikhani MY, Mirsalehian A, Aslani MM: Detection of typical and atypical enteropathogenic
Escherichia coli (EPEC) in Iranian children with and without diarrhoea. J Med Microbiol 2006,
55:1159–1163. [PubMed: 16914644]
22. Ud-Din A, Wahid S: Relationship among Shigella spp. And enteroinvasive Escherichia coli (EIEC)
and their differentiation. Brazilian J Microbiol 2014, 45:1131–1138.

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 9

23. Moreno ACR, Filho AF, Gomes T do AT, Ramos STS, Montemor LPG, Tavares VC, Filho L dos S,
Irino K, Martinez MB: Etiology of childhood diarrhea in the northeast of Brazil: significant
Author Manuscript

emergent diarrheal pathogens. Diagn Microbiol Infect Dis 2010, 66:50–57. [PubMed: 18508227]
24. Turhanoglu M, Gulsun S, Onur A, Bilman FB: The frequency of Escherichia coli (EPEC, ETEC,
EIEC and serotypes) Shigella, rotavirus and parasite agents among children with acute
gastroenteritis in Southeast Anatolia, Turkey. African J Microbiol Res 2012, 6.
25. Pasqua M, Michelacci V, Di Martino ML, Tozzoli R, Grossi M, Colonna B, Morabito S, Prosseda
G: The intriguing evolutionary journey of enteroinvasive E. coli (EIEC) toward pathogenicity.
Front Microbiol 2017, 8:1–12. [PubMed: 28197127]
26. Gomes TAT, Elias WP, Scaletsky ICA, Guth BEC, Rodrigues JF, Piazza RMF, Ferreira LCS,
Martinez MB: Diarrheagenic Escherichia coli. Brazilian J Microbiol 2016, doi:10.1016/
j.bjm.2016.10.015.
27. Van Den Beld MJC, Reubsaet FAG: Differentiation between Shigella, enteroinvasive Escherichia
coli (EIEC) and noninvasive Escherichia coli. Eur J Clin Microbiol Infect Dis 2012, 31:899–904.
[PubMed: 21901636]
28. Pavlovic M, Luze A, Konrad R, Berger A, Sing A, Busch U, Huber I: Development of a duplex
Author Manuscript

real-time PCR for differentiation between E. coli and Shigella spp. J Appl Microbiol 2011,
110:1245–1251. [PubMed: 21332893]
29. Dhakal R, Wang Q, Lan R, Howard P, Sintchenko V: Novel multiplex PCR assay for identification
and subtyping of enteroinvasive escherichia coli and differentiation from Shigella based on target
genes selected by comparative genomics. J Med Microbiol 2018, 67:1257–1264. [PubMed:
29969087]
30. Nataro JP, Yikang D, Cookson S, Cravioto A, Savarino SJ, Guers LD, Levine MM, Tacket CO:
Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated. J Infect Dis 1995,
171:465–468. [PubMed: 7844392]
31. Mathewson JJ, Johnson PC, Dupont HL, Satterwhite JK, Winsor DK: Pathogenicity of
enteroadherent Escherichia coli in adult volunteers. J Infect Dis 1986, 154:524–527. [PubMed:
3525699]
32. Nataro JP, Kaper JB, Robins-Browne R, Prado V, Vial P, Levine MM: Patterns of adherence of
diarrheagenic escherichia coli to HEp-2 cells. Pediatr Infect Dis J 1987, 6:829–831. [PubMed:
3313248]
Author Manuscript

33. Jensen BH, Olsen KEP, Struve C, Krogfelt KA, Petersen AM: Epidemiology and clinical
manifestations of enteroaggregative Escherichia coli. 2014, 27:614–630.
34. Dudley EG, Thomson NR, Parkhill J, Morin NP, Nataro JP: Proteomic and microarray
characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative
Escherichia coli. Mol Microbiol 2006, 61:1267–1282. [PubMed: 16925558]
35. Moon JY, Park JH, Kim YB: Molecular epidemiological characteristics of virulence factors on
enteroaggregative E. coli. FEMS Microbiol Lett 2005, 253:215–220. [PubMed: 16257141]
36. Nishi J, Sheikh J, Mizuguchi K, Luisi B, Burland V, Boutin A, Rose DJ, Blattner FR, Nataro JP:
The export of coat protein from enteroaggregative Escherichia coli by a specific ATP-binding
cassette transporter system. J Biol Chem 2003, 278:45680–45689. [PubMed: 12933818]
37. Boisen N, Scheutz F, Rasko DA, Redman JC, Persson S, Simon J, Kotloff KL, Levine MM, Sow S,
Tamboura B, et al.: Genomic characterization of enteroaggregative Escherichia coli from children
in Mali. J Infect Dis 2012, 205:431–444. [PubMed: 22184729]
38. Servin AL: Pathogenesis of Afa/Dr Diffusely Adhering Escherichia coli. Clin Microbiol Rev 2005,
Author Manuscript

18:264–292. [PubMed: 15831825]

39. Le Bouguénec C, Servin AL: Diffusely adherent Escherichia coli strains expressing Afa/Dr
adhesins (Afa/Dr DAEC): Hitherto unrecognized pathogens. FEMS Microbiol Lett 2006,
256:185–194. [PubMed: 16499605]
40. Tacket CO, Moseley SL, Kay B, Losonsky G, Levine MM: Challenge studies in volunteers using
Escherichia coli strains with diffuse adherence to HEp-2 cells. J Infect Dis 1990, 162:550–2.
[PubMed: 2197345]
41. Scaletsky ICA, Silva MLM, Trabulsi LR: Distinctive patterns of adherence of enteropathogenic
Escherichia coli to HeLa cells. Infect Immun 1984, 45:534–536. [PubMed: 6146569]

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 10

42. Hernandes RT, Elias WP, Vieira MAM, Gomes TAT: An overview of atypical enteropathogenic
Escherichia coli. FEMS Microbiol Lett 2009, 297:137–149. [PubMed: 19527295]
Author Manuscript

43. Van Loy CP, Sokurenko E V, Moseley SL: The major structural subunits of Dr and F1845 fimbriae
are adhesins. Infect Immun 2002, 70:1694–1702. [PubMed: 11895931]
44. Muniesa M, Hammerl JA, Hertwig S, Appel B, Brüssow H: Shiga toxin-producing Escherichia coli
O104:H4: A new challenge for microbiology. Appl Environ Microbiol 2012, 78:4065–4073.
[PubMed: 22504816]
45. Soysal N, Mariani-Kurkdjian P, Smail Y, Liguori S, Gouali M, Loukiadis E, Fach P, Bruyand M,
Blanco J, Bidet P, et al.: Enterohemorrhagic Escherichia coli hybrid pathotype O80:H2 as a new
therapeutic challenge. Emerg Infect Dis 2016, 22:1604–1612. [PubMed: 27533474]
46. Johura FT, Parveen R, Islam A, Sadique A, Rahim MN, Monira S, Khan AR, Ahsan S, Ohnishi M,
Watanabe H, et al.: Occurrence of hybrid Escherichia coli strains carrying Shiga toxin and heat-
stable toxin in livestock of Bangladesh. Front Public Heal 2017, 4:287.
47. Nyholm O, Halkilahti J, Wiklund G, Okeke U, Paulin L, Auvinen P, Haukka K, Siitonen A:
Comparative genomics and characterization of hybrid Shigatoxigenic and enterotoxigenic
Escherichia coli (STEC/ETEC) strains. PLoS One 2015, 10.
Author Manuscript

48. Dutta S, Pazhani GP, Nataro JP, Ramamurthy T: Heterogenic virulence in a diarrheagenic
Escherichia coli: Evidence for an EPEC expressing heat-labile toxin of ETEC. Int J Med
Microbiol 2015, 305:47–54. [PubMed: 25465159]
49. Díaz-Jiménez D, García-Meniño I, Herrera A, García V, López-Beceiro AM, Alonso MP, Blanco J,
Mora A: Genomic characterization of Escherichia coli isolates belonging to a new hybrid aEPEC/
ExPEC pathotype O153:H10-A-ST10 eae-beta1 cccurred in meat, poultry, wildlife and human
diarrheagenic samples. Antibiotics 2020, 9:192.
50. Robins-Browne RM, Holt KE, Ingle DJ, Hocking DM, Yang J, Tauschek M: Are Escherichia coli
pathotypes still relevant in the era of whole-genome sequencing? Front Cell Infect Microbiol 2016,
6:1–9. [PubMed: 26870699]
51. Liu Y, Cao Y, Wang T, Dong Q, Li J, Niu C: Detection of 12 common food-borne bacterial
pathogens by TaqMan real-time PCR using a single set of reaction conditions. Front Microbiol
2019, 10:222. [PubMed: 30814987]
52. Wongboot W, Okada K, Chantaroj S, Kamjumphol W, Hamada S: Simultaneous detection and
quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay. J
Author Manuscript

Microbiol Methods 2018, 151:76–82. [PubMed: 29928913]

53. Zhi S, Szelewicki J, Ziebell K, Parsons B, Chui L: General detection of Shiga toxin 2 and
subtyping of Shiga toxin 1 and 2 in Escherichia coli using qPCR. J Microbiol Methods 2019,
159:51–55. [PubMed: 30772308]
54. Huang RSP, Johnson CL, Pritchard L, Hepler R, Ton TT, Dunn JJ: Performance of the Verigene®
enteric pathogens test, Biofire FilmArray™ gastrointestinal panel and Luminex xTAG®
gastrointestinal pathogen panel for detection of common enteric pathogens. Diagn Microbiol Infect
Dis 2016, 86:336–339. [PubMed: 27720206]
55 *. Baghdadi JD, Coffey KC, Leekha S, & Johnson JK, Diekema DJ, Morgan DJ: Diagnostic
Stewardship for Comprehensive Gastrointestinal Pathogen Panel Tests. Curr Infect Dis Rep 2020,
22:1–15. [PubMed: 31933158] [This review summarizes results from widely-used diagnostic GI
pathogen panels and recommends strategies for diagnostic stewardship in order to improve their
implementation.]
56 **. Yang J, Zhang N, Lv J, Zhu P, Pan X, Hu J, Wu W, Li S, Li H: Comparing the performance of
Author Manuscript

conventional PCR, RTQ-PCR, and droplet digital PCR assays in detection of Shigella. Mol Cell
Probes 2020, 51:101531. [PubMed: 32062018] [This study demonstrated that ddPCR was a faster
and more sensitve method than conventional PCR or qPCR for detecting EIEC/Shigella gene
targets in food]
57. Lertsethtakarn P, Silapong S, Sakpaisal P, Serichantalergs O, Ruamsap N, Lurchachaiwong W,
Anuras S, Platts-Mills JA, Liu J, Houpt ER, et al.: Travelers’ diarrhea in Thailand: A quantitative
analysis using TaqMan® Array Card. Clin Infect Dis 2018, 120:67.
58. Lurchachaiwong W, Serichantalergs O, Lertsethtakarn P, Ruamsap N, Srijan A, Oransathid W,
Khemnu N, Vesely BA, Demons ST, Waters NC, et al.: Enteric etiological surveillance in acute

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 11

diarrhea stool of United States Military Personnel on deployment in Thailand, 2013-2017. Gut
Pathog 2020, 12.
Author Manuscript

59 *. Rogawski ET, Liu J, Platts-Mills JA, Kabir F, Lertsethtakarn P, Siguas M, Khan SS, Praharaj I,
Murei A, Nshama R, et al.: Use of quantitative molecular diagnostic methods to investigate the
effect of enteropathogen infections on linear growth in children in low-resource settings:
longitudinal analysis of results from the MAL-ED cohort study. Lancet Glob Heal 2018,
6:e1319–e1328.[This study used TAC to detect 29 enteropathogens in stool samples collected
during the MAL-ED study and estimated associations between pathogens and child growth. They
determined that asymptomatic carriage of EAEC and other enteropathogens had negative impacts
on child growth.]
60. Richard SA, Mccormick BJ, Murray-Kolb LE, Lee GO, Seidman JC, Mahfuz M, Ahmed T,
Guerrant RL, Petri WA, Rogawski ET, et al.: Enteric dysfunction and other factors associated with
attained size at 5 years: MAL-ED birth cohort study findings. Am J Clin Nutr 2019, 110:131–138.
[PubMed: 31127812]
61. Lalani T, Tisdale MD, Liu J, Mitra I, Philip C, Odundo E, Reyes F, Simons MP, Fraser JA, Hutley
E, et al.: Comparison of stool collection and storage on Whatman FTA® Elute cards versus frozen
stool for enteropathogen detection using the TaqMan® Array Card PCR assay. PLoS One 2018,
Author Manuscript

13.
62. Platts-Mills JA, Mccormick BJJ, Kosek M, Pan WK, Checkley W, Houpt ER, Network TM-E:
Methods of analysis of enteropathogen infection in the MAL-ED cohort study. Clin Infect Dis ®
2014, 59:233–241.
63. Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, Verweij JJ, Taniuchi M, Sobuz SU, Haque
R, et al.: A laboratory-developed TaqMan Array Card for simultaneous detection of 19
enteropathogens. 2013, doi:10.1128/JCM.02658-12.
64. Platts-Mills JA, Liu J, Rogawski ET, Kabir F, Lertsethtakarn P, Siguas M, Khan SS, Praharaj I,
Murei A, Nshama R, et al.: Use of quantitative molecular diagnostic methods to assess the
aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a
reanalysis of the MAL-ED cohort study. Lancet Glob Heal 2018, 6:e1309–e1318.
65. Liu J, Platts-Mills JA, Juma J, Kabir F, Nkeze J, Okoi C, Operario DJ, Uddin J, Ahmed S, Alonso
PL, et al.: Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in
children: a reanalysis of the GEMS case-control study. Lancet 2016, 388:1291–1301. [PubMed:
Author Manuscript

27673470]
66. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, McCormick BJJ, McGrath M,
Olortegui MP, Samie A, et al.: Pathogen-specific burdens of community diarrhoea in developing
countries: A multisite birth cohort study (MAL-ED). Lancet Glob Heal 2015, 3:e564–e575.
67 **. Diaz MH, Waller JL, Theodore MJ, Patel N, Wolff BJ, Benitez AJ, Morris T, Raghunathan PL,
Breiman RF, Whitney CG, et al.: Development and implementation of multiplex TaqMan Array
Cards for specimen testing at child health and mortality prevention surveillance site laboratories.
Clin Infect Dis 2020, 69:S311–S321.[CHAMPs laboratories in Africa and Asia developed and
implemented a TAC assay for testing postmortem samples for >100 pathogen gene targets,
including 8 associated with DEC].
68. Joensen KG, Tetzschner AMM, Iguchi A, Aarestrup FM, Scheutz F: Rapid and easy in silico
serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J Clin Microbiol
2015, 53:2410–2426. [PubMed: 25972421]
69. Larsen M V, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-
Pontén T, Ussery DW, Aarestrup FM, et al.: Multilocus sequence typing of total-genome-
Author Manuscript

sequenced bacteria. J Clin Microbiol 2012, 50:1355–1361. [PubMed: 22238442]

70. Van Hoek AHAM, Van Veldhuizen JNJ, Friesema I, Coipan C, Rossen JWA, Bergval IL, Franz E:
Comparative genomics reveals a lack of evidence for pigeons as a main source of stx 2f -carrying
Escherichia coli causing disease in humans and the common existence of hybrid Shiga toxin-
producing and enteropathogenic E. coli pathotypes. BMC Genomics 2019, 20:271. [PubMed:
30953471]
71. Montealegre MC, Talavera Rodríguez A, Roy S, Hossain MI, Islam MA, Lanza VF, Julian TR:
High genomic diversity and heterogenous origins of pathogenic and antibiotic-resistant

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 12

Escherichia coli in household settings represent a challenge to reducing transmission in low-

income settings. mSphere 2020, 5.
Author Manuscript

72 *. Brown E, Dessai U, McGarry S, Gerner-Smidt P: Use of whole-genome sequencing for food

safety and public health in the United States. Foodborne Pathog Dis 2019, 16:441–450.
[PubMed: 31194586] [This review of genome sequencing applications for foodborne pathogens
includes information on number of E. coli genomes that have been uploaded to PulseNet and
GenomeTrakr.]
73. Besser JM, Carleton HA, Trees E, Stroika SG, Hise K, Wise M, Gerner-Smidt P: Interpretation of
whole-genome sequencing for enteric disease surveillance and outbreak investigation. Foodborne
Pathog Dis 2019, 16:504–512. [PubMed: 31246502]
74. Hazen TH, Donnenberg MS, Panchalingam S, Antonio M, Hossain A, Mandomando I, Ochieng
JB, Ramamurthy T, Tamboura B, Qureshi S, et al.: Genomic diversity of EPEC associated with
clinical presentations of differing severity. Nat Microbiol 2016, 1:1–9.
75. Steyert SR, Sahl JW, Fraser CM, Teel LD, Scheutz F, Rasko DA: Comparative genomics and stx
phage characterization of LEE-negative Shiga toxin-producing Escherichia coli. Front Cell Infect
Microbiol 2012, 2:133. [PubMed: 23162798]
Author Manuscript

76. Nadon C, Van Walle I, Gerner-Smidt P, Campos J, Chinen I, Concepcion-Acevedo J, Gilpin B,

Smith AM, Kam KM, Perez E, et al.: Pulsenet International: Vision for the implementation of
whole genome sequencing (WGS) for global foodborne disease surveillance. Eurosurveillance
2017, 22.
77. Timme RE, Sanchez Leon M, Allard MW: Utilizing the public GenomeTrakr database for
foodborne pathogen traceback In Methods in Molecular Biology. . Humana Press Inc.; 2019:201–
212.
78 *. Hazen TH, Nagaraj S, Sen S, Permala-Booth J, Del Canto F, Vidal R, Barry EM, Bitoun JP, Chen
WH, Tennant SM, et al.: Genome and functional characterization of colonization factor antigen I-
and CS6-encoding heat-stable enterotoxin- only enterotoxigenic Escherichia coli reveals lineage
and geographic variation. mSystems 2019, 4:329–347.[This comparative genomics study of
diptherial ETEC found that the CFA/I and CS6 colonization factors were associated with
significant geographical and phylogenetic diversity for sta+ lt− strains.]
79 *. Hazen TH, Rasko DA: The complete genome of the atypical enteropathogenic Escherichia coli
archetype isolate E110019 highlights a role for plasmids in dissemination of the type III secreted
Author Manuscript

effector ESPT. Infect Immun 2019, 87.[This study describes the whole genome sequence of an
outbreak-associated aEPEC strain and the multiple unique plasmids that encode a unique type-III
secreted effector gene.]
80 *. Bernabeu M, Sánchez-Herrero JF, Huedo P, Prieto A, Hüttener M, Rozas J, Juárez A: Gene
duplications in the E. coli genome: Common themes among pathotypes. BMC Genomics 2019,
20:1–11. [PubMed: 30606130] [This study identified several duplicated genes in a high
percentage of DEC and extraintestinal E. coli isolates. Some gene duplications were pathotype or
strain-specific and may play a role in virulence.]
81 *. Bai X, Zhang J, Ambikan A, Jernberg C, Ehricht R, Scheutz F, Xiong Y, Matussek A: Molecular
characterization and comparative genomics of clinical hybrid Shiga toxin-producing and
enterotoxigenic Escherichia coli (STEC/ETEC) strains in Sweden. Sci Rep 2019, 9:1–9.
[PubMed: 30626917] [This study characterized STEC/ETEC hybrid DEC strains using molecular
and comparative genomics methods and found considerable virulence, serotype, and stx subtype
diversity amongst hybrid strains.]
82. Haarmann N, Berger M, Kouzel IU, Mellmann A, Berger P: Comparative virulence
Author Manuscript

characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative
Escherichia coli. Int J Med Microbiol 2018, 308:912–920. [PubMed: 29941383]
83. Loman NJ, Constantinidou C, Christner M, Chan JZM, Quick J, Weir JC, Quince C, Smith GP,
Betley JR, Aepfelbacher M, et al.: A culture-independent sequence-based metagenomics approach
to the investigation of an outbreak of shiga-toxigenic Escherichia coli O104:H4. JAMA - J Am
Med Assoc 2013, 309:1502–1510.
84 **. Pena-Gonzalez A, Soto-Giro MJ, Smith S, Sistrunk J, Montero L, Pa M, Ortega E and acute;a,
Hatt JK, Cevallos W, Trueba G, et al.: Metagenomic signatures of gut infections caused by
different Escherichia coli pathotypes. Appl Environ Microbiol 2019, 85.[This study recovered

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 13

high quality E. coli metagenome assembled genomes from shotgun metagenome and illustrated
that the etiological agent of diarrheal disease can be diagnosed using gut microbiome tools.]
Author Manuscript

85. Cheng M, Cao L, Ning K: Microbiome big-data mining and applications using single-cell
technologies and metagenomics approaches toward precision medicine. Front Genet 2019, 10.
86. Schnoes AM, Brown SD, Dodevski I, Babbitt PC: Annotation error in public databases:
Misannotation of molecular function in enzyme superfamilies. PLoS Comput Biol 2009, 5.
87. Pires SM, Fischer-Walker CL, Lanata CF, Devleesschauwer B, Hall AJ, Kirk MD, Duarte ASR,
Black RE, Angulo FJ: Aetiology-specific estimates of the global and regional incidence and
mortality of diarrhoeal diseases commonly transmitted through food. PLoS One 2015,
10:e0142927. [PubMed: 26632843]
88. Shah N, Tang H, Doak TG, Ye Y: Comparing bacterial communities inferred from 16S rRNA gene
sequencing and shotgun metagenomes In Biocomputing 2011. . WORLD SCIENTIFIC;
2011:165–176.
89. Pop M, Paulson JN, Chakraborty S, Astrovskaya I, Lindsay BR, Li S, Bravo HC, Harro C, Parkhill
J, Walker AW, et al.: Individual-specific changes in the human gut microbiota after challenge with
enterotoxigenic Escherichia coli and subsequent ciprofloxacin treatment. BMC Genomics 2016,
Author Manuscript

17:1–11. [PubMed: 26818753]

90. Jovel J, Patterson J, Wang W, Hotte N, O’Keefe S, Mitchel T, Perry T, Kao D, Mason AL, Madsen
KL, et al.: Characterization of the gut microbiome using 16S or shotgun metagenomics. Front
Microbiol 2016, 7:459. [PubMed: 27148170]
91 *. Hamner S, Brown BL, Hasan NA, Franklin MJ, Doyle J, Eggers MJ, Colwell RR, Ford TE:
Metagenomic profiling of microbial pathogens in the Little Bighorn River, Montana. Int J
Environ Res Public Health 2019, 16:1097.[This study used metagenomic sequencing to identify
EAEC, EHEC, and EPEC in a Montana river.]
92. Leonard SR, Mammel MK, Lacher DW, Elkins CA: Strain-level discrimination of Shiga toxin-
producing Escherichia coli in spinach using metagenomic sequencing. PLoS One 2016, 11.
93. Satinsky BM, Gifford SM, Crump BC, Moran MA: Use of Internal Standards for Quantitative
Metatranscriptome and Metagenome Analysis. Elsevier Inc.; 2013.
94. Lin Y, Gifford S, Ducklow H, Schofield O, Cassara N: Towards quantitative microbiome
community profiling using internal standards. Appl Environ Microbiol 2019, 85.
Author Manuscript

95. Loman NJ, Quick J, Simpson JT: A complete bacterial genome assembled de novo using only
nanopore sequencing data. Nat Methods 2015, 12:733–735. [PubMed: 26076426]
96. Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, Jin Q: VFDB: a reference database for bacterial
virulence factors. Nucleic Acids Res 2005, 33:D325–D328. [PubMed: 15608208]
97. Mirhoseini A, Amani J, Nazarian S: Review on pathogenicity mechanism of enterotoxigenic
Escherichia coli and vaccines against it. Microb Pathog 2018, 117:162–169. [PubMed: 29474827]
98. Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H: Molecular
mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect 2010, 12:89–98.
[PubMed: 19883790]
99. Nguyen RN, Taylor LS, Tauschek M, Robins-Browne RM: Atypical enteropathogenic Escherichia
coli infection and prolonged diarrhea in children. Emerg Infect Dis 2006, 12:597–603. [PubMed:
16704807]
100. Vasco K, Graham JP, Trueba G: Detection of zoonotic enteropathogens in children and domestic
animals in a semirural community in ecuador. Appl Environ Microbiol 2016, 82:4218–4224.
[PubMed: 27208122]
Author Manuscript

101. Nguyen Y, Sperandio V: Enterohemorrhagic E. coli (EHEC) pathogenesis. Front Cell Infect
Microbiol 2012, 2:90. [PubMed: 22919681]
102. Newell DG, La Ragione RM: Enterohaemorrhagic and other Shiga toxin-producing Escherichia
coli (STEC): Where are we now regarding diagnostics and control strategies? Transbound Emerg
Dis 2018, 65:49–71. [PubMed: 29369531]
103. Karch H, Bielaszewska M, Bitzan M, Schmidt H: Epidemiology and diagnosis of Shiga toxin-
producing Escherichia coli infections In Diagnostic Microbiology and Infectious Disease. .
Elsevier; 1999:229–243.

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
 Jesser and Levy Page 14

104. Fagan PK, Hornitzky MA, Bettelheim KA, Djordjevic SP: Detection of Shiga-like toxin (stx 1
and stx 2 ), Intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC
Author Manuscript

hlyA) genes in animal feces by multiplex PCR. Appl Environ Microbiol 1999, 65:868–872.
[PubMed: 9925634]
105. Parsot C: Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS
Microbiol Lett 2005, 252:11–18. [PubMed: 16182469]
106. Harrington SM, Dudley EG, Nataro JP: Pathogenesis of enteroaggregative Escherichia coli
infection. FEMS Microbiol Lett 2006, 254:12–18. [PubMed: 16451173]
107. Rogawski ET, Guerrant RL, Havt A, Lima IFN, Medeiros PHQS, Seidman JC, McCormick BJJ,
Babji S, Hariraju D, Bodhidatta, et al.: Epidemiology of enteroaggregative Escherichia coli
infections and associated outcomes in the MAL-ED birth cohort. PLoS Negl Trop Dis 2017,
11:1–17.
Author Manuscript
Author Manuscript
Author Manuscript

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KEY POINTS
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• Current diarrheagenic E. coli (DEC) pathotype definitions are based on

molecular detection of pathotype-specific virulence genes, but these have not
been well defined for all pathotypes.

• Existing and emerging PCR and sequence-based technologies continue to

move forward efforts to diagnose and study DEC pathotypes.

• Additional sequencing efforts are needed for DEC pathotypes of global

importance.

• Pathotype categorization is imperfect but useful for understanding differences

in DEC pathogenicity, transmission, clinical presentation, and epidemiology.
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Table 1.

Characteristics of DEC pathotypes

Other diagnostic
Pathotype Clinical description Pathogenesis Epidemiology Diagnostic genes Transmission Reference(s)
methods
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Commonly diagnosed in
children in LMIC settings
Loose/watery stools, Relies on adhesins and
and traveler’s diarrhea in The lt and/or sta Human-to-human
ETEC may be accompanied plasmid-encoded n/a [15,97,98]
industrialized nations; hemolysins fecal-oral transmission
by fever and vomiting enterotoxins
asymptomatic carriage is
common
Commonly diagnosed in
Virulence genes on the Human-to-human
children in LMIC settings; tEPEC strains also
LEE pathogenicity island The LEE pathogenicity fecal-oral
Mild to severe loose/ aEPEC has been linked to have the plasmid-
EPEC cause attaching and island-encoded eae transmission; animal- [20,99,100]
watery stools persistent diarrhea lasting encoded bfp bundle-
effacing lesions on adhesin to-human transmission
>14 days; asymptomatic forming pilus gene
enterocytes for aEPEC
carriage is common
The Shiga toxin stx gene
Serotyping, specific Human-to-human and
Mild to severe loose/ defines STEC and
Shiga toxin inhibits Associated with foodborne and differential media animal-to-human
watery stools; EHEC additional targets may be
STEC/ protein synthesis and disease outbreaks, often and molecular assays fecal-oral
causes bloody stools used, including genes [101-104]
EHEC causes host cell death in linked to leafy greens, milk, for EHEC strains, transmission; cattle are
and can advance to encoded by the LEE-
the large intestine or beef products especially EHEC considered a major
life-threatening HUS pathogenicity island also
O157:H7 reservoir
present in EPEC
Enteroinvasive virulence
Mild to severe loose/ mechanism involves EIEC and Shigella
watery stools, rarely crossing the intestinal Commonly diagnosed in isolates can be
EIEC/ accompanied by fever, epithelial barrier, killing LMIC settings and The gene for the type-III differentiated using Human-to-human
[22,25,27,105]
Shigella tenesmus, blood, macrophages, invading traveler’s diarrhea in effector protein ipaH biochemical fecal-oral transmission
mucus, and leukocytes enterocytes, intracellular industrialized nations characteristics or
in the stool and cell-to-cell molecular assays
propagation
The plasmid-encoded Human-to-human
Mild loose/watery Associated with childhood
transcriptional activator Characteristic fecal-oral
stools, may be Colonization is facilitated diarrhea in LMICs;
aggR, pathogenicity stacked-brick or transmission; EAEC
accompanied by fever, by fimbriae, followed by inconsistent results in adult
EAEC island-associated gene honeycomb pattern in has been isolated from [9,33,35,106,107]
nausea, tenesmus, biofilm formation and challenge studies and
aaiC, and the plasmid- microscopic HEp-2 animals but

Curr Opin Infect Dis. Author manuscript; available in PMC 2021 October 01.
borborygmi, or mucus toxin release asymptomatic carriage is
encoded dispersin cell assay transmission has not
in the stool common
transporter aatA been established
Associated with childhood
Adhesins trigger Genes in the afa, dra, or
diarrhea in LMICs; adult
signaling pathways that daa adhesin operons;
Mild loose/watery challenge studies failed to Human-to-human
DAEC promote cell lesions, loss cross-reactivity with HEp-2 cell assay [8]
stools cause diarrhea and fecal-oral transmission
of intestinal microvilli EAEC genes frequently
asymptomatic carriage is
and inflammation reported
common.
Page 16
 Jesser and Levy Page 17

Table 2.

Blastn results for DEC pathotype-specific genes against all complete E. coli genomes (n=1,060) and all E. coli
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draft assemblies (n=18,195) in the NCBI Genome Database (accessed March 9, 2020).

Number of E. Number of total

Number of
coli draft diarrheal deaths
Marker complete E.coli
Pathotype Gene description genomes in children <5
a genomes with
gene with blastn c
b (%)
blastn hits b
hits
eltA 24 474
Heat-labile enterotoxin, subunits A and B
ETEC eltB 24 465 23,649.8 (4.7)
sta Heat-stable enterotoxin 27 279

eaeA LEE-encoded intimin protein 9 159

EPEC 11,284.3 (2.3)
d Plasmid-encoded bundle-forming pilus 1 91
bfpA
aggR Plasmid-encoded transcriptional activator 17 216
EAEC Not measured
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aaiC Pathogenicity island-encoded secreted protein 2 127

afaE-I 7 70
DAEC Afimbrial adhesin subunit, human-specific variants Not measured
afaE-III 0 17

EIEC/Shigella ipaH pINV plasmid-encoded type-III effector protein 2 128 e

54,905.5 (11)

stx1A 126 2104

stx1B 126 2095

STEC/EHEC Shiga-like toxin variants, subunits A and B Not measured
stx2A 202 2970
stx2B 202 2438

a
Reference sequences downloaded from the Virulence Factors Database [96]
b
>90% query coverage and >90% sequence identity
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c
From Global Burden of Disease Study 2015 [3], only reports values for ETEC, EPEC and Shigella
d
present in typical EPEC (tEPEC), absent in atypical EPEC (aEPEC)
e
for Shigella only
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